Cell Biology - Scripps Research Institute · Cell Biology Overview M embers of the Department of...

Cell Biology

Electron cryomicroscopy and single-particle analysis were

used to determine the 30-Å structure of the self-assembling

coat protein complex-II cage nanoparticle. Shown are the

2-fold (bottom), 3-fold (middle), and 4-fold (top) fold sym-

metry axes of the biologically unprecedented cuboctahedron

structure responsible for directing cargo selection and mem-

brane curvature during endoplasmic reticulum vesicle bud-

ding. Reprinted from Stagg, S.M., Gurkan, C., Fowler, D.M.,

LaPointe, P., Foss, T.R., Potter, C.S., Carragher, B., Balch,

W.E. Structure of the Sec13/31 COPII coat cage. Nature

439:234, 2006. This work is a collaboration between the

laboratories of William Balch, Ph.D., Bridget Carragher, Ph.D,

and Clinton Potter, B.S., at the National Resource for

Automated Molecular Microscopy.

Gaudenz Danuser, Ph.D., Associate Professor

Dinah Leorke, Ph.D., Research Associate

James Lim, Graduate Student

Department of Cell Biology

D E P A R T M E N T O F

C E L L B I O L O G Y

S T A F F

Sandra L. Schmid, Ph.D.*Professor and Chairman

Francisco Asturias, Ph.D.**Associate Professor

William E. Balch, Ph.D.*Professor

Kristin Baldwin, Ph.D.***Assistant Professor

Bridget Carragher, Ph.D.**Associate Professor

Benjamin Cravatt, Ph.D.****ProfessorDirector, Helen L. Dorris

Child & Adolescent Neuro-Psychiatric DisorderInstitute

Gaudenz Danuser , Ph.D.**Associate Professor

Philip E. Dawson, Ph.D.***** Associate Professor

Velia Fowler, Ph.D.**Professor

Martin Friedlander, M.D.,Ph.D.

Professor

Larry R. Gerace, Ph.D.*Professor

Shelley Halpain, Ph.D.*** Associate Professor

Natasha Kralli, Ph.D.Associate Professor

Peter Kuhn, Ph.D.**Associate Professor

David Loskutoff, Ph.D.Professor EmeritusMari Manchester, Ph.D.**Associate Professor

Stephen P. Mayfield,Ph.D.*****

ProfessorAssociate Dean of Graduate

Studies

Mark Mayford, Ph.D.***Associate Professor

Lindsey Miles, Ph.D.Associate Professor

Ronald A. Milligan, Ph.D.**ProfessorDirector, Center for

Integrative Biosciences

Ulrich Müller*** Professor

Ardem Patapoutian, Ph.D.†

Associate Professor

Clinton Potter , B.S.**Associate Professor

James Quigley, Ph.D. Professor

Lisa Stowers, Ph.D.††

Assistant Professor

Heidi Stuhlmann, Ph.D.Associate Professor

Kevin F. Sullivan, Ph.D.†††

University of IrelandGalway, Ireland

Peter N.T. Unwin, Ph.D.**Professor

Clare Waterman-Storer,Ph.D.**

Associate Professor

Elizabeth Winzeler, Ph.D.†

Associate Professor

John R. Yates III, Ph.D.Professor

Mark J. Yeager, M.D., Ph.D.Professor

A D J U N C T A P P O I N T M E N T S

Alan Bell, B.S.C.S.Xerox Palo Alto Research

CenterPalo Alto, California

Richard Bruce, Ph.D.Xerox Palo Alto Research


Douglas Curry, B.S. (E.E.C.S.)Xerox Palo Alto Research


Bertil Daneholt, M.D.Karolinska InstitutetStockholm, Sweden

Scott Elrod, Ph.D.Xerox Palo Alto Research


Mark Ginsberg, M.D.University of CaliforniaSan Diego, California

David Goldberg, Ph.D.Xerox Palo Alto Research


Xiaohua Gong, Ph.D.University of CaliforniaBerkeley, California

Klaus Hahn, Ph.D.University of North CarolinaChapel Hill, North Carolina

Eric Peeters, Ph.D.Xerox Palo Alto Research


S T A F F S C I E N T I S T S

Michael Bracey, Ph.D.

Anchi Cheng, Ph.D.

Elena Deryugina, Ph.D.

Robert Fischer, Ph.D.

Elizabeth Wilson, Ph.D.

S E N I O R R E S E A R C H

A S S O C I A T E S

Brian Adair, Ph.D.

Barbara Calabrese, Ph.D.

Mark Daniels, Ph.D.

Jeremiah Joseph, Ph.D.

Edward Korzus, Ph.D.†††

University of CaliforniaRiverside, California

Matthew Ritter, Ph.D.

Martin Schwander, Ph.D.

Gina Story, Ph.D.†††

Washington UniversitySt. Louis, Missouri

Defne Yarar, Ph.D.

Andries Zijlstra, Ph.D.

R E S E A R C H A S S O C I A T E S

Jessica Alexander, Ph.D.

Geza Ambrus-Aikelin, Ph.D.

Veronica Ardi, Ph.D.

Angelique Aschrafi, Ph.D.††††

Andrea Bacconi, Ph.D.

Hongdong Bai, Ph.D.

Kent Baker, Ph.D.

Claudia Barros, Ph.D.

Maria Beligni, Ph.D.

Richard Belvindrah, Ph.D.

Edward Brignole III, Ph.D.

C E L L B I O L O G Y 2 0 0 6 T H E S C R I P P S R E S E A R C H I N S T I T U T E 1 9

Florence Brunel, Ph.D.

Anja Bubeck, Ph.D.†††

Gen-Probe, Inc.San Diego, California

Gang Cai, Ph.D.

Gregory Cantin, Ph.D.

Eric Carlson, Ph.D.

Aurelia Cassany, Ph.D.

Yuriy Chaban, Ph.D.

Pablo Chamero, Ph.D.

Emily Chen, Ph.D.

Ihsiung Chen, Ph.D.†††

CODA GenomicsLaguna Hills, California

Yei Hua Chen, Ph.D.

Charmian Cher, Ph.D.

Smita Chitnis, Ph.D.

Esther Choi, Ph.D.

Parag Chowdhury, Ph.D.

Jill Chrencik, Ph.D.

Michael Churchill, Ph.D.

Francesco Conti, Ph.D.

Judith Coppinger, Ph.D.

Kaustuv Datta, Ph.D.

Leif Dehmelt, Ph.D.

Ajay Dhaka, Ph.D.

Anouk Dirksen, Ph.D.

Meng-Qui Dong, Ph.D.

Michael Dorrell, Ph.D.

Kelly A. Dryden, Ph.D.

Jerome Dupuy, Ph.D.

Anna Durrans, Ph.D.

Samer Eid, Ph.D.†††

Merck ResearchLaboratories, NeuroscienceDrug Discovery

West Point, Pennsylvania

Michael Fitch, Ph.D.†††

Tanabe ResearchLaboratories U.S.A.

San Diego, California

Santos Franco, Ph.D.

Margaret Gardel, Ph.D.

Maria Gonzalez, Ph.D.†††

Rincon PharmaceuticalsLa Jolla, California

Jorg Grandl, Ph.D.

Nicolas Grillet, Ph.D.

Cemal Gurkan, Ph.D.††††

Johannes Hewel, Ph.D.

Michael Hock, Ph.D.

Ke Hu, Ph.D.

Michael Huber, Ph.D.

Darren Hutt, Ph.D.

Eric Hwang, Ph.D.

Khuloud Jaqaman, Ph.D.

Anass Jawhari, Ph.D.††††

Lin Ji, Ph.D.

Nobutaka Kato, Ph.D.

Claire Kidgell, Ph.D.††††

Katsuhiro Kita, Ph.D.

Kevin Koehntop, Ph.D.

Jenny Kohler, Ph.D.

Atanas Koulov, Ph.D.

Paul LaPointe, Ph.D.

Nicole Lazarus, Ph.D.

Donmienne Leung, Ph.D.†††

Applied Molecular EvolutionSan Diego, California

John Lewis, Ph.D.†††

Dalhousie UniversityHalifax, Nova Scotia, Canada

Lujian Liao, Ph.D.

Maria Lillo, Ph.D.†††

University of SalamancaSalamanca, Spain

Jennifer Lin, Ph.D.

Ryan Littlefield, Ph.D.†††

University of WashingtonSeattle, Washington

Dinah Loerke, Ph.D.

Darren Logan, Ph.D.

Bingen Lu, Ph.D.

Matthias Machacek, Ph.D.

Kalotina Machini, Ph.D.

Mark Madsen, Ph.D.

Valentina Marchetti, Ph.D.

Julia Marin-Navarro, Ph.D.

Michael Matho, Ph.D.

Naoki Matsuo, Ph.D.

Daniel McClatchy, Ph.D.

Caroline McKeown, Ph.D.

Marcel Mettlen, Ph.D.

Helena Mira, Ph.D.

Jennifer Mitchell, Ph.D.

Machiko Muto, Ph.D.

Andromeda Nauli, Ph.D.

Jacobus Neels, Ph.D.

Sherry Niessen, Ph.D.

Silvia Ortega-Gutierrez,Ph.D.

Lesley Page, Ph.D.

Ana Maria Pasaperi-Limon,Ph.D.

Olivier Pertz, Ph.D.†††

University of CaliforniaSan Diego, California

Barbie Pornillos, Ph.D.

Anita Pottekat, Ph.D.

Judith Prieto, Ph.D.

Natalie Prigozhina, Ph.D.†††

Vala Sciences, Inc.La Jolla, California

Thomas Pucadyil, Ph.D.

Rajesh Ramachandran,Ph.D.

Vandana Ramachandran,Ph.D.

Abbas Razvi, Ph.D.

Leon Reijmers, Ph.D.

Anna Reynolds, Ph.D.

Edwin Romijn, Ph.D.†††

Philips Scientific EquipmentDivision

Eindhoven, the Netherlands

Cristian Ruse, Ph.D.

Mohsen Sabouri-Ghomi,Ph.D.

Alan Saghatelian, Ph.D.†††

Harvard UniversityCambridge, Massachusetts

Kumar Saikatendu, Ph.D.

Tomoyo Sakata, Ph.D.

2 0 C E L L B I O L O G Y 2 0 0 6 T H E S C R I P P S R E S E A R C H I N S T I T U T E

Cleo Salisbury, Ph.D.

Ian Schneider, Ph.D.

Christina Schroeder, Ph.D.

Stephan Sieber, Ph.D.†††

Ludwig-Maximilians UniversityMunich, Germany

Pratik Singh, Ph.D.

Scott Stagg, Ph.D.

Mark Surka, Ph.D.

Patricia Szainer, Ph.D.

Claire Tiraby Nguyen, Ph.D.

Tuija Uusitalo, Ph.D.†††

University of HelsinkiHelsinki, Finland

Valerie Uzzell, Ph.D.

John Venable, Ph.D.

Josep Villena, Ph.D.

Xiaodong Wang, Ph.D.†††

Medical University of OhioToledo, Ohio

Kari Bradtke Weber, Ph.D.

Eranthie Weerapana, Ph.D.

BinQing Wei, Ph.D.

Scott Westenberger, Ph.D.

Ann Wheeler, Ph.D.

Torsten Wittmann, Ph.D.†††

University of San FranciscoSan Francisco, California

James Wohlschlegel, Ph.D.

Catherine Wong, Ph.D.

Aaron Wright, Ph.D.

Lihua Wu, Ph.D.†††

Department of ImmunologyScripps Research

Ge Yang, Ph.D.

Masahiro Yasuda, Ph.D.†††

University of MichiganAnn Arbor, Michigan

Rie Yasuda, Ph.D.†††

Osaka UniversityOsaka, Japan

Zhongmin Zou, Ph.D.†††

Institute of Combined Injuryof PLA, Third MilitaryMedical University

Chongqing, P.R. China

S C I E N T I F I C A S S O C I A T E S

Hilda Edith Aguilar de Diaz,M.D.

Alexei Brooun, Ph.D.

Claire Delahunty, Ph.D.

Mohammed El-Kalay, Ph.D.

Tinglu Guan, Ph.D.

Anand Kolatkar, Ph.D.

* Joint appointment in the

Department of Molecular Biology

** Joint appointment in the Center

for Integrative Molecular

Biosciences

*** Joint appointment in the

Institute for Childhood and

Neglected Diseases

**** Joint appointments in the

Department of Chemistry, the

Skaggs Institute for Chemical

Biology, and the Helen L. Dorris

Child and Adolescent Neuro-

Psychiatric Disorder Institute

***** Joint appointment in the Skaggs

Institute for Chemical Biology

† Joint appointments in the

Institute for Childhood and

Neglected Diseases and the

Genomics Institute of the Novartis

Research Foundation

†† Joint appointments in the Helen

L. Dorris Child and Adolescent

Neuro-Psychiatric Disorder

Institute

††† Appointment completed; new

location shown

†††† Appointment completed


Cell Biology Overview

Members of the Department of Cell Biology con-tinue to excel in the rich environment of ScrippsResearch, and their successes are being rec-

ognized by others. Clare Waterman-Storer, who launchedher independent career at Scripps Research, received theNational Institutes of Health Director’s Pioneer Awardthis year, along with a 5-year grant to support her researchprogram. Dr. Waterman-Storer was 1 of 13 scientistschosen from more than 800 applicants for this extremelyprestigious award; the Director’s Pioneer Award recog-nizes leading scientists in the United States and investsin their potential to make important advances in bio-medical research. Also this year John Yates receivedthe prestigious Christian B. Anfinsen Award from theProtein Society, which recognizes significant technicalachievements in the field of protein science. GaudenzDanuser, Martin Friedlander, Dr. Waterman-Storer, and Ihave given plenary addresses recognizing notable scien-tific achievements and leadership in our respective fields.Finally, Dr. Danuser and Natasha Kralli were promotedto associate professor, and Stephen Mayfield, who alsoserves as associate dean of Graduate Studies, was pro-moted to full professor—positions befitting their acade-mic and scientific accomplishments.

A unique attribute of Scripps Research that distin-guishes it from academic institutions whose faculty mustmeet diverse educational obligations is its ability to buildresearch efforts around areas of strength, ensuring criti-cal mass and leadership in important areas of biomed-ical research. Although the department is proud of thesuccess of its individual scientists, we believe that thissuccess reflects in part the strong synergies that havedeveloped as we have built on areas of strength withincell biology. Synergy is defined as 2 or more groupsworking together in such a way that the result is greaterthan the sum of their individual capabilities. Althoughthere are numerous foci of synergy driving innovationand research in the department, I highlight here the 3 areas that represent the strengths on which we willcontinue to build.

An early and unique strength of our department, whichsynergizes with the structural efforts in the Departmentof Molecular Biology, is the use of electron cryomicro-scopy to provide structural insights into the workings ofcomplex, multisubunit cellular machines. FranciscoAsturias, Bridget Carragher, Clint Potter, Ron Milligan,Nigel Unwin, Mark Yeager, and their colleagues havebuilt an internationally preeminent center for electroncryomicroscopy. Together, these groups are developingnew methodologies, solving important structures, andtraining the next generation of electron cryomicroscopystructural biologists, not only among students and fellowsat Scripps Research, but through popular intensive sum-mer courses offered to students from around the world.These synergistic interactions have driven unprecedentedproductivity. Many important structures have been solvedin the past year, including (1) the chloroplast ribosometo reveal functionally important differences between itand its bacterial progenitor (Dr. Milligan in collaborationwith members of Dr. Mayfield’s laboratory), (2) the intactinfectious P22 bacteriophage to reveal the mechanismsof viral DNA transfer into the host cell (Drs. Carragherand Potter in collaboration with Jack Johnson in theDepartment of Molecular Biology), (3) the coat proteincomplex II cage to reveal a unique architecture for deform-ing a membrane and collecting cargo molecules into trans-port vesicles (Drs. Carragher and Potter in collaborationwith members of Bill Balch’s laboratory), (4) the struc-ture of DNA polymerase epsilon, providing new insightinto its interaction with its DNA template (Dr. Asturias),(5) the structure of a minus-end directed kinesin to revealthe mechanism of its unusual directionality (Dr. Milliganin collaboration with Ron Vale at the University of Cali-


Sandra Schmid, Ph.D.

fornia, San Francisco), and (6) the structure of an inte-grin complexed to its substrate revealing the activatedform of this cell adhesion molecule (Dr. Yeager). Theseremarkable accomplishments reveal the accelerated paceof electron cryomicroscopy structural biology made pos-sible through the relatively recent expansion of theseefforts within the Center for Integrative Molecular Bio-sciences, and the resulting innovations in technologyand efforts to automate this historically slow and tedioustechnology. The importance of these efforts, under theleadership of Drs. Carragher and Potter, has been recog-nized with funding from the National Center for ResearchResources and designated as a National Resource forAutomated Molecular Microscopy.

A second area of synergy, which has been built inassociation with the Institute for Childhood Diseases, isin cellular and molecular neurobiology. Kristen Baldwinrecently joined these efforts from Columbia Universityafter completing her postdoctoral training with RichardAxel, the recipient of the 2004 Nobel Prize in Physiol-ogy and Medicine for his work on olfaction. Using theolfactory system as a model, Dr. Baldwin plans to dis-sect the mechanisms governing neuronal diversity andthe establishment of neural connectivities that enable usto process and respond to complex sensory input. Shejoins a group of investigators at the Institute for Child-hood Diseases that includes Shelley Halpain, Mark May-ford, Uli Mueller, Ardem Patapoutian, and Lisa Stowers,who work on diverse but complementary aspects ofneuronal development, sensory perception (especiallytouch, smell, and hearing), the establishment of neu-ronal circuitry, and higher-order functions of learningand memory. Their combined expertise allows them totackle the complexities of how the brain is wired forhigher-order functioning. The outcomes of their studieshave important implications for childhood diseases suchas autism, as well as for mental retardation, schizophre-nia, and neurodegenerative diseases such as Alzheimer’sand Parkinson’s.

A third area of synergy on which we plan to build isin the quantitative spatial and temporal analyses of higher-order cellular processes, such as cell migration, signaltransduction, the establishment of polarity, and intra-cellular trafficking. These efforts, also being carried outwithin the Center for Molecular Biosciences, are spear-headed by Dr. Danuser, Velia Fowler, and Dr. Waterman-Storer. Traditionally, cell biologists have focused theirefforts on dissecting a single cellular process or a singlepiece of the cellular machinery, often in isolation from

the cell. The work of Drs. Danuser and Waterman-Storeris revealing the complex molecular and physical inter-actions between multiple moving parts of the cell thatare required for directed cell locomotion. Dynamic inter-actions and interdependencies between the actin cyto-skeleton and the endocytic machinery are being revealedin collaboration with scientists in my laboratory. Spatialand temporal regulation of signaling events that directcellular behavior are being analyzed in collaboration withGary Bokoch in the Department of Immunology. Thesophisticated microscopy, image analysis, and mathe-matical modeling techniques being developed in theCenter for Molecular Biosciences, combined with innova-tions in molecular biology, are enabling cell biologists tomanipulate and study the complex behavior of whole cells,rather than the traditional and more limited “divide andconquer” approaches of the past.

Although the Department of Cell Biology at ScrippsResearch is clearly leading in these important areas ofresearch and innovation, in this fast-paced and compet-itive arena if you’re not moving forward, you’ll quicklyslip backward. Therefore, we hope to continue to buildon these areas of strength with the recruitment of tal-ented and creative faculty who bring new and comple-mentary expertise to our efforts and who will contributeto and benefit from the synergistic environments wehave established.


INVESTIGATORS’ REPORTS

Structural Characterization ofMacromolecular MachinesF.J. Asturias, Y. Chaban, J. Brown, E. Brignole, G. Cai

We use state-of-the-art electron microscopy andimage analysis techniques to determine the3-dimensional structures of macromolecular

complexes involved in a variety of cellular processes,including DNA transcription, DNA replication, chromatinmodification and remodeling, and fatty acid synthesis.Macromolecular electron microscopy is an ideal tech-nique for these studies because it requires only a smallamount of material and the conditions for preparingsamples are physiologically relevant. Images of indi-vidual macromolecules are recorded and then computa-tionally combined to obtain structures of low to moderate(25–10 Å) resolution. These structures are often inter-preted by docking atomic resolution structures of com-ponent subunits in the lower resolution map of an entirecomplex. Our ultimate goal is to use a combination ofbiochemical and structural information to reveal themechanism by which a macromolecular complex car-ries out its function.

In our current research on DNA transcription andits regulation, we are analyzing the basal machineryand assembly of the RNA polymerase II preinitiationcomplex. We are also studying complexes involved inthe regulation of transcription during initiation and inprevious steps in which the structure of chromatin isaltered to control access to DNA. We are particularlyinterested in the structure and function of Mediator, acomplex that plays a central role in regulating tran-scription in eukaryotes at the time transcription begins.We have developed a reproducible protocol for purify-ing Mediator that will enable us to pursue biochemicaland structural studies to get to the heart of the mech-anism of regulation by Mediator. We are also gearingup to use fluorescence microscopy to validate the invivo relevance of our in vitro studies.

In the past year we also made progress in analyz-ing the structure and mechanism of DNA polymerase ε(Pol ε). We used electron microscopy and single-parti-cle image analysis to calculate a 16-Å resolution of thepolymerase, the first of a multisubunit eukaryotic DNApolymerase. We were able to determine the location of

2 of the 4 subunit components of Pol ε and to docu-ment and measure changes in the relative orientationof the 2 large domains that constitute the structure.Although no atomic resolution structures of the com-ponent subunits were available to help in interpretingthe electron microscopy map, we used the structuralinformation to design template elongation assays thatresulted in a model for interaction of Pol ε with DNAthat explains the intrinsic processivity and capacity ofPol ε to interact with a variety of templates (Fig. 1).


F i g . 1 . Structure of Pol ε and a model for its interaction with DNA.

Electron microscopy and single-particle image analysis were used

to calculate the structure of the polymerase at a resolution of 16 Å.

The structure of the catalytic Pol2 subunit was calculated indepen-

dently, as was the structure of the Pol2–Dpb2 subunit complex.

Image analysis also revealed changes in the relative orientation of the

Pol2 and Dpb2-Dpb3-Dpb4 domains made possible by a flexible

connection. These changes in relative orientation might result in a

conformation that would allow access of the DNA to the active-site

cleft (top). A return of the tail to its normal conformation after entry

of double-stranded DNA into the active-site cleft would result in close

interaction of the nucleic acid with the extended tail domain (bottom).

This mode of interaction with DNA would explain the intrinsic proces-

sivity of Pol ε and the involvement of the Dpb3–Dpb4 subunit com-

plex in double-stranded DNA binding. The processivity dependence

of the length of the double-stranded primer region revealed by elon-

gation assays would be explained by the requirement for a minimal

length of double-stranded DNA to ensure proper interaction with the

full length of the extended tail domain in the Pol ε structure.

Finally, we continue to investigate the role that con-formational changes play in the function of mammalianfatty acid synthase (FAS), the enzyme responsible forthe synthesis of long-chain fatty acids. In this truemacromolecular assembly line, the different enzymesinvolved in the synthesis of fatty acids have fused intoa single polypeptide chain that includes 6 catalytic and1 acyl carrier protein domains. Using a novel approachin which FAS point mutants were imaged in the pres-ence of substrates (effectively pausing the enzyme at agiven catalytic step), we were able to determine anFAS structure that led to a revised model for FASorganization. That model has now been confirmed bya recently published partial x-ray structure of FAS. Asa result of the molecular flexibility that appears to beessential for the function of FAS, 2 of the FAS domainswere not observed in the x-ray structure. Further elec-tron microscopy analysis of FAS will reveal the locationof all domains and provide information about the vari-ety of conformational states that make possible themultitude of interdomain interactions required for thefunction of the enzyme.

PUBLICATIONSAsturias, F.J., Cheung, I., Sabouri, N., Chilkova, O., Wepplo, D., Johansson, E.Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electronmicroscopy. Nat. Struct. Mol. Biol. 13:35, 2006.

Takagi, Y., Chadick, J.Z., Davis, J.A., Asturias, F.J. Preponderance of free Mediatorin the yeast Saccharomyces cerevisiae. J. Biol. Chem. 280:31200, 2005.

Chemical Biology ofConformational Disease and Membrane Traffic

W.E. Balch, Y. An, C. Chen, J. Conkright-Johnson, D. Fowler,

C. Gurkan, D. Hutt, A. Koulov, P. LaPointe, J. Matteson,

A. Nauli, L. Page, H. Plutner, A. Pottekat, A. Razvi, S. Stagg,

P. Szajner, I. Yonemoto

Amajor challenge is to understand and treat themany protein-misfolding diseases that affecthuman health, including cystic fibrosis, emphy-

sema, type 2 diabetes, and amyloidosis. Theseabnormalities are classified as membrane-traffickingconformational diseases because a defect in proteinfolding at some stage of the eukaryotic secretory path-way results in loss of activity or protein aggregation. Akey concern is to determine the underlying defect in

protein folding and how that defect affects the abilityof the protein to function normally within the contextof the cell’s intracellular transport machinery or in theextracellular environment of the host.

Our broad objective is to define the molecularbasis for the trafficking of normal and misfolded pro-teins through the secretory pathway of eukaryotic cells.We use chemical, structural, biological, and bioinfor-matics approaches.

Eukaryotic cells are highly compartmentalized; eachcompartment of the exocytic and endocytic pathwaysprovides a unique chemical landscape in which proteinfunction and folding may be modulated. Movementbetween these compartments involves the activity ofboth anterograde and retrograde transport tubules andvesicles. Many conformational diseases are a conse-quence of dysfunction at different stages of this trans-port pathway or outside the cell.

Transport through the secretory pathway involves aselective mechanism in which cargo molecules are con-centrated into carrier vesicles. Vesicle-mediated trans-port is regulated by a diverse group of small GTPasesbelonging to the Ras superfamily. Each of these mole-cules acts as a “molecular sensor” to regulate differentsteps in the reversible assembly of vesicle coats andtargeting-fusion complexes. During export from the firstcompartment of the secretory pathway, the endoplasmicreticulum, coat recruitment to budding sites involvesactivation of the GTPase Sar1. After activation, thecytosolic coat components Sec23/24 and Sec13/31form the coatomer complex II coat (COPII) that poly-merizes to promote budding from the surface of theendoplasmic reticulum. This machinery directs exitfrom the endoplasmic reticulum of proteins encodedby nearly one third of the genome in eukaryotes.

Recently, in collaboration with C. Potter and B. Car-ragher, Department of Cell Biology, we solved the 2-dimensional electron cryomicroscopy structure of theSec13/31 cage (Fig. 1). This cage is a self-assemblingnanoparticle that collects cargo by assembling into apolymer scaffold that interacts with an adaptor proteincomplex bound to “exit codes” found on the cytoplas-mic domains of cargo and cargo receptors. These exitcodes bind to a multivalent adaptor platform found onthe surface of Sec24 facing the lipid layer. With J.R.Yates, Department of Cell Biology, we are using state-of-the-art proteomics (multidimensional protein identificationtechnology or MudPIT) to identify unknown componentsinvolved in cargo selection.

C E L L B I O L O G Y 2 0 0 6 2 5

After budding and fusion of COPII transport vesiclesfrom the endoplasmic reticulum, targeting and fusion ofthe vesicles to generate the next compartment of thesecretory pathway, the Golgi apparatus, require a differ-ent class of Ras-like GTPases that belong to the Rabfamily. Members of the large Rab family (>70 mem-bers) act as molecular switches that assemble com-plexes involved in vesicle tethering and fusion. Usinga bioinformatics approach involving hierarchial cluster-ing and mRNA expression profiling (microarray), wefound that each Rab GTPase executes targeting andfusion decisions at a distinct step in the exocytic orendocytic pathway. By integrating the interactions ofmultiple distinct effectors at each step, Rab GTPasesact as hubs to define the highly distinctive membrane

architecture of eukaryotic cells found in different tis-sues. This systems biology approach provides for thefirst time a global view of membrane traffic from thetop down, integrating form with function.

Of particular importance is our characterization ofthe structure of the Rab1 tether p115, done in collabora-tion with I.A. Wilson, Department of Molecular Biology.The structure reveals a superhelical coiled coil multi-valent assembly platform that facilitates Rab-dependentmaturation of tethering-fusion complexes. In addition,Rab proteins are recycled for use in multiple rounds oftether assembly. We recently showed the surprisingimportance of the Hsp90 chaperone system in Rabrecycling after vesicle fusion.

Many mutation disrupt cargo traffic from the endo-plasmic reticulum by preventing proper protein foldingduring synthesis, resulting in loss of recognition by theCOPII selection machinery. Other protein conformationaldiseases have mutations that disrupt function at latersteps of the secretory pathway and outside the cell innew chemical environments that can alter the proteinfold. In collaboration with J. Kelly, Department ofChemistry, we are studying the link between traffick-ing defects and the protein-folding energetics of anumber of conformational diseases, including cysticfibrosis, hereditary childhood emphysema, Gaucherdisease, familial amyloidosis of Finnish type, Parkin-son’s disease, and transthyretin amyloidosis. Theseanalyses have led to a new understanding of the func-tion of the endoplasmic reticulum in normal physiol-ogy, suggesting that this compartment functions as acapacitor for protein folding and human evolution. Ouranalysis of cystic fibrosis has revealed that system-widemodification of the chaperone folding pathways (thechaperone) can alter the steady-state energetic poolsof unfolded and folded macrostates (conformationalpopulations) that allow for rescue of the traffickingdefect and restore the function of chloride channels atthe cell surface.

Through a multidisciplinary approach that com-bines the tools of chemistry, biology, systems biology,bioinformatics, and structure, we hope to gain criticalinsight into the fundamental principles of cargo traf-ficking and the basis for a variety of inherited transportdiseases. Knowledge of the function of these cargoselection pathways will enable the development ofsmall-molecule chemical chaperones to encourageexport and stability of misfolded proteins, leading torestoration of normal cellular function.


F i g . 1 . Structure of the self-assembling COPII cage. Illustrated are

the 3 different symmetry-related views of the Sec13/31 complex that

self-assembles to form unprecedented cuboctahedron geometry on

the surface of the endoplasmic reticulum to form a molecular scaf-

fold (cage) that collects cargo for export. By coordinating cargo con-

centration with membrane curvature and fission, the cage can

generate a transit vesicle that mobilizes cargo to the cell surface.

Reprinted from Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe,

P., Foss, T.R., Potter, C.S., Carragher, B., Balch, W.E. Structure of

the Sec13/31 COPII coat cage. Nature 439:234, 2006.

PUBLICATIONSBannykh, S.I., Plutner, H., Matteson, J., Balch, W.E. The role of ARF1 and RabGTPases in polarization of the Golgi stack. Traffic 6:803, 2005.

Chen, C.Y., Balch, W.E. The Hsp90 chaperone complex regulates GDI-dependentRab recycling. Mol. Biol. Cell 17:3494, 2006.

Chen, C.Y., Sakisaka, T., Balch, W.E. Use of Hsp90 inhibitors to disrupt GDI-dependent Rab recycling. Methods Enzymol. 403:339, 2005.

Fowler, D.M., Koulov, A.V., Alory-Jost, C., Marks, M.S., Balch, W.E., Kelly, J.W.Functional amyloid formation within mammalian tissue. PLoS Biol. 4:e6, 2006.

Gurkan, C., Balch, W.E. Recombinant production in baculovirus-infected insectcells and purification of the mammalian Sec13/Sec31 complex. Methods Enzymol.404:58, 2005.

Gurkan, C., Lapp, H., Alory, C., Su, A.I., Hogenesch, J.B., Balch, W.E. Large-scaleprofiling of Rab GTPase trafficking networks: the membrome. Mol. Biol. Cell16:3847, 2005.

Gurkan, C., Lapp, H., Hogenesch, J.B., Balch, W.E. Exploring trafficking GTPasefunction by mRNA expression profiling: use of the SymAtlas Web-application andthe Membrome datasets. Methods Enzymol. 403:1, 2005.

Kelly, J.W., Balch, W.E. The integration of cell and chemical biology in proteinfolding. Nat. Chem. Biol. 2:224, 2006.

Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.J., Venable, J., Yates, J., Kelly, J.W.,Balch, W.E. Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. EmboJ. 24:4124, 2005.

Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, P., Foss, T.R., Potter, C.S., Carragher, B., Balch, W.E. Structure of the Sec13/31 COPII coat cage. Nature439:234, 2006.

Suk, J.Y., Zhang, F., Balch, W.E., Linhardt, R.J., Kelly, J.W. Heparin acceleratesgelsolin amyloidogenesis. Biochemistry 45:2234, 2006.

Wang, X., Venable, J., LaPointe, P., Hutt, D.M., Koulov, A.V., Coppinger, J., Gurkan,C., Kellner, W., Matteson, J., Plutner, H., Riordan, J.R., Kellly, J.W., Yates, J.R. III,Balch, W.E. Hsp90 cochaperone rescue of misfolding disease. Cell, in press.

Wiseman, R.L., Balch, W.E. A new pharmacology: drugging stressed folding path-ways. Trends Mol. Med. 11:347, 2005.

Molecular Mechanisms ofOlfactory Perception and NeuralCircuit FormationK.K. Baldwin, S. Tate, B. Fields, S. Ghosh

In mammals, the sense of smell is critical for sur-vival. Scents trigger suckling at birth, distinguishfood from poison, provide warning of predators, and

identify attractive mates. A primary goal of neurobiol-ogy is to discover how neural circuits link these typesof sensory inputs to appropriate behavioral outputs.Surprisingly little is known about how neural circuitsspecific to one set of inputs are organized or built.

We take advantage of the unique architecture andgenetic tractability of the mouse olfactory system tostudy specific olfactory circuits at the first 2 levels of pro-

cessing. Our goal is to genetically label the neurons thatrespond to specific odors in the nose and the olfactorybulb, to trace the projections of the neurons into the cor-tical regions where inputs converge, and to identify themolecular mechanisms that govern the formation of spe-cific neural circuits. We anticipate that our findings willreveal mechanisms common to neural circuit formationthroughout the brain and provide insight into geneticbases of human cognitive and behavioral disorders.C L O N I N G M I C E F R O M N E U R O N S

In contrast to gene activation in other sensory sys-tems, odorant receptor genes are activated by stochasticmechanisms. Stochastic gene activation in the immunesystem is due to irreversible DNA rearrangements. Wesearched for chromosomal rearrangements by cloningmice from the nuclei of olfactory sensory neurons. Wefound that odorant receptor choice is reversible, as isneuronal differentiation. We will now clone mice fromother types of neurons to determine whether irrevers-ible chromosomal alterations accompany neuronal diver-sification in the brain.V I S U A L I Z I N G O L F A C T O R Y I N P U T S T O T H E B R A I N

A major challenge is to understand how olfactoryinformation is integrated in the olfactory cortex. Animportant first step is to describe the anatomy of thesecond-order circuit. This endeavor has been hinderedby the lack of specific promoters for the output neurons(mitral cells) of the olfactory bulb. We identified a genethat is expressed specifically in mitral cells and haveproduced mice in which subsets of these cells expressfluorescent proteins. We use confocal and 2-photonmicroscopy to map the projections of individual mitralcells into the brain. By visualizing the second-orderolfactory circuit, we can begin to understand how thebrain recognizes odors.N E U R A L D I V E R S I T Y A N D C I R C U I T F O R M A T I O N

Although genes that regulate axon guidance andlarge-scale brain patterning have been identified, thegenes that endow neurons with the precise patterns ofneuronal synaptic connectivity remain enigmatic. Wereasoned that genes expressed in subsets of mitral cellswould be good candidates to direct the formation ofspecific neuronal circuits. Using bioinformatics andsingle-cell gene profiling, we have identified about 70genes that can be used to subdivide mitral cells intodifferent classes. We will use gene targeting to test therole of these genes in neural circuit formation.

One class of genes that diversifies mitral cells is thelarge family of approximately 60 clustered genes for pro-


tocadherins. Protocadherins are expressed throughoutthe nervous system. Intriguingly, each neuron seems toexpress a distinct combination of several protocadherinproteins. We have produced mice that do not express onesubfamily of protocadherins. These mice have behavioralabnormalities consistent with defects in neuronal func-tion. We are investigating the cellular and physiologicconsequences of loss of protocadherin diversity.

Automated Molecular Imaging

B. Carragher, C.S. Potter, A. Cheng, D. Fellmann, G. Lander,

S. Mallick, P. Mercurio, J. Pulokas, J. Quispe , S. Stagg,

C. Yoshioka

During the past decade, electron cryomicroscopyhas emerged as a powerful method for deter-mining the structure of large macromolecular

complexes. Elucidating the structure and mechanismof action of these “molecular machines” is an emergingfrontier in understanding how the information in thegenome is transformed into cellular activities. Examplesof the machines include ribosomes, transcription com-plexes, track-motor complexes, and membrane-embed-ded pumps and channels.

In electron cryomicroscopy, the macromolecularspecimen is preserved in a thin layer of vitreous (glassy)ice and imaged in an electron microscope by using lowdoses of electrons. The low signal-to-noise ratio of theresulting images means that averaging is required torecover the signal and reconstruct a 3-dimensionalmap of the structure.

In 2002, we established the National Resource forAutomated Molecular Microscopy (NRAMM) to develop,test, and apply technology for automating the processesinvolved in using electron cryomicroscopy to solvemacromolecular structures. The goal of automation isnot only to facilitate the process of molecular micros-copy, although this facilitation is a welcome benefit,but also to expand the scope of accessible problemsand push experimental frontiers by making possibleinvestigations deemed too difficult or high risk becauseof the considerable effort involved in using manualmethods. An additional goal of automation is to enablemuch higher throughput of data and thus improve res-olution for single-particle reconstructions by increasingthe numbers of particles that contribute to the average3-dimensional map. Another mission of NRAMM is to

use the infrastructure developed to open up the some-times esoteric practices of electron cryomicroscopy toa much wider group of researchers, including investi-gators in cell biology, x-ray crystallography, and mate-rials science.

During the past 3 years, the new techniques andtechnologies that we developed included a new gridsubstrate designed to improve quality and throughputfor vitreous ice specimens; a prototype of a roboticgrid-handling system used for screening; Leginon, anautomated system for microscope control and imageacquisition; a relational database that tracks and man-ages data acquired by Leginon and tools for viewingand delivering the data via Web browsers; and ACE, aprogram for the automated measurement and correctionof contrast transfer function. These technologies allcontributed in demonstrating the potential for automatedhigh-throughput data acquisition and analysis in anexperiment in which images of more than 280,000particles of GroEL, a molecule involved in protein fold-ing, were acquired in a single 25-hour session at themicroscope and subsequently subjected to completelyautomated procedures to reconstruct a 3-dimensionalmap to a resolution better than 8 Å (Fig. 1).

These technological developments have beendesigned for and used in a number of collaborativeresearch projects, including reconstruction of a mini-mal coatomer complex II cage and reconstruction ofan intact infectious P22 virion. The infrastructure hasalso, in accordance with our mission, made electroncryomicroscopy accessible to a much wider commu-


F i g . 1 . More than 280,000 particles of GroEL were acquired

from a single grid by using Leginon in a period of 25 hours. These

particles were sorted by ice thickness and used to reconstruct a

3-dimensional density map to a resolution of approximately 8 Å.

Visualization by Scott Stagg and Mike Pique.

nity, leading to publications from research groups inchemistry, x-ray crystallography, materials science,and industry. NRAMM currently provides support formore than 35 collaborative and service projects, andthe Leginon software, including the database, has beendistributed to about 30 laboratories outside ScrippsResearch. We are also distributing ACE, a variety ofother software packages, and the novel grid substrates.These efforts are complemented by training activitiesthat include small-group training, a biennial largetraining course in electron cryomicroscopy, and smallworkshops focused on various aspects of automation.

An additional project, sponsored by the NationalScience Foundation, is the development of automateddata collection techniques for imaging serial sections byusing an electron microscope. Understanding the finestructure of cells and cellular components contributesto a more profound understanding of cellular functionand intracellular or intercellular interactions. In orderto visualize these large, complex structures in 3 dimen-sions at resolutions sufficient to observe structure onthe nanoscale, the cells must be cut into sections andthen examined by using a transmission electron micro-scope. Acquiring high-magnification images of a longseries of sections is difficult and extremely labor inten-sive. The region of interest in each section must betracked across sections and across grids, a processthat requires examining the sections at a variety ofscales before acquiring high-magnification images ofinteresting areas. Multiscale imaging of this sort isnot straightforward because the image formed by anelectron microscope shifts and rotates as the magnifi-cation is changed. The overall task of reconstructing a3-dimensional volume from a set of serial sections ischallenging and time consuming, and the number oflarge-scale reconstructions has been limited to a fewspectacular examples. Our objectives are to design,develop, and implement a software application toautomate the task of acquiring high-magnificationimages of specific regions of the cell across tens tohundreds of serial sections.

PUBLICATIONSCheng, A., Fellmann, D., Pulokas, J., Potter, C.S., Carragher, B. Does contaminationbuildup limit throughput for automated cryoEM? J. Struct. Biol. 154:303, 2006.

Fellmann, D., Banez, R., Carragher, B., Potter, C.S. Temperature monitoring of anEM environment. Microsc. Today 14:24, January 2006.

Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, P., Foss, T.R., Potter, C.S., Car-ragher, B., Balch, W.E. Structure of the Sec13/31 COPII coat cage. Nature439:234, 2006.

Stagg, S.M., Lander, G.C., Pulokas, J., Fellmann, D., Cheng, A., Quispe, J.D.,Mallick, S.P., Avila, R.M., Carragher, B., Potter, C.S. Automated cryoEM dataacquisition and analysis of 284,742 particles of GroEL. J. Struct. Biol., in press.

Suloway, C., Pulokas, J., Fellmann, D., Cheng, A., Guerra, F., Quispe, J., Stagg,S., Potter, C.S., Carragher, B. Automated molecular microscopy: the new Leginonsystem. J. Struct. Biol. 151:41, 2005.

Regulation ofCytomechanochemical SystemsG. Danuser, A. Bacconi, J. Dorn, K. Jaqaman, L. Ji,

J. Kunken, D. Loerke, M. Machacek, A. Matov, M. Sabouri,

K. Thompson, G. Yang

We study how force-generating molecularmachines are spatially and temporally regu-lated to mediate complex cell functions,

including migration, division, and intracellular transportof organelles and vesicles. Specifically, we investigatethe relationships between assembly and contraction ofthe actin cytoskeleton and the dynamic coupling of actinfilaments with other components of the cytoskeletonduring cell migration. We also study how assemblyand disassembly of microtubules and motor-driven slid-ing of microtubule bundles are orchestrated to symmet-rically segregate replicated DNA from the dividingmother cell into 2 daughter cells.

In the past year, we expanded our research pro-gram with 2 new, collaborative projects. In the firstproject, we aim to establish the requirements for localregulation of cortical actin mechanics during endocy-tosis. In the second, we are analyzing the modes ofinteraction between microtubule plus end– and minusend–directed motor families in vesicle transport alongneuronal axons.

To examine molecular systems, we develop com-putational models to predict the relationship betweenthe dynamics of molecular-level component processesand cellular-level outputs. Subsequently, we validatethe models and estimate unknown parameters by fit-ting the parameters to measurements of cell dynam-ics. The challenges in such data-driven, multiscalemodeling are 2-fold: the precise and complete charac-terization of cell dynamics in space and time and theimplementation of numerical tools for fitting cellular-level data to models with molecular resolution.

In our studies of cell migration, we made 2 majoradvancements. First, in collaboration with C. Waterman-Storer, Department of Cell Biology, we extended fluores-


cent speckle microscopy to accomplish an integrated,correlative multiparameter analysis of cytoskeletondynamics. We can now determine accurately how cellmovements depend on molecular processes such asthe assembly, disassembly, and transport of cytoskele-ton components or adhesions, and we can use biosen-sor probes to visualize activation of signals. We recentlyused our analysis framework in studies in which we dis-sected the roles of several signaling cascades in theregulation of cell motility and the involvement of adhe-sion molecules in the transient, integrin-mediated cou-pling of the actin cytoskeleton to the extracellular matrix.

A second breakthrough was achieved in our effortto reconstruct intracellular force distributions from lightmicroscopic measurements of cytoskeleton deformation.We established a unique method to probe the relation-ship between spatially distributed force generation andthe resulting cell morphologic outputs, for example,during cell migration. We will use this tool to dissectthe mechanism of force regulation by signals and iden-tify feedback interactions between force transductionand signal activation, which are a central element inmolecular systems control, not only in cell motility butalso in a broad set of other cell functions.

To study chromosome segregation, we use fluores-cent speckle microscopy to analyze the dynamics ofmicrotubule scaffolds associated with the spindle appa-ratus in animal cells and 3-dimensional, high-resolutionlight microscopy to analyze the dynamics of single chro-mosomes in yeast. The first approach should reveal howmicrotubule assembly and disassembly across the spin-dle are coregulated with motor-mediated generation offorce. The second approach should allow us to identifythe functions of proteins in the kinetochore, a molecu-lar complex that regulates the attachment of chromo-somes to spindle microtubules.

We have developed fully automated, image-basedapproaches of unprecedented sensitivity for investiga-tions of phenotype microtubule and chromosome dynam-ics. In collaboration with E.D. Salmon, University ofNorth Carolina, T. Kapoor, Rockefeller University, andP. Sorger, Massachusetts Institute of Technology, we areusing the data obtained to systematically characterizethe involvement of spindle- and kinetochore-associatedproteins in the regulation of chromosome motionthroughout the cell cycle.

PUBLICATIONSCameron, L.A., Yang, G., Cimini, D., Canman, J.C., Kisurina-Evgenieva, O.,Khodjakov, A., Danuser, G., Salmon, E.D.S. Kinesin 5-independent poleward fluxof kinetochore microtubules in Ptk1 cells. J. Cell Biol. 173:173, 2006.

Danuser, G. Coupling the dynamics of two actin networks: new views on themechanics of cell protrusion. Biochem. Soc. Trans. 33:1250, 2005.

Danuser, G., Waterman-Storer, C.M. Quantitative fluorescent speckle microscopyof cytoskeleton dynamics. Ann. Rev. Biophys. Biomol. Struct. 35:361, 2006.

deRooij, J., Kerstens, A., Danuser, G., Schwartz, M.A., Waterman-Storer, C.M.Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J.Cell Biol. 171:153, 2005.

Dorn, J.F., Jaqaman, K., Rines, D.R., Jelson, G.S., Sorger, P.K., Danuser, G. Yeastkinetochore microtubule dynamics analyzed by high-resolution three-dimensionalmicroscopy. Biophys. J. 89:2834, 2005.

Ji, L., Danuser, G. Tracking quasi-stationary flow of weak fluorescent features byadaptive multi-frame correlation. J. Microsc. 220:150, 2005.

Lussi, J.W., Tang, C., Kuenzi, P.A., Staufer, U., Csucs, G., Voros, J., Danuser, G.,Hubbell, J.A., Textor, M. Selective molecular assembly patterning at the nanoscale:a novel platform for producing protein patterns by electron-beam lithography onSiO2/indium tin oxide-coated glass substrates. Nanotechnology 16:1781, 2005.

Machacek, M., Danuser, G. Morphodynamic profiling of protrusion phenotypes.Biophys. J. 90:1439, 2006.

Meijering, E., Smal, I., Danuser, G. Tracking in molecular bioimaging. IEEE SignalProcess. Mag. 23:46, May 2006.

Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman-Storer, C.M., Danuser, G.Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithe-lial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J.89:3456, 2005.

Shah, S., Yang, G., Danuser, G., Goldstein, L.S.B. Axonal transport: imaging andmodeling of a neuronal process. Springer Lecture Notes in Physics. In: The NobelSymposium. Springer, New York, in press.

Yang, G., Matov, A., Danuser, G. Reliable tracking of large scale dense antiparallelparticle motion for fluorescence live cell imaging. In: Proceedings of the 2005 IEEEComputer Society Conference on Computer Vision and Pattern Recognition (CVPR’05)Workshops. IEEE Computer Society, Washington, DC, 2005, Vol. 3, p. 138.

Synthetic Protein ChemistryP.E. Dawson, A. Dirksen, F. Brunel, M. Churchill, F. Hansen,E. Lempens, N. Metanis, T. Shekhter, T. Tiefenbrunn

We use chemical synthesis to design and engi-neer proteins with novel structures and func-tions. We continue to develop methods to

link fully unprotected peptides and carbohydrates vianative and nonnative linkages. During the past year,we focused on synthesizing mimics of the HIV envelopeprotein gp41, the oxidoreductase enzyme glutaredoxin,a glycosylated form of monocyte chemoattractant pro-tein-3, and carbohydrate-binding proteins. We are alsousing these chemoselective ligation reactions to labelproteins such as thrombin and nanoparticle quantumdots. Overall, our goal is to use synthetic chemistry tounderstand the molecular basis of protein structureand function.S E L E N O G L U T A R E D O X I N

Selenoenzymes have a central role in maintainingcellular redox potential. These enzymes have selenylsul-


fide bonds in their active sites that catalyze the reductionof peroxides, sulfoxides, and disulfides. The preparationof enzymes containing selenocysteine is experimentallychallenging. As a result, little is known about the kineticrole of selenols in enzyme active sites, and the redoxpotential of a selenylsulfide or diselenide bond in a pro-tein has not been experimentally determined.

To fully evaluate the effects of selenocysteine onoxidoreductase redox potential and kinetics, we synthe-sized glutaredoxin 3 (Grx3; Fig. 1) and all 3 seleno-

cysteine variants of the enzyme’s conserved 11CXX14Cactive site. Grx3, Grx3(C11U), and Grx3(C14U) hadredox potentials of –193, –259 and –273 mV, respec-tively. The position of redox equilibrium betweenGrx3(C11U-C14U) (–308 mV) and thioredoxin (–270mV) suggests a possible role for diselenide bonds inbiological systems. Kinetic analysis indicated that thelower redox potentials of the selenocysteine variants isdue primarily to the greater nucleophilicity of the active-site selenium rather than to the role of the seleniumas either a leaving group or a “central atom” in theexchange reaction. The 100- to 10,000-fold increase

in the rate of thioredoxin reduction by the seleno-Grx3analogs indicates that compared with their sulfide coun-terparts, oxidoreductases containing either selenylsulfideor diselenide bonds can have physiologically compatibleredox potentials and enhanced reduction kinetics.

H I V V A C C I N E D E S I G N

The transmembrane protein gp41 is an attractivetarget for the development of an HIV vaccine. We arecollaborating with M.B. Zwick and D.R. Burton, Depart-ment of Immunology, and I.A. Wilson, Department ofMolecular Biology, to design peptides that mimic thegp41 epitopes of known neutralizing antibodies. Themembrane-proximal external region of gp41 containsseveral neutralizing epitopes, including 4E10 and Z13e1.On the basis of our previous work on 4E10, we designedand synthesized peptides to map the Z13 epitope andperformed an alanine scan to identify key elementswithin the sequence. Structural constraints are beingintroduced into the peptides to obtain an antigen capa-ble of eliciting both 4E10- and Z13e1-like antibodies.

To better mimic the molecular environment ofnative gp41, we plan to introduce steric constraintssuch as polyethylene glycol and carbohydrates. Tomimic the viral membrane, we have appended atransmembrane helix and have incorporated the pep-tide into soluble lipid bilayers. Recently, neutralizingantibodies to the N-heptad repeat of gp41 have beendiscovered. We designed and synthesized 3-helix bun-dles to mimic this region of gp41, and we are usingthem to identify these epitopes and map the key bind-ing interactions.

PUBLICATIONSBrunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R.,Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutraliz-ing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006.

Cremeens, M.E., Fujisaki, H., Zhang, Y., Zimmermann, J., Sagle, L.B., Matsuda,S., Dawson, P.E., Straub, J.E., Romesberg, F.E. Efforts toward developing directprobes of protein dynamics. J. Am. Chem. Soc. 128:6028, 2006.

Delehanty, J.B., Medintz, I.L., Pons, T., Brunel, F.M., Dawson, P.E., Mattoussi, H.Self-assembled quantum dot-peptide bioconjugates for selective intracellular deliv-ery. Bioconjug. Chem. 17:920, 2006.

Medintz, I.L., Clapp, A.R., Brunel, F.M., Tiefenbrunn, T., Uyeda, H.T., Chang,E.L., Deschamps, J.R., Dawson, P.E., Mattoussi, H. Proteolytic activity monitoredby fluorescence resonance energy transfer through quantum-dot-peptide conju-gates. Nat. Mater. 5:581, 2006.

Sagle, L.B., Zimmermann, J., Matsuda, S., Dawson, P.E., Romesberg, F.E. Redox-coupled dynamics and folding in cytochrome c. J. Am. Chem. Soc. 128:7909. 2006.

Yamamoto, N., Takayanagi, A., Sakakibara, T., Dawson, P.E., Kajihara, Y. Highlyefficient synthesis of sialylglycopeptides overcoming unexpected aspartimide forma-tion during activation of Fmoc-Asn(undecadisialyloligosaccharide)-OH. TetrahedronLett. 47:1341, 2006.


F i g . 1 . Chemical synthesis of Grx3 by using folding to accelerate

the ligation reaction. Selective oxidation of the active-site disulfide

allowed alkylation of the cysteine residue at the ligation site.

Regulation of Actin Dynamics inMorphogenesis and Development

V.M. Fowler, T. Fath, R.S. Fischer, C. McKeown, J. Moyer,

R. Nowak, J. Palomique, K. Weber

Regulation of actin dynamics at the ends of fila-ments determines the organization and turnoverof actin cytoskeletal structures and is critical

for cell motility and architecture and actin-based mor-phogenetic processes in development. We focus on thetropomodulin family of proteins that cap the pointedends of actin filaments. Tropomodulins are a conservedfamily of proteins of about 40 kD that bind to tropo-myosin and actin. The tropomodulins are expressed ina tissue-specific and developmentally regulated fash-ion in vertebrates, flies, and worms.

In vertebrates, the tropomodulin 1 isoform is asso-ciated with stable architectural arrays of actin filamentssuch as thin filaments in striated muscle myofibrils andactin filaments in the membrane skeleton of red bloodcells (RBCs) and on the lateral membranes of the fibercells of the eye lens. Previous research indicated thattropomodulin 1 regulates the dynamics of actin pointedends and thus the length and stability of thin filamentsin myofibrils of cultured cardiac muscle cells. Tropo-modulin 3, the isoform in the cytoplasm, is associatedwith dynamic actin filaments in the lamellipodia ofcrawling endothelial cells, where it is a negative regu-lator of cell migration.

Our goal is to tie the molecular and cellular regula-tion of the dynamics of actin pointed ends by tropo-modulins to the in vivo functions of the proteins inactin-based morphogenetic processes in development.We use mouse genetic models to study the function oftropomodulins in myofibril assembly and cardiac develop-ment, the biogenesis and stability of the RBC membraneskeleton, and the morphogenesis and transparency offiber cells in the eye lens.

The structure and function of tropomodulins is bestunderstood for tropomodulin 1, which consists of 2domains: an unstructured, flexible N-terminal domainand a compact, folded C-terminal domain composedof 5 leucine-rich repeats. The N-terminal domain bindstropomyosin and is regulated by tropomyosin to captropomyosin-actin pointed ends with nanomolar affinity.The C-terminal domain caps actin pointed ends withsubmicromolar affinity and is unaffected by tropomyosin.

Despite the high level of sequence conservation(~70%) among vertebrate tropomodulins, comparisons oftheir actin-binding activities reveals that tropomodulin 3,but not tropomodulin 1, binds actin monomers andnucleates actin filament assembly in addition to cappingpointed ends. Tropomodulin 3 can be chemically cross-linked to actin in a 1:1 complex, providing a tool toidentify the amino acids at the tropomodulin 3–actinbinding interface. Initial results from tryptic digestionand mass spectrometry indicate that tropomodulin 3interacts with actin monomers via a unique interface onthe actin and on the tropomodulin 3. Site-directed muta-genesis plus structural and functional interaction studiesare in progress to further define the tropomodulin3–actin binding interface and to develop tropomodulinmutants for studies of cellular functions in vivo.

To investigate the in vivo function of tropomodulin 1in myofibril assembly and cardiac development, we areusing mice that lack the gene for this tropomodulin.We showed previously that myofibril assembly in theheart is grossly aberrant in the embryos of thesemutants, leading to aborted cardiac development andthe death of embryos between days 9 and 10 of devel-opment. To investigate the primary defect in myofibrilassembly, we examined nascent myofibrils on myocytemembranes in embryos at 4–5 days of development,before the appearance of gross cardiac abnormalities.

In wild-type embryos, the earliest myofibrils con-tain 1–3 sarcomeres in tandem with regularly spacedZ bodies and continuous F-actin, indicative of unregu-lated filament lengths. Such sarcomere structures arenever observed in the absence of tropomodulin 1;instead, α-actinin and F-actin are present in rodlike,aberrant Z disc structures on myocyte membranes. Thisfinding suggests that tropomodulin 1 has a novel earlyfunction in the organization of Z discs into sarcomeres.More recently, we found that cardiac development failsspecifically at the stage of looping morphogenesis, atan earlier stage than that observed in all other micethat lack genes for contractile proteins. We are testingthe hypotheses that defective myofibril assembly inabsence of tropomodulin 1 may lead directly or indi-rectly to aberrant cell-cell contacts or polarity, and/orto defective cell proliferation, leading to failure of loop-ing morphogenesis.

To investigate the consequences in RBCs of delet-ing the gene for tropomodulin 1, we prevented deathin the embryos of the mutant mice by expressing atropomodulin 1 transgene solely in the heart. The result


was viable mice with no tropomodulin 1 in their RBCs.Hematologic analyses revealed that these mice had acompensated mild hemolytic anemia, with increasedreticulocytosis and RBCs that were abnormally variablein size in blood smears. Measurements of mechanicalstability and deformability indicated that tropomodulin1–deficient RBCs were less deformable and more fragilethan normal RBCs. Western blotting indicated increasedlevels of tropomodulin 3 in the tropomodulin 1–defi-cient RBCs.

Using these tropomodulin 1–deficient RBCs, wecan test the effects of tropomodulin 3 on the lengthand dynamics of actin filaments and the consequencesfor stability of the membrane skeleton, RBC survival,and function in vivo. Mice with the transgene also pro-vide an opportunity to examine the function of tropo-modulin 1 in vivo in other tropomodulin 1–expressingcells and tissues such as the eye lens, neurons, andkidney. We are also producing mice that lack the genefor tropomodulin 3 to obtain mice with RBCs deficientin both tropomodulin 1 and tropomodulin 3 to assessthe consequences of complete lack of tropomodulin onRBC structure and function.

PUBLICATIONSFowler, V.M., McKeown, C.R., Fischer, R.S. Nebulin: does it measure up as aruler? Curr. Biol. 16:R18, 2006.

Angiogenesis-DependentDisease and Membrane Protein TopogenesisM. Friedlander, E. Aguilar, E. Banin, F. Barnett, R. Bautchek,

M. Dorrell, M. El-Kalay, S.F. Friedlander, S. Hanekamp,

R. Jacobson, A. Johnson, V. Machetti, M. Ritter, L. Scheppke,

J. Trombley, H. Uusitalo-Jarvinen, V. Marchetti, W. Ruf

A N G I O G E N E S I S - D E P E N D E N T D I S E A S E

Most diseases that cause catastrophic loss ofvision do so as a result of abnormal growthof blood vessels. Similarly, tumors depend on

a blood supply for their growth and use these new ves-sels as an avenue for metastasis. Blood vessels them-selves can generate tumors (e.g., hemangiomas) whenthe growth and organization of vascular endothelialcells is not properly controlled. Our goal is to under-stand the mechanisms of ocular neovascularization innormal and pathologic situations.

We use a neonatal mouse retina model to identifyregulators of developmental angiogenesis and under-stand endothelial guidance mechanisms. In addition,in a long-standing collaboration with D.A. Cheresh,University of California, San Diego, we are using thissystem to evaluate the role of integrins in this process.In collaboration with P.R. Schimmel, Department ofMolecular Biology, we found that fragments of tryptophantRNA synthetase are potent angiostatics that signifi-cantly reduce retinal neovascularization. The synthetasefragments are also angiostatic in vivo when deliveredby a cell-based method.

Most recently, we used combination therapy to showthat targeting multiple, distinct angiogenic pathwayswith fragments of tryptophan tRNA synthetase andantagonists of integrins and vascular endothelial cellgrowth factor provides highly synergistic, potent angio-static activity. Although this therapeutic approach shouldbe useful in the treatment of diseases in which com-plete inhibition of angiogenesis is desirable, it may notbe efficacious in the treatment of ischemic retinal dis-ease. In ischemic retinal disease, relief of hypoxia byvascular reconstruction, rather than destruction, may bethe desired outcome.

To examine possible therapies for diseases of reti-nal ischemia, we explored the potential usefulness ofstem cells derived from the bone marrow of adult micefor cell-based delivery of angiostatic and neurotrophicsubstances and for the trophic actions of the cellsthemselves in vascular and neuronal degenerative dis-eases. We found that both lineage-negative hemato-poietic progenitors and CD44hi-expressing myeloidprogenitors specifically target activated retinal astro-cytes, incorporate into and around new vessels, and,in a mouse model of retinal degeneration, rescue andstabilize a degenerating retinal vasculature.

We also showed that both types of stem cells havea profound neurotrophic effect when injected into eyesof mice with inherited retinal degeneration; not only isthe vasculature rescued in these mice but photorecep-tors and visual function are also preserved. The stemcells also rescue retinal vasculature subject to hypoxicstress and may be useful in the treatment of ischemicretinal abnormalities such as diabetic retinopathy andretinopathy of prematurity. The mechanism of rescueis not clear, but it is related to high levels of heat-shockproteins found in these populations of cells. We alsodefined a role for CD44hi-derived microglia in facilitat-ing vascular recovery in models of retinal ischemia.


Glioblastoma multiforme is an incurable brain tumorthat is usually fatal within 1 year after diagnosis. Weare using gene therapy and a rat model of this diseaseto study the efficacy of an antiangiogenic approach intreating these tumors. Hemangiomas are endothelialtumors that proliferate rapidly and later involute spon-taneously. We are using DNA microarrays to studychanges in gene expression as hemangiomas progress.Our goal is to identify (1) new targets for therapy forthese tumors and (2) novel regulators of angiogenesis.In a collaboration with G.R. Nemerow, Department ofImmunology, we used pseudotyped adenovirus to selec-tively target specific cell types in the retina. By usingthe appropriate fiber type, we can deliver transgenesto cells, such as photoreceptors, that ordinarily are nottargeted by adenovirus.M E M B R A N E P R O T E I N T O P O G E N E S I S

We are also studying the mechanism whereby pro-teins are asymmetrically integrated into cell membranes.In addition to studies of membrane protein topogene-sis at the molecular level, we are studying defects inprotein processing and insertion that occur in severaldegenerative diseases of the eye. In collaboration withK. Philipson, University of California, Los Angeles, weare investigating the topology of the cardiac sodium-cal-cium exchanger. On the basis of hydropathy analysis ofthe amino acid sequence, the exchanger is proposedto contain 12 hydrophobic segments, the first of whichis a cleaved signal sequence. Using a variety of reporterdomains (glycosylation sites, epitopes, and proteo-lytic cleavage sites), we analyzed the topology of theexchanger both in vitro and in oocyte expression systems.Because nearly all other polytopic eukaryotic membraneproteins do not have cleaved signal sequences, we areinvestigating the putative role of such a sequence inthe insertion and targeting of these exchangers.

Our results indicate that the native, cleaved N-ter-minal signal sequence is not necessary for insertion ofa functional exchanger into the cell membrane. In con-trast, the photoreceptor exchanger does not have acleaved N-terminal signal sequence. If the N-terminal65 amino acids are deleted, translocation of the N ter-minus of the protein is disrupted, but the remainder ofthe exchanger is integrated into the membrane. We arealso using large-scale genomic analysis to study trans-genic mice in which mutated exchanger is expressedand mice that lack the gene for the exchanger.

PUBLICATIONSBanin, E., Dorrell, M.I., Aguilar, E., Ritter, M.R., Aderman, C.M., Smith, A.C.H.,Friedlander, J., Friedlander, M. T2-TrpRS inhibits preretinal neovascularization andenhances physiological vascular regrowth in OIR as assessed by a new method ofquantification. Invest. Ophthalmol. Vis. Sci. 47:2125, 2006.

Dorrell, M., Uusitalo-Jarvinen, H., Aguilar, E., Friedlander, M. Ocular angiogene-sis; basic mechanisms and therapeutic advances. Surv. Ophthalmol., in press.

Dorrell, M.I., Friedlander, M. Mechanisms of endothelial cell guidance and vascularpatterning in the developing mouse retina. Prog. Retin. Eye Res. 25:277, 2006.

Friedlander, M. Stem cells and retinal disease. In: Retina, 4th ed. Ryan, S.J. (Editor-in-Chief). St. Louis, Mosby, 2006, Vol. 1, p 23.*

Friedlander, S.F., Ritter, M.R., Friedlander, M. Recent progress in our understanding ofthe pathogenesis of infantile hemangiomas. Lymphat. Res. Biol. 3:219, 2005.

Jin, H., Aiyer, A., Su, J., Borgstrom, P., Stupack, D., Friedlander, M., Varner, J. Ahoming mechanism for bone marrow-derived progenitor cell recruitment to the neo-vasculature. J. Clin. Invest. 116:652, 2006.

Ritter, M., Aguilar, E., Banin, E., Scheppke, L., Uusitalo-Jarvinen, H., Friedlander, M.Three-dimensional in vivo imaging of the mouse ocular vasculature during develop-ment and disease. Invest. Ophthalmol. Vis. Sci. 46:3021, 2005.

Ritter, M., Banin, E., Aguilar, E.A., Dorrell, M.I., Moreno, S.K., Friedlander, M.Myeloid progenitors differentiate into microglia and promote vascular repair in amodel of ischemic retinopathy. J. Clin. Invest., in press.

Ritter, M., Friedlander, M. Integrins in ocular angiogenesis. In: Ocular Angiogenesis:Diseases, Mechanisms, and Therapeutics. Tobran-Tink, J., Barnstable, C. (Eds.).Humana Press. Totowa, NJ, 2006, p. 279.

Ritter, M., Reinisch, J., Friedlander, S.F., Friedlander, M. Myeloid cells in infantilehemangioma. Am. J. Pathol. 168:621, 2006.

Nucleocytoplasmic Transportand Role of the Nuclear Lamina in Higher Level Nuclear OrganizationL. Gerace, G. Ambrus-Aikelin, A. Aschrafi, J. Bednenko,

A. Bubeck, A. Cassany, B. Chen, E. Choi, K. Datta, T. Guan,

M. Huber, K. Kanelakis

The nuclear envelope is a specialized domain ofthe endoplasmic reticulum that forms the bound-ary of the nucleus in eukaryotic cells. The enve-

lope consists of inner and outer nuclear membranes, thenuclear lamina, and nuclear pore complexes (NPCs).The nuclear lamina, a protein meshwork lining theinner nuclear membrane, provides a structural scaffoldfor the nuclear envelope and an anchoring site at thenuclear periphery for chromatin. NPCs are large supra-molecular assemblies that span the nuclear envelopeand serve as channels for molecular transport betweenthe nucleus and the cytoplasm. We are using a combina-tion of biochemical, structural, and functional approachesto investigate NPCs and the lamina.


N U C L E O C Y T O P L A S M I C T R A N S P O R T M E C H A N I S M S

Transport of protein and RNA through NPCs is anenergy-dependent process mediated by nucleocyto-plasmic shuttling receptors of the karyopherin β fam-ily. Karyopherins bind to transport signals on proteinor RNA cargo molecules, and the receptor-cargo com-plexes are translocated through the NPC by receptorbinding to a group of NPC proteins (nucleoporins) thatcontain phenylalanine-glycine amino acid motifs. Thedirectionality of nuclear transport is determined largelyby the small GTPase Ran, which directly interacts withkaryopherins and thereby regulates cargo binding.Conformational flexibility of karyopherins is thought tobe fundamental to their dynamic interactions withcargo, Ran, and nucleoporins.

We are using in vitro assays with digitonin-perme-abilized cells to analyze the molecular events that specifytranslocation of cargo-receptor complexes through NPCs.Recently, using site-directed mutagenesis of importin β,the prototypical nuclear import receptor, we character-ized 2 distinct binding sites in importin β for nucleo-porins containing the phenylalanine-glycine motif anddefined mutational hot spots for cargo binding. A majorgoal is to determine how the conformational dynamicsof importin β are linked to discrete transport steps. Tothis end, we are complementing structure-function stud-ies with analysis involving small-molecule inhibitors.

In a related project, we are analyzing nuclear importof the adenovirus genome, which consists of a 36-kbdouble-stranded DNA molecule. Results from our invitro transport studies indicate that adenovirus DNAtransport is driven by import signals on DNA-associatedproteins. Our characterization of multiple import signalsin adenovirus protein VII and the tight association ofthe protein with the genome suggest that this viral pro-tein may be the protein adaptor involved in the DNAimport. Nuclear import of protein VII involves severalof the major cellular importins, suggesting that adeno-virus has evolved to use redundant import pathwaysto ensure efficient nuclear delivery of its genome.

We also are analyzing nuclear export of HIV type 1mRNA mediated by the viral regulatory protein Rev. Revpolymerizes on a cis-acting sequence of viral mRNAs,providing a platform for assembly of nuclear export fac-tors. We are using proteomics combined with a perme-abilized cell assay for Rev-dependent HIV mRNA exportto functionally characterize the proteins assembled onthe Rev platform. This project is part of a larger collabo-ration with a research team at Scripps Research to iden-

tify small-molecule inhibitors of Rev transport and func-tion; the goal is to find compounds for developing newdrugs to inhibit HIV replication in humans.N U C L E A R L A M I N A A N D H I G H E R L E V E L N U C L E A R

O R G A N I Z A T I O N

The nuclear lamina in vertebrates contains a poly-mer of 2–4 related intermediate filament proteins calledlamins, which are associated with a number of trans-membrane proteins of the inner nuclear membrane.The lamina plays essential roles in nuclear structureand functions, as indicated by the recent findings thatmore than 15 inherited diseases in humans, includingseveral muscular dystrophies, are caused by mutationsin lamins or lamina-associated transmembrane proteins.The involvement of the lamina in disease is thought tobe linked to its roles in nuclear integrity, cell signaling,and gene expression. Until recently, only about 12transmembrane proteins specific to the nuclear enve-lope had been identified.

To determine the full complement of proteins in thenuclear envelope, we carried out a proteomics analysisof the nuclear envelope of rodent liver cells in collabo-ration with J.R. Yates, Department of Cell Biology. Weidentified 67 novel putative nuclear envelope trans-membrane proteins. Almost all members of this groupthat we have examined are authentic components ofthe nuclear envelope.

Currently, we are analyzing nuclear envelope trans-membrane proteins in muscle, because this is the tis-sue most sensitive to disruption of lamina function bydisease-causing mutations. Using transcriptional pro-filing of cultured myoblasts, we found that the genesfor 6 of the nuclear envelope transmembrane proteinsare strongly upregulated in myoblast differentiation. Thegenes also are highly expressed in muscle in adults,consistent with a role of the genes in muscle differen-tiation and/or maintenance. We have confirmed thatthese nuclear envelope transmembrane proteins areauthentic nuclear envelope proteins; we are using genesilencing approaches to analyze their requirement inmuscle cell function. Our goals are to identify novelgenes that may have a role in human muscular dystro-phies and to further elucidate how the protein networkconsisting of lamins and associated transmembraneproteins directs nuclear structure and functions.

PUBLICATIONSOspina, J.K., Gonsalvez, G.B., Bednenko, J., Darzynkiewicz, E., Gerace, L., Matera,A.G. Cross-talk between snurportin1 subdomains. Mol. Biol. Cell 16:4660, 2005.

Schirmer, E.C., Gerace, L. The nuclear membrane proteome: extending the enve-lope. Trends Biochem. Sci. 30:551, 2005.


Wodrich, H., Cassany, A., D’Angelo, M.A., Guan, T., Nemerow, G., Gerace, L.Adenovirus core protein pVII is translocated into the nucleus by multiple importreceptor pathways. J. Virol. 80:9608, 2006.

Organization and Function ofthe Neuronal CytoskeletonS. Halpain, J. Braga, B. Calabrese, L. Dehmelt, E. Hwang,J. Koehler, K. Spencer

During the past year, we made significant progressin research on the development and regenerationof neurons. In 2 main projects, we focused on

cytoskeletal proteins of nerve cells, key proteins thatunderlie the structure and morphologic flexibility requiredby neurons for transmitting, storing, and processingsynaptic signals. We used biochemical, molecular bio-logical, and microscopy-based approaches to understandthe function of these molecules. Fluorescence time-lapseimaging of living neurons is an important tool that weused to uncover structure-function relationships for cyto-skeletal proteins and the consequences of the dysfunc-tion of the proteins. The results of these projects havecontributed to our understanding of molecular eventsin normal brain development and in regeneration ofneuronal structure after injury and disease.M I C R O T U B U L E - A S S O C I A T E D P R O T E I N S

One project concerns microtubule-associated pro-teins (MAPs). These proteins are important in regulat-ing the assembly and stability of microtubules and theinteractions of microtubules with other components ofthe cytoskeleton. We found that one microtubule-bindingprotein, MAP2, also directly binds actin filaments andinduces filament bundling. Using fluorescence-basedtime-lapse imaging and high-resolution confocal micros-copy, we tracked the behaviors of microtubules and actinfilaments in living neuronal cells with normal and mutantforms of MAP2.

Recently, we found that the microtubule-based molec-ular motor dynein plays a key role in transporting micro-tubules toward the cell periphery. This dynein-dependentactivity provides a key force that pushes the cell mem-brane outward during neurite initiation. Currently, weare using proteomic approaches and high-content, micros-copy-based screening technology to identify other cyto-skeletal proteins and signal transduction pathwayscrucial to the initiation of neurites.D E N D R I T I C S P I N E S

A second project concerns the regulation of den-dritic spines, specialized cellular protrusions that form

the receptive, postsynaptic element at glutamate syn-apses. Spines become lost or dysmorphic in many typesof mental retardation and in psychiatric conditions suchas chronic depression and schizophrenia. Furthermore,spines are vulnerable to injury in diseases such as strokeand epilepsy, in which excessive release of glutamatecan induce neuronal injury and subsequent cell death(a condition termed excitotoxicity). Understanding howspines form, what regulates their stability, and how theyrecover from injury is therefore of therapeutic interestfor several neurologic conditions.

Our most recent results suggest a neuroprotectiverole for spines, because preventing the collapse of den-dritic spines attenuates neuronal cell death induced by asubsequent lethal stimulus. The spine cytoskeleton iscomposed mainly of actin filaments. We discovered thatactin filaments in spines are rapidly broken down withinminutes of an injury-inducing stimulus. However, thisdamage to the spine can be rapidly reversed withinminutes under appropriate conditions, indicating forthe first time that spines can regrow after they collapse.

We also discovered that the membrane-associatedprotein myristoylated alanine-rich C kinase substrate(MARCKS), a major substrate of the signaling enzymeprotein kinase C, is a key molecule in the regulationof spine shape and stability. Alterations in MARCKSare implicated in Alzheimer ’s disease and in majordepression. We have extensively characterized theeffects of either depleting MARCKS or overexpressingvarious mutant forms of MARCKS in hippocampal neu-rons. The results revealed several key insights intoMARCKS function and novel forms of synaptic plastic-ity in young neurons.

PUBLICATIONSCalabrese, B., Wilson, M.S., Halpain, S. Development and regulation of dendriticspine synapses. Physiology (Bethesda) 21:38 2006.

Halpain, S., Dehmelt, L. MAP1 family proteins. Genome Biol., in press.

Function of Nuclear Receptorsin Stress and MitochondrialHomeostasisA. Kralli, J. Cardenas, B. Hazen, M.B. Hock, F. Jaramillo, C. Tiraby-Nguyen, J. Villena

We are interested in the molecular mechanismsthat relay metabolic stress signals to a net-work of transcriptional regulators and the


ensuing transcriptional outputs that mediate adaptivemetabolic responses to the stress signals. In particular,we focus on the coactivators peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) andPGC-1β and the orphan nuclear receptors of the estro-gen-related receptor (ERR) subfamily, which controlmitochondrial biogenesis and energy homeostasis. Ourgoals are to elucidate the biology of this specific tran-scriptional network, understand how deregulation of thenetwork leads to disease, and ultimately identify thecomponents of the network that are most suitable fordrug intervention to counteract metabolic disease.

R E G U L A T I O N O F T H E P G C - 1 / E R R N E T W O R K

Levels of PGC-1α and PGC-1β change in responseto signals that relay metabolic needs. The coactivatorsthen relay such signals, via interactions with ERRs andother factors, to regulate the expression of specific tar-get genes. We are interested in the mechanisms thatregulate PGC-1s at the postranslational level via cova-lent modifications of or interaction with other proteins,and thereby control the properties of the PGC-1/ERRnetwork. This past year, in collaboration with M. Stall-cup, University of Southern California, we showed thatPGC-1α is methylated by the protein arginine methyl-transferase 1 and that this methylation increases theactivity of PGC-1α. As a result, protein arginine methyl-transferase 1 and PGC-1α act cooperatively to activateERRα and to induce ERRα target genes with roles inmitochondrial biogenesis. Currently, we are investigat-ing the molecular mechanisms by which methylationregulates PGC-1α activity.

R O L E O F E R R αα I N M I T O C H O N D R I A L F U N C T I O N

Our previous studies suggested that the effects ofPGC-1α and PGC-1β on mitochondrial biogenesis aremediated primarily by ERRα. To determine the physio-logic relevance of ERRα for mitochondrial function, weare studying brown adipose tissue. This tissue is richin mitochondria, it expresses high levels of ERRα, and itsfunction is readily assayed in the context of the wholeorganism. Compared with wild-type mice, mice lack-ing ERRα have a decrease in the expression of genesassociated with oxidative phosphorylation, lipid oxida-tion, and the tricarboxylic acid cycle. Morphologic analy-sis by electron microscopy revealed that brown adiposetissue from mice lacking ERRα has reduced mitochon-drial density and increased lipid accumulation. Whenexposed to cold, mice lacking ERRα had impaired adap-tive thermogenesis, despite normal induction of theuncoupling protein UCP-1. Adipocytes isolated from

the mice had reduced oxidative capacity, suggesting thatthe effect of ERRα on mitochondrial function is cellautonomous. This research establishes for the first timethat ERRα is an important component of the regulatorynetwork that supports high levels of mitochondrial bio-genesis and oxidative metabolism in vivo.

Interestingly, whereas ERRα is an important down-stream effector of PGC-1α in the stimulation of mitochon-drial biogenesis and oxidative capacity, it counteractsthe ability of PGC-1α to induce gluconeogenic genesin hepatocytes. Notably, it is required for proper sup-pression of gluconeogenic enzymes in the liver of fedmice. Mitochondrial dysfunction has been implicatedas an underlying cause of insulin resistance and type2 diabetes. Moreover, derepression of hepatic gluconeo-genesis contributes to high glucose levels in diabetes.The opposing effects of ERRα on genes important formitochondrial oxidative capacity vs genes important ingluconeogenesis suggest that enhancing ERRα activitycould have beneficial effects on glucose metabolismin patients with diabetes via 2 distinct mechanisms:increasing mitochondrial oxidative capacity in peripheraltissues and suppressing inappropriate glucose produc-tion in the liver.

PUBLICATIONSCartoni, R., Léger, B., Hock, M.B., Praz, M., Crettenand, A., Pich, S., Ziltener, J.,Luthi, F., Dériaz, O., Zorzano, A., Gobelet, C., Kralli, A., Russell A.P. Mitofusins1/2 and ERRα expression are increased in human skeletal muscle after physicalexercise. J. Physiol. 567(Pt. 1):349, 2005.

Herzog, B., Cardenas, J., Hall, R.K., Villena, J.A., Budge, P.J., Giguère, V.,Granner, D.K., Kralli, A. Estrogen-related receptor α is a repressor of phospho-enolpyruvate carboxykinase gene transcription. J. Biol. Chem. 281:99, 2006.

Teyssier, C., Ma, H., Emter, R., Kralli, A., Stallcup, M.R. Activation of nuclearreceptor coactivator PGC-1α by arginine methylation. Genes Dev. 19:1466, 2005.

Structural and FunctionalProteomicsP. Kuhn, J. Nieva,* E. Abola, A. Brooun, R. Bruce, C. Chen,

J. Chrencik, P. Clark, S. Coon, J. Dupuy, S. Foster, J. Joseph,

L. Kim, A. Kolatkar, M. Kraus, N. Lazarus, M. Leach,

D. Marrinucci, E. Rayon, K. Saikatendu, V. Subramanian,

A. Tang, M. Yadav

* Department of Molecular and Experimental Medicine, Scripps Research

During the past 3 years, we have focused on 4major research programs: detection of cancercells in circulation, structural proteomics of

cancer drug targets, structural and functional proteomics


of the coronavirus that causes severe acute respira-tory syndrome (SARS-CoV), and development of novelapproaches in miniaturization, integration, and automa-tion to lower the overall cost of moving from the synthe-sis of genes to examination of the structures encodedby the genes. These programs involve collaborationswith R.C. Stevens, Department of Molecular Biology;other researchers at Scripps Research; and scientistsat the Palo Alto Research Center and Lyncean Technolo-gies in Palo Alto, California, the Novartis Institutes forBiomedical Research, Cambridge, Massachusetts, anddeCODE biostructures, Bainbridge Island, Washington.D E T E C T I N G R A R E C E L L S I N T H E C I R C U L A T I O N

Many clinically important cells in blood occur at fre-quencies of less than 1 cell per 1 million cells. Detectingand characterizing these rare cells requires the develop-ment of new technology that operates with exceptionalspecificity. Scientists at the Scripps-PARC Institute forAdvanced Biomedical Sciences have developed aninstrument, based on fiber-optic array scanning technol-ogy (FAST), that provides rapid and accurate identifica-tion of rare cells in the circulation in humans. Potentialclinical applications include finding circulating tumorcells, circulating endothelial cells, and circulating fetalcells in the maternal circulation.

Malignant cells from solid tumors begin to circulateat the earliest stages in cancer formation. Because thecirculating cells are quite rare, technology to detect andcharacterize them can be valuable in screening for can-cer and in guiding individualized cancer therapy. Duringthe past year, we used FAST to accurately enumeratecirculating cancer cells in blood samples obtained frompatients with metastatic breast or lung cancer (Fig. 1).In collaboration with J. Kroener, Scripps Clinic, we foundthat accurate detection of circulating tumor cells byFAST can be used to predict prognosis in patients withmetastatic breast cancer. We are developing moleculartools to characterize these rare tumor cells after theyhave been detected. Our goal is to determine their tis-sue of origin, their potential anatomic destination, andtheir metastatic potential.S T R U C T U R A L P R O T E O M I C S A N D D R U G D I S C O V E R Y

We are collaborating with the Novartis Institutes forBiomedical Research, Cambridge, Massachusetts, ina project to understand and modulate therapeuticallyrelevant protein-protein interactions. Analysis of aninitial subset of 5 therapeutically relevant protein-pro-tein interaction pairs will provide the guiding principlesfor the selection of a feasible set of drug targets to beexamined. We used a high-throughput approach to

rapidly identify ideal constructs for expression, crystalli-zation, and biophysical studies. The results are provid-ing general insights into the range of different physicalinteractions for a given protein-protein interaction.

The Eph-ephrin interaction is particularly interest-ing, because it has been implicated in cancer progres-sion and in pathologic forms of angiogenesis. We havesolved the crystal structure of the ligand-binding domainof EphB4 in complex with an antagonistic peptide thatinhibits ephrin binding and has antitumorigenic prop-erties in vivo. We have also solved the cocrystal struc-ture of an EphB4–ephrin-B2 complex. Structural andbiophysical analysis are providing the first insights intohow we can modulate pathways involved in tumorigene-sis and angiogenesis that rely on EphB4–ephrin-B2signaling.These results will deepen our understandingof the basic biology behind protein-protein interactionsand aid in the development of novel therapeuticapproaches for modulating protein-protein interactions.

S T R U C T U R A L A N D F U N C T I O N A L P R O T E O M I C S

A N A L Y S I S O F S A R S - C o V

We are generating a structure-function-interactionmap of the SARS-CoV proteome and its interactionswith the host cell to provide a comprehensive set oftargets for rational, structure-based drug and vaccinedesign. We use bioinformatics, structural biology, geneticmethods, and functional assays.


F i g . 1 . An image of a single circulating tumor cell from a

patient with breast cancer identified from a background of 50 mil-

lion peripheral blood mononuclear cells by using a FAST cytometer.

So far, we have determined the structures of 7SARS-CoV proteins. We used crystallography for 3nonstructural proteins, nuclear magnetic resonance for3 other nonstructural proteins, and electron cryomi-croscopy for a large surface glycoprotein spike. Thesestudies are providing important information on the for-mation of the replicase complex, transcription, andRNA-processing events unique to the SARS-CoV lifecycle. A total of 7 other proteins have been success-fully expressed in soluble, folded forms, and theirstructures are being determined. Together, the resultsfrom these studies should enable identification ofcompounds that may be effective agents for treatmentof infections caused by SARS-CoV.A C C E L E R A T E D T E C H N O L O G I E S C E N T E R F O R G E N E

T O 3 D S T R U C T U R E

Scientists at the Accelerated Technologies Centerfor Gene to 3D Structure are simultaneously develop-ing, operating, and deploying 3 key technologies toimprove the costs of using x-ray crystallography todetermine the structure of experimental proteins. Thefirst technology, computer-aided design of expression-optimized synthetic genes and protein constructs forcrystallography, improves the success rate for geneisolation and allows researchers to engineer the genesequence of interest to be optimized for protein produc-tion in a desired heterologous expression system. Thesecond technology, microfluidic plug-based nanovolumeprotein crystallization in microcapillaries for in situ x-ray screening and data collection, is economical andgreatly broadens the range of useful quantities of pro-teins required for crystal growth. This technology alsoallows for fine control over chemical gradients in crystalgrowth, thereby expanding the coverage of crystalliza-tion space without consuming large quantities of protein.

The third technology, the compact light source, isa tunable laboratory x-ray source with peak intensityat x-ray wavelengths that span selenium anomalousabsorbance. Having a tunable laboratory x-ray sourcein the same facility where a crystal inventory is heldwill greatly enhance the ability to efficiently solve newprotein crystal structures.

The future integration of such technologies in a sin-gle facility at Scripps Research will enable efficient genedesign for improved protein production, small-volumecrystallization with in situ x-ray diffraction screening,and tunable x-ray data collection in a single laboratory.Our target focus is the structural elucidation of humanproteins of biomedical relevance (or eukaryotic homo-

logs thereof) that belong to superfamilies of integralmembrane proteins and transcription factors.

PUBLICATIONSChrencik, J.E., Brooun, A., Kraus, M.L., Recht, M.I., Kolatkar, A.R., Han, G.W.,Seifert, J.M., Widmer, H., Auer, M., Kuhn, P. Structural and biophysical character-ization of the EphB4–ephrinB2 protein-protein interaction and receptor specificity.J. Biol. Chem. 281:28185, 2006.

Chrencik, J.E., Brooun, A., Recht, M.I., Kraus, M.L., Koolpe, M., Kolatkar, A.R.,Bruce, R.H., Martiny-Baron, G., Widmer, H., Pasquale, E.B., Kuhn, P. Structureand thermodynamic characterization of the EphB4/ephrin-B2 antagonist peptidecomplex reveals the determinants for receptor specificity. Structure 14:321, 2006.

Hsieh, H.B., Marrinucci, D., Bethel, K., Curry, D.N., Humphrey, M., Krivacic,R.T., Kroener, J., Kroener, L., Ladanyi, A., Lazarus, N., Kuhn, P., Bruce, R.H.,Nieva, J. High speed detection of circulating tumor cells. Biosens. Bioelectron.21:1893, 2006.

Joseph, J.S., Saikatendu, K.S., Subramanian, V., Neuman, B.W., Brooun, A.,Griffith, M., Moy, K., Yadav, M.K., Velasquez, J., Buchmeier, M.J., Stevens, R.C.,Kuhn, P. Crystal structure of nonstructural protein 10 from the severe acute respi-ratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J.Virol. 80:7894, 2006.

Marrinucci, D.C., Bethel, K., Bruce, R.H., Curry, D.N., Hsieh, B., Humphrey, M.,Krivacic, R.T., Kroener, J., Kroener, L., Ladanyi, A., Lazarus, N.H., Nieva, J.,Kuhn, P. Case study of the morphologic variation of circulating tumor cells. Hum.Pathol., in press.

Neuman, B.W., Stein, D.A., Kroeker, A.D., Chruchill, M.J., Kim, A.M., Kuhn, P.,Dawson, P., Moulton, H.M., Bestwick, R.K., Iverson, P.L., Buchmeier, M.J. Inhibi-tion, escape, and attenuated growth of severe acute respiratory syndrome coron-avirus treated with antisense morpholino oligomers. J. Virol. 79:9665, 2005.

Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson,M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural geno-mics of the severe acute respiratory syndrome coronavirus: nuclear magnetic reso-nance structure of the protein nsP7. J. Virol. 79:12905, 2005.

Ratia, K., Saikatendu, K.S., Santarsiero, B.D., Barretto, N., Baker, S.C., Stevens,R.C., Mesecar, A.D. Severe acute respiratory syndrome coronavirus papain-likeprotease: structure of a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S.A. 103:5717, 2006.

Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K.,Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structuralbasis of severe acute respiratory syndrome coronavirus ADP-ribose-1′′-phosphatedephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005.

Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W.W., Roach L.S., Roach, R.F.,Ismagilov, F., Kuhn, P., Stevens, R.C. In situ data collection and structure refinementfrom microcapillary protein crystallization. J. Appl. Crystallogr. 38:900, 2005.

Vascular Imaging and TumorTargeting With Virus-BasedNanoparticlesM. Manchester, G. Destito, M. Estrada, M.J. Gonzalez,

K. Koudelka, E. Powell, C. Rae, P. Singh, D. Thomas

Current treatment of cancer typically involveschemotherapies that have severe adverse effects.The requirement that patients must withstand the

toxic effects of treatment often limits the effectivenessof the therapy. Further, many promising anticancer


compounds that are highly effective in vitro are tootoxic to be used in vivo.

The ability to specifically target therapies to the siteof a developing tumor while avoiding healthy tissue isan important goal for cancer research. Similarly, atremendous need exists to identify, image, and monitortumors, particularly at early stages and during treatment.Recently, “smart” nanoparticles, which combine thesemultiple targeting, imaging, and drug delivery functions,have been developed. Therapies based on nanoparticleshave tremendous potential to increase the sensitivityand specificity of diagnostic imaging and treatment.Many different classes of nanoparticles are currently indevelopment, including dendrimers, liposomes, para-magnetic nanoparticles, and quantum dots.

We focus on virus-based nanoparticles as platformsfor the development of tissue-specific targeting andimaging agents in vivo. Two of the viruses we study arecowpea mosaic virus (CPMV) and canine parvovirus.C P M V A S A N O V E L B I O M O L E C U L A R S E N S O R F O R

V A S C U L A R I M A G I N G

CPMV is an icosahedral, 31-nm particle that isproduced easily and inexpensively in black-eyed peaplants. In contrast to the structure of most other nano-materials, the structure of the CPMV capsid is definedand can be engineered to display peptides or proteinsin controlled orientations on particle surfaces via eithergenetic manipulation of the viral genome or by chemicalattachment to the particle surface. CPMV is bioavail-able and nontoxic, and the capsids are highly stableto temperature, pH, and the conditions required forchemical reactions.

By conjugation to surface lysine residues, CPMVcan be labeled with fluorophores at high densities,resulting in an extremely bright, nontoxic material thatis an outstanding tool for imaging vasculature in liveanimals. Working with H. Stuhlmann, Department ofCell Biology, we showed that CPMV can be used toeffectively image the complete vasculature in theembryos of several species and that it is superior toother imaging particles such as lectins, fluorescentdextrans, or polystyrene microspheres.

CPMV particles have also been highly useful in high-lighting angiogenesis in developing tumors. Uptake ofparticles into endothelial cells occurs, yielding a brightimaging signal that can be used to differentiate betweenarterial and venous vessels. Such endothelial uptake ismediated by a cellular membrane protein, and uptakecan be observed in the endothelium of a variety of

healthy tissues as well as in disease states such asatherosclerosis.T U M O R T A R G E T I N G W I T H V I R U S - B A S E D

N A N O P A R T I C L E S

We have designed virus-based nanoparticles thatcan specifically target tumors in vivo. In collaborationwith M.G. Finn, Department of Chemistry, we bioconju-gated CPMV to tumor ligands such as transferrin andfolic acid, whose receptors are upregulated on meta-bolically active tumor cells. The targeted particles hada high degree of specificity for the tumor ligand and foruptake by tumor cells. In a separate study, we showedthat canine parvovirus, which has a natural affinity forthe transferrin receptor and thus for tumor cells, couldspecifically deliver small molecules to tumor cells.

These studies will allow the further design of anti-tumor agents that can provide localized, highly specificimaging and therapy in vivo. Use of virus-based nanopar-ticles may help us visualize and eliminate small tumorsbefore the tumors have a chance to metastasize. Inaddition, the ability of the particles to focus toxic effectsto the site of the malignant cells, thereby expandingthe range of effective therapies that can be used invivo, holds great promise for reducing cancer-relatedmorbidity and mortality.

PUBLICATIONSHsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A.,Reddy, V.S. Characterization of polymorphism displayed by the coat proteinmutants of tomato bushy stunt virus. Virology 349:222, 2006.

Lewis, J.D., Destito, G., Zijlstra, A., Gonzalez, M.J., Quigley, J.P., Manchester,M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat.Med. 12:354, 2006.

Manchester, M., Singh, P. Virus-based nanoparticles (VNPs): platform technologiesfor diagnostic imaging. Adv. Drug Dev. Rev., in press.

Rae, C.S., Khor, I.W., Wang, Q., Destito, G., Gonzalez, M.J., Singh, P., Thomas,D.M., Estrada, M.N., Powell, E., Finn, M.G., Manchester, M. Systemic traffickingof plant virus nanoparticles in mice via the oral route. Virology 343:224, 2005.

Scobie, H.M., Thomas, D., Marlett, J.M., Destito, G., Wigelsworth, D.J., CollierR.J., Young J.A.T., Manchester, M. A soluble receptor decoy protects rats againstanthrax lethal toxin challenge. J. Infect. Dis. 192:1047, 2005.

Sen Gupta, S., Kuzelka, J., Singh, P., Lewis, W.G., Manchester, M., Finn, M.G.Accelerated bioorthogonal conjugation: a practical method for the ligation of diversefunctional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16:1572,2005.

Singh, P., Destito, G., Schneemann, A., Manchester, M. Canine parvovirus-likeparticles, a novel nanomaterial for tumor targeting. J. Nanobiotechnol. 4:2, 2006.

Singh, P., Gonzalez, M.J., Manchester, M. Viruses and their uses in nanotechnol-ogy. Drug Dev. Res., in press.


Translational Regulation inChloroplasts and Expression ofHuman Monoclonal Antibodiesin Eukaryotic Algae

S.P. Mayfield, D. Barnes, A. Manuell, M. Beligni,

J. Marin-Navarro, M. Muto, M. Tran, D. Siefker, R. Henry

Gene expression in chloroplasts is primarily con-trolled during the translation of plastid mRNAsinto proteins, and understanding how this pro-

cess is regulated is key to understanding plant develop-ment and function. Controlling chloroplast translationis also an essential component in optimizing the pro-duction of human therapeutic proteins in algae.

Using proteomics and bioinformatics analyses, weidentified the set of proteins that function in chloroplasttranslation. These studies indicated that the core trans-lational apparatus of chloroplasts is highly related tothat of bacteria but that chloroplasts have incorporatedadditional protein components that allow more com-plex regulatory mechanisms. Some of these additionalcomponents are ribosomal proteins; others are proteintranslation factors. Chloroplast mRNAs also contain anumber of RNA regulatory elements that are not foundin bacteria, as well as conserved RNA elements, suchas ribosome-binding sequences, but even these con-served elements appear to function in chloroplast trans-lation differently than in bacterial translation. The uniquecomponents of chloroplast translation provide the oppor-tunity for regulation of chloroplast translation, for exam-ple in response to exposure to light, that cannot beachieved in simpler bacterial systems.

To better understand translation in plants, we areexamining the structure of both the chloroplast andcytoplasmic ribosomes from Chlamydomonas reinhardtii,a unicellular photosynthetic eukaryote. Using electroncryomicroscopy and single-particle reconstruction, wedetermined the structure of the C reinhardtii cytoplas-mic 80S ribosome and found that it is nearly identicalto ribosomes from animals, including the human 80Sribosome. Parallel proteomics analysis supported thisfinding. We also determined the structure of the chlo-roplast ribosome to 20 Å and found that although it isconserved with bacterial 70S ribosomes, it has largeunique structural domains, as predicted by our pro-teomics analysis.

Compared with bacterial 70S ribosomes, the chlo-roplast ribosome has unique structural domains locatedprimarily on the small ribosomal subunit (Fig. 1). This

finding is supported by our proteomics results thatindicated that the mass of the small (30S) subunit ofthe chloroplast ribosome is 25% larger than the bac-terial 30S subunit. Chloroplast-unique structures arefound on the solvent side of the small subunit; thelarge subunit interacting face is similar to that in bac-terial ribosomes.

The largest of the chloroplast-unique domains occursin the vicinity of the mRNA exit tunnel but also extendsup alongside the head and down across the platform.This structure has multiple lobes and possibly spans theentire solvent-exposed face of the chloroplast small ribo-somal subunit, connecting with a small region of chlo-roplast-unique density below the shoulder. Anotherdistinct region of chloroplast-unique density is locatedin the beak region of the ribosome, near the mRNAentrance tunnel. These chloroplast-unique ribosomalstructures are poised to interact with chloroplastmRNAs early in message recognition, a key point fortranslational regulation. These studies have revealedthe structural basis from which we can pursue identi-fication of the molecular and biochemical interactionsof mRNAs, translation factors, and the chloroplastribosome that result in regulated translation of chloro-plast mRNAs.


F i g . 1 . Structure of the chloroplast ribosome from C reinhardtii

calculated to 20-Å resolution. Structures identified as chloroplast

unique through comparison with bacterial ribosomes are labeled.

The chloroplast-unique structures are found on the path of mRNA

through the ribosome, which is labeled as mRNA entrance and exit

tunnels. These structures are positioned for interaction with mRNAs

before and during translation; such interactions most likely are

used to select and position mRNA for initiation of translation and

to increase the rate and fidelity of mRNA translation.

In addition to these basic studies on translation, wehave developed a system for the expression of recom-binant proteins, including human therapeutic proteins,in C reinhardtii chloroplasts. We have expressed a num-ber of mammalian proteins, including human mono-clonal antibodies. We recently expressed 83K7C, ahuman monoclonal antibody that binds to protectiveantigen 83 of anthrax toxin. We showed that 83K7Cassembles in the cell to form functional antibodies thatbind the antigen and thus potentially could block thetoxic effects of Bacillus anthracis during infection. Wealso showed that this algal-based system can be usedto produce high levels of a number of other proteinswith potential human therapeutic value.

These studies indicate that eukaryotic algae havetremendous potential for the expression of recombinanthuman therapeutic proteins, because algae can be growneconomically at large scale. Our continued genetic, bio-chemical, and structural studies should lead to a greaterunderstanding of the mechanism of chloroplast translationand enable us to design appropriate transgenes to achievehigher levels of expression of therapeutic proteins.

PUBLICATIONSBarnes, D., Franklin, S., Schultz, J., Henry, R., Brown, E., Coragliotti, A., May-field, S.P. Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to theexpression of Chlamydomonas reinhardtii chloroplast genes. Mol. Genet. Genomics274:625, 2005.

Fletcher, S.P., Muto, M., Mayfield, S.P. Optimization of recombinant proteinexpression in the chloroplast of green algae. In: Transgenic Microalgae as GreenCell Factories. León, R., Gaván, A., Fernández, E. (Eds.). Landes Bioscience,Austin, TX, in press.

Manuell, A.L., Mayfield, S.P. A bright future for Chlamydomonas. Genome Biol.7:327, 2006.

Manuell, A.L., Yamaguchi, K., Haynes, P.A., Milligan, R.A., Mayfield, S.P. Compo-sition and structure of the 80S ribosome from the green alga Chlamydomonas rein-hardtii: 80S ribosomes are conserved in plants and animals. J. Mol. Biol.351:266, 2005.

Molecular Basis of CognitiveFunction and DysfunctionM. Mayford, E. Korzus, G.J. Reijmers, M. Yasuda, R. Yasuda,

S. Miller, N. Matsuo

The ability to remember is perhaps the most sig-nificant and distinctive feature of our cognitivelife. We are who we are in large part because of

what we have learned and what we remember. Impair-ments in learning and memory are a component of dis-orders that affect human beings throughout life, from

childhood forms of mental retardation to psychiatricdisorders such as schizophrenia with onsets in lateadolescence and early adulthood to diseases of agingsuch as Alzheimer’s disease. We use genetic manipu-lation in mice to investigate the molecular eventsinvolved in learning and memory.C A L C I U M S I G N A L I N G A N D M E M O R Y

We know relatively little at a molecular level abouthow the brain stores new information. One hypothesis,which we tested, is that calcium-regulated changes inthe strength of synaptic connections between nerve cellscan store information. The enzyme calcium/calmodu-lin-dependent protein kinase is abundant at synapsesand when activated by calcium can strengthen synapticconnections. We used genetic manipulations in miceto indiscriminately activate this kinase at all synapsesin the entorhinal cortex, a part of the brain that isimportant for memory and is affected in the earlieststages of Alzheimer’s disease in humans.

We found not only that the formation of new memo-ries is impaired but also that previously establishedmemories could be erased. If memories are stored asprecise patterns of synaptic weights, then the indiscrim-inate strengthening of synapses might be expected toerase memories in a manner similar to the way writingall 1’s in computer memory will erase previously storedinformation. We also examined where calcium/calmodu-lin-dependent protein kinase functions within cells. Wefound that the synthesis of this kinase from RNA locatedspecifically at synapses is necessary for the stabilizationof memories that last several months.G E N E T I C M O D E L S O F D I S E A S E

The determination of the complete sequences ofthe mouse and human genomes indicates that humansare highly similar to mice at the genetic level. Oneapproach for investigating a genetic disease in humansis to introduce the same mutations into mice to producea model of the disease for better understanding of themolecular pathology and for testing possible treatments.

Rubenstein-Taybi syndrome is a developmental andcognitive disorder that results from mutation in thegene CBP. We produced a strain of mice with a defectin CBP and found that the mice were impaired in severallearning and memory tasks. More important, we showedthat these impairments were not due to problems indevelopment of the brain because they could be reversedby providing a normally functioning CBP gene to adultmice. The protein encoded by CBP chemically modifieshistones to allow the expression of a large variety of other


genes. We found that the memory deficits in the micecould be reversed by treatment with a drug that targetsthis histone-modifying function, suggesting a possibletreatment for Rubenstein-Taybi syndrome and possiblyother cognitive disorders.

M O L E C U L A R A N A T O M Y O F M E M O R Y

When humans learn new information, they use onlya tiny fraction of the neurons in the brain. One of thedifficulties in studying memory is an inability to identifyand specifically manipulate those neurons that partici-pate in a particular memory trace. We developed agenetic technique for use in mice that enables us tospecifically introduce genetic changes into neuronsthat are activated by behavioral stimuli. We are usingthis approach to introduce marker proteins that enableus to see the connections between neurons that havebeen activated during learning.

We have used this approach to study extinction, aprocess used in the treatment of phobias by which mem-ories are weakened by repeated exposure to a relevantstimulus. We found that the neurons originally activatedby a fearful stimulus were no longer activated afterextinction. This finding suggests that extinction train-ing actually erases or interferes with some componentof the original memory trace.

PUBLICATIONSColvis, C.M., Pollock, J.D., Goodman, R.H., Impey, S., Dunn, J., Mandel, G.,Champagne, F.A., Mayford, M., Korzus, E., Kumar, A., Renthal, W., Theobald,D.E., Nestler, E.J. Epigenetic mechanisms and gene networks in the nervous sys-tem. J. Neurosci. 25:10379, 2005.

Reijmers, L.G., Coats, J.K., Pletcher, M.T., Wiltshire, T., Tarantino, L.M., Mayford,M. A mutant mouse with a highly specific contextual fear-conditioning deficit found inan N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Learn. Mem. 13:143, 2006.

Yasuda, M., Mayford, M.R. CaMKII activation in the entorhinal cortex disrupts pre-viously encoded spatial memory. Neuron 50:309, 2006.

Regulation of the PlasminogenActivation SystemL.A. Miles, A. Baik, J.W. Mitchell, H. Bai, F.J. Castellino,*

R.J. Parmer**

* University of Notre Dame, Notre Dame, Indiana

** University of California, San Diego, California

Assembly of plasminogen and plasminogen acti-vators on cell surfaces is a key control point forpositive regulation of cell-surface proteolytic

activity necessary in physiologic and pathologic pro-cesses. Plasminogen-binding sites are markedly upreg-

ulated when monocytoid cells undergo apoptosis. There-fore, we are investigating the ability of plasminogen tomodulate monocyte apoptosis.

We cultured monocytoid cells (freshly isolated humanmonocytes and U937 cells) in plasminogen-deficientserum and induced the cells to undergo apoptosis byusing either TNF-α or cycloheximide. When induced inthe presence of increasing concentrations of plasminogen,apoptosis was inhibited in a dose-dependent manner; fullinhibition occurred at a concentration of plasminogenequal to its normal physiologic concentration. Treatmentwith plasminogen also markedly reduced intranucleo-somal DNA fragmentation and the active caspase-3,caspase-8, and caspase-9 induced by TNF-α or bycycloheximide.

Because monocytoid cells synthesize plasminogenactivators, we examined the role of plasmin proteolyticactivity in the antiapoptotic effects of plasminogen. Aplasminogen active-site mutant did not recapitulate thecytoprotective effect of wild-type plasminogen. In addi-tion, the antiapoptotic activity of plasminogen wasblocked by increasing concentrations of α2-antiplasmin,with full reversal at a 2-µM concentration of α2-antiplas-min, suggesting that the cytoprotective effect of plasmin-ogen requires activation of plasminogen to plasmin.Furthermore, antibodies against protease-activated recep-tor 1 blocked the antiapoptotic effects of plasminogen.

Our results suggest that plasminogen protects mono-cytic cells from apoptosis via a mechanism that requiresthe proteolytic activity of plasmin and that the protectionis mediated by protease-activated receptor 1. Becausemonocyte apoptosis regulates inflammation and ather-osclerosis, these results provide insight into a novel rolefor plasminogen in these processes.

An emerging area of research has indicated a novelrole for the plasminogen activation system in regulatingthe release of neurotransmitters. Prohormones, secretedby cells within the sympathoadrenal system, are proc-essed by plasmin to bioactive peptides that mediatefeedback inhibition of secretagogue-stimulated releaseof neurotransmitters. Catecholaminergic cells of thesympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasminis enhanced in the presence of catecholaminergic cells,and the enhancement requires binding of plasmin(ogen)to cellular receptors. Consequently, modulation of thelocal cellular fibrinolytic system of catecholaminergiccells results in substantial changes in catecholaminerelease. However, mechanisms for enhancing prohor-


mone processing and cell-surface molecules that medi-ate the enhancement on catecholaminergic cells havenot been investigated.

We found that plasminogen activation was enhancedmore than 6.5-fold on catecholaminergic cells. Treat-ment with carboxypeptidase B decreased cell-dependentplasminogen activation by almost 90%, suggesting thatthe binding of plasminogen to proteins exposing C-ter-minal lysines on the cell surface is required to promoteplasminogen activation. Using a novel strategy of targetedspecific proteolysis with carboxypeptidase B combinedwith a proteomics approach involving 2-dimensional gelelectrophoresis, radioligand blotting, and tandem massspectrometry, we identified catecholaminergic plasmino-gen receptors required for enhancing plasminogen acti-vation. Two major plasminogen-binding proteins thatexposed C-terminal lysines on the cell surface containedamino acid sequences corresponding to β- and γ-actin.A monoclonal antibody to actin inhibited cell-dependentplasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggestthat forms of actin expressed on the cell surface bindplasminogen, thereby promoting plasminogen activa-tion and increased prohormone processing leading toinhibition of neurotransmitter release.

PUBLICATIONSMitchell, J.W., Baik, N., Castellino, F.J., Miles, L.A. Plasminogen inhibits TNFαapoptosis in monocytes. Blood 107:4383, 2006.

Structure and Action ofMolecular MachinesR.A. Milligan, J. Chappie, P. Chowdhury, R. Coleman, T. Dang,

S. Falke,* E. Gogol,** M.B. Lee, S. Mulligan, M. Reedy,***

M.K. Reedy,*** A.B. Ward, E.M. Wilson-Kubalek, C. Yoshioka

* William Jewel College, Liberty, Missouri

** University of Missouri, Kansas City, Missouri

*** Duke University Medical Center, Durham, North Carolina

Macromolecular assemblies may be composed offrom 2 to perhaps scores of proteins and are thefunctional units—the molecular machines—of

the cell. We use electron cryomicroscopy and imageanalysis to study the structure and mechanism of actionof several of these machines. We combine the 3-dimen-sional maps calculated from electron images of themachines with biochemical data and high-resolutionx-ray structures of the individual components to pro-

vide insight into the operation of the machines. Duringthe past year, we continued our work on members of themyosin and kinesin superfamilies, microtubule-stabi-lizing proteins, and membrane proteins.

Although the mechanism of plus end–directed, pro-cessive motion by conventional kinesins is now wellunderstood, the mechanism by which members of thekinesin 14 class move toward the minus ends of micro-tubules is not. Likewise, in the myosin superfamily,how nucleotide-mediated conformational changes inthe motor domain of class VI myosins result in “back-ward” motility is not known. We are elucidating themolecular mechanisms of these more unusual membersof the myosin and kinesin superfamilies. (Movies show-ing the motions of conventional myosin and kinesin canbe viewed at www.scripps.edu/milligan/projects.html.)

The kinesin Ncd belongs to the kinesin 14 class ofmotor proteins. Compared with the situation with plusend–directed kinesins, the nature and timing of thestructural changes that underlie the motility of kinesin14 motors are poorly understood. We used electroncryomicroscopy and image analysis to calculate 3-dimen-sional maps of Ncd bound to microtubules in variousstages in its mechanochemical cycle. The maps revealeda minus end–directed rotation of approximately 70° ofa coiled coil mechanical element of microtubule-boundNcd upon ATP binding. In parallel with these struc-tural studies, our collaborators, N. Endres and R. Valeat the University of California, San Francisco, showedthat extending or shortening this mechanical elementrespectively increases or decreases movement velocity,without affecting ATPase activity. These results indicatethat as with other kinesins, the force-producing con-formational change of Ncd occurs upon ATP bindingbut, unlike the situation with other kinesins, involvesthe swing of a rigid, lever arm–like mechanical ele-ment similar to that described for myosins.

Whereas most kinesins move along intact micro-tubules, members of the kinesin 13 class, such asKinI, destabilize and depolymerize microtubules anddo not appear to have motile properties. We foundthat a KinI fragment consisting of only the conservedmotor core is necessary and sufficient for ATP-depen-dent depolymerization. The motor core binds alongmicrotubules in all nucleotide states, but in the pres-ence of a nonhydrolyzable ATP analog, depolymerizationalso occurs. Structural characterization of the analog-induced depolymerization products provided a snapshotof the disassembly machine at the microtubule ends.


Our data indicate that whereas conventional kinesinsuse the energy of ATP binding to execute a power strokethat results in unidirectional motion along the microtu-bule surface, KinIs at the ends of microtubules use theenergy to bend the underlying protofilament, therebydestabilizing the microtubule lattice and leading tomicrotubule depolymerization. Furthermore, when themotor core is associated with the microtubule wall, thecore is stalled in a weakly bound, nucleotide-free state.Progression to the strongly bound, ATP-containing stateis possible only when the KinI encounters a microtubuleend, where it can catalyze deformation of protofilamentsand disassembly of microtubules. The unusual mechano-chemical coupling of this kinesin provides an elegantmechanistic basis for its microtubule-depolymerizingactivity. Our current research focuses on understandingthe role of the second head in these dimeric molecules.

The protein doublecortin is expressed in migratingand differentiating neurons. In humans, mutations inthis protein disrupt brain development, causing lissen-cephaly. Although doublecortin is associated with andstabilizes the microtubule cytoskeleton, it has no homol-ogy with other microtubule-binding proteins such asMAP2 or tau. We found that doublecortin preferentiallynucleates and binds to 13-protofilament microtubules.This specificity was explained when we discovered thatthe protein binds in the valleys between the protofila-ments of the microtubule wall. This binding site isunique and appears to be ideally located for microtu-bule stabilization. In this location, doublecortin mostlikely contributes to both the longitudinal and the lat-eral interactions that stabilize the microtubule wall.We are now investigating the binding of proteins thattrack with the tips of dynamic microtubules.

In collaboration with G. Chang, Department ofMolecular Biology, we have grown well-ordered arraysof several membrane proteins that are involved in mul-tidrug resistance. These arrays, helical tubes and 2-dimensional crystals of membrane-embedded proteins,are suitable for structural studies via electron micros-copy. In one instance, we trapped a drug transporterin various stages of its mechanistic cycle and withsubstrates bound. We anticipate that 3-dimensionalelectron microscopy maps of membrane-embeddedtransporters in various states, together with the high-resolution x-ray structures of the detergent-solubilizedprotein, will provide insights into the mechanisms usedto transport metabolites and drugs across membranes.

In other studies, we developed a general methodfor helical crystallization of proteins on lipid tubules

that we are using to study the virulence factor per-fringolysin O from Clostridium perfringens. Perfringo-lysin O is a cytolysin, an important class of proteinsthat oligomerize and embed within membranes as partof the proteins’ lytic functions. We obtained helicalcrystals of wild-type and several mutant forms of thecytolysin on nickel-lipid tubules. Three-dimensionalmaps of these proteins derived from images of thehelical crystals will be used to complement our stud-ies of pore formation by perfringolysin O on lipid lay-ers. These studies will provide a better understandingof the pathogenic function of cytolysins. Additionalstudies involving tubular crystallization of membraneproteins and other bacterial toxins are opening up prom-ising new areas for future research.

PUBLICATIONSDang, T.X., Milligan, R.A., Tweten, R.K., Wilson-Kubalek, E.M. Helical crystalliza-tion on nickel-lipid nanotubes: perfringolysin O as a model protein. J. Struct. Biol.152:129, 2005.

Endres, N.F., Yoshioka, C., Milligan, R.A., Vale, R.D. A lever-arm rotation drivesmotility of the minus-end-directed kinesin Ncd. Nature 439:875, 2006.

Manuell, A.L., Yamaguchi, K., Haynes, P.A., Milligan, R.A., Mayfield, S.P. Compo-sition and structure of the 80S ribosome from the green alga Chlamydomonas rein-hardtii: 80S ribosomes are conserved in plants and animals. J. Mol. Biol.351:266, 2005.

CNS Development andMechanosensory PerceptionU. Müller, C. Barros, R. Belvindrah, F. Conti, S. Franco,

N. Grillet, S. Hankel, P. Kazmierczak, R. Radakovits,

C. Ramos, A. Reynolds, A. Sczaniecka, M. Schwander,

S. Webb

Adisproportionately large number of genes in thegenomes of vertebrates encode cell recognitionmolecules that mediate cell-cell interactions and

interactions between cells and the extracellular matrix.This finding most likely reflects an evolutionary trendtoward increasingly more complex cellular interactions inhigher metazoans. The highest diversity of such interac-tions occurs in the CNS, where thousands of differentneuronal subtypes are connected into defined neuronalcircuits. We use mouse genetics, genomics, cell biol-ogy, biochemistry, and imaging technology to analyze thefunction of cell recognition molecules during the develop-ment of neuronal circuits in the CNS. In another project,we are elucidating the mechanisms by which cell recogni-tion molecules contribute to mechanosensory perception.


F O R M A T I O N O F C O R T I C A L S T R U C T U R E S I N T H E C N S

The establishment of the 3-dimensional cytoarchitec-ture of the nervous system depends on interactions ofreceptors on neuronal cells with molecules presentedwithin the extracellular matrix and by neighboring cells.Integrins are a class of neuronal receptors that mediateinteractions with glycoproteins secreted by the extracellu-lar matrix and with membrane-anchored counterreceptors.

Recently, we found that integrins cooperate withsecreted signaling molecules such as sonic hedgehogand Reelin to regulate important steps during CNSdevelopment, such as cell proliferation and formationof neuronal layers during the development of the cerebraland cerebellar cortex. We are identifying the downstreamsignaling pathways activated by integrins during corti-cal development. We are also studying signaling inter-actions between integrins and other receptors such asreceptor tyrosine kinases. Finally, we have extendedour studies to the analysis of integrin functions in theCNS in adults.C E L L R E C O G N I T I O N M O L E C U L E S , M E C H A N O S E N S O R Y

P E R C E P T I O N , A N D D E A F N E S S

Mechanosensation, the transduction of mechanicalforce into an electrochemical signal, allows living organ-isms to detect touch, hear, register movement andgravity, and sense changes in cell volume and shape.In mammals, the hair cells of the inner ear are theprinciple mechanosensors for the detection of soundand movement. Hair cells elaborate stereocilia thatcontain mechanosensitive ion channels. The stere-ocilia of a hair cell are interconnected by extracellularbridges into a bundle and are situated next to special-ized extracellular matrix assemblies. Sound waves orhead movements lead to deflection of the stereociliabundle, changes in the ion permeability of the mech-anosensitive channels, and depolarization of the haircells. The molecules that regulate development andfunction of hair cells are poorly defined.

Because defects in hair cells cause inherited formsof deafness, we use human and mouse genetics as aguideline to identify and study molecules that regulatethe development and function of mechanosensory haircells. Currently, about 70 genes have been identifiedin which mutations lead to deafness. Many of thesegenes encode molecules secreted into the extracellularmatrix and membrane-anchored cell adhesion molecules.Mutations in the genes for the cell adhesion moleculecadherin 23 in mice and humans cause deafness. Ourfindings provide strong evidence that cadherin 23 is a

component of the so called tip-link, which has beenpredicted to transmit force onto mechanically gatedion channels in the stereocilia of hair cells. We areanalyzing the function of cadherin 23, proteins thatinteract with this cell adhesion molecule, and proteinsencoded by additional “deafness” genes for mechano-transduction. We are also doing genetic screens in miceto identify novel recessive deafness traits. Using thisstrategy, we have already identified several novel genesthat may be associated with deafness.

PUBLICATIONSBarros, C., Müller, U. Cell adhesion in nervous system development: integrin func-tions in glial cells. In: Integrins and Development. Danen, E.H.J. (Ed.). Landes Bio-science, Austin, TX, 2006, p. 185.

Belvindrah, R., Müller, U. Integrin signaling and central nervous system develop-ment. In: Extracellular Matrix in Development and Disease. Miner, J.H. (Ed.). Else-vier, St. Louis, 2005, p. 153. Advances in Developmental Biology andBiochemistry; Vol. 15.

Belvindrah, R., Nalbant, P., Chuanyue, W., Bokoch, G.M., Müller, U. Integrin-linked kinase regulates Bergmann glial differentiation during cerebellar develop-ment. Mol. Cell. Neurosci., in press.

Escher, P., Lacazette, E., Courtet, M., Blindenbacher, A., Landmann, L., Beza-kova, G., Lloyd, K., Müller, U., Brenner H.R. Synapses form in skeletal muscleslacking neuregulin receptors. Science 308:1920, 2005.

Naylor, M.J., Li, N., Cheung, J., Lowe, E.T., Lambert, E., Marlow, R., Wang, P.,Schatzmann, F., Wintermantel, T., Schuetz, G., Clarke, A.R., Müller, U., Hynes,N.E., Streuli, C.H. Ablation of β1 integrin in mammary epithelium reveals a keyrole for integrin in glandular morphogenesis and differentiation. J. Cell Biol.171:717, 2005.

Senften, M., Schwander, M., Kazmierczak, P., Lillo, C., Shin, J.B., Hasson, T.,Geleoc, G.S.G., Gillespie, P.G., Williams, D., Holt, J.R., Müller, U. Physical andfunctional interaction between protocadherin 15 and myosin VIIa in mechanosen-sory hair cells. J. Neurosci. 26:2060, 2006.

Molecular Mechanisms ofThermosensationA. Patapoutian, M. Bandell, A. Dhaka, A. Dubin, T. Earley,J. Grandl, H. Hu, L. Macpherson, T. Miyamoto, V. Uzzell

We are interested in the molecular descriptionof the function of sensory neurons. Of the 5popularly characterized senses—sight, hearing,

taste, smell, and touch—touch is among the most var-ied and least understood. Within this sense is the abilityto sense mechanical forces, chemical stimuli, and tem-perature, and the molecules that mediate this abilityhave been a long-standing mystery. Temperature sensa-tion in particular has received relatively little attentionfrom biologists and yet is critical for interactions withthe environment.

We recently discovered proteins that may enablesensory neurons to convey information about tempera-


ture. These proteins are ion channels activated by spe-cific changes in temperature; thus they act as the molec-ular thermometers of the body. Specifically, our resultshave led to the identification and characterization of 1novel warm-activated transient-receptor potential (TRP)channel, TRPV3 (33°C threshold) and 2 novel cold-acti-vated TRP channels, TRPM8 (25°C threshold) andTRPA1 (ANKTM1, 17°C threshold). We found thatTRPM8 is also the receptor for the compound menthol,providing a molecular explanation of why mint flavorsare typically perceived as cooling. Furthermore, wediscovered that TRPA1 is activated by cinnamaldehyde,allicin (garlic), and other compounds with a burning sen-sory quality, consistent with a role of TRPA1 in thedetection of noxious cold sensations. Together these tem-perature-activated channels represent a new subfamily ofTRP channels that we have dubbed thermoTRPs.

In agreement with a role in initiating temperature sen-sation, most of the thermoTRPs are normally found insubsets of neurons in dorsal root ganglia. A surprisinglydistinct expression pattern was observed for TRPV3, thewarm receptor. In mice, high levels of TRPV3 occur solelyin skin keratinocytes, suggesting that skin cells might beable to “sense” temperature and then communicate thisinformation to neurons in dorsal root ganglia. How tem-perature information is coded from the skin to the spinalcord is not well understood, and we are using a varietyof approaches to answer this question. For example, datafrom mice lacking the gene for TRPV3 suggest thatTRPV3 is indeed required for proper heat sensation invivo, reinforcing a role of skin in thermosensation.

All organisms have a need for thermosensation.Because some invertebrate species are more amenableto genetic studies than mammals are, we asked whethernonvertebrates also use thermoTRPs to sense tempera-ture. We showed that the Drosophila ortholog of TRPA1is an ion channel activated by warm temperatures, sug-gesting an evolutionarily conserved role of TRP chan-nels in temperature sensing. In collaborative effortswith P. Garrity, Massachusetts Institute of Technology,Cambridge, Massachusetts, and W. Shafer, Universityof California, San Diego, we are using genetic studiesto examine the role of TRPA family members in inver-tebrate species.

Another key question is what makes thermoTRPstemperature sensitive whereas other TRPs are not?Answering this question requires insight into the funda-mental biophysical mechanism of how temperature acti-vates ion channels. Our ongoing structure-functionexperiments, including mutagenesis and chimeric protein

analysis of the thermoTRPs, will provide important cluesabout how cold or heat activates these ion channels.

Our long-term goal is to synthesize an integratedpicture of sensory neuron function. By identifying theproteins that initiate the molecular cascade leading totemperature perception, we have provided the basis forprobing the foundation of the sense of temperature. Wenow have the opportunity to extend these insights intoimportant areas of human health, such as pain patho-physiology. For example, TRPA1 is a potential targetfor treating pain, and we are identifying small-moleculeinhibitors of TRPA1 in collaboration with scientists atthe Genomics Institute of the Novartis Research Founda-tion, San Diego, California. Therefore, the approacheswe are using will yield insights into the basic biologyof the peripheral nervous system and may also havean effect on novel treatments for pain.

PUBLICATIONSBandell, M., Dubin, A.E., Petrus, M.J., Orth, A., Mathur, J., Hwang, S.W., Pat-apoutian, A. High-throughput random mutagenesis screen reveals TRPM8 residuesspecifically required for activation by menthol. Nat. Neurosci. 9:493, 2006.

Dhaka, A., Viswanath, V., Patapoutian, A. TRP ion channels and temperature sen-sation. Annu. Rev. Neurosci. 29:135, 2006.

Macpherson, L.J., Geierstanger, B.H., Viswanath, V., Bandell, M., Eid, S.R.,Hwang, S.W., Patapoutian, A. The pungency of garlic: activation of TRPA1 andTRPV1 in response to allicin. Curr. Biol. 15:929, 2005.

Functional Proteins in TumorMetastasis and AngiogenesisJ.P. Quigley, E.I. Deryugina, A. Zijlstra, J.P. Partridge,

T. Kupriyanova, M. Madsen, V. Ardi, M.C. Subauste

We have established a number of in vivo modelsystems that can recapitulate the major cellu-lar and tissue events that occur during tumor

metastasis and angiogenesis. The model systems allowquantitative measurements, microscopic analysis in realtime, biochemical and immunologic probing, and directmolecular and therapeutic interventions.

Recently, use of small interfering RNA moleculesdirected against specific expressed genes and applieddirectly into the models provided insights into the con-tributory role of the gene products in tumor disseminationand neovascularization. In addition, use of subtractiveimmunization, which is used to generate unique func-tion-blocking monoclonal antibodies, in combination withimmunoproteomics enables us to identify specific anti-genic molecules that are functionally active in metas-


tasis and angiogenesis. Finally, use of activity-basedprotein profiling, in collaboration with B.F. Cravatt,Department of Cell Biology, enables us to detect, iso-late, and identify active proteolytic enzymes that aredistinctively and differentially activated during metas-tasis and angiogenesis.M E T A S T A S I S

Selected human tumor cells inoculated onto thechorioallantoic membrane of developing chick embryosform primary tumors on the membrane in 4–7 days. Asmall percentage of the cells in the primary tumor dis-seminate through the vasculature and within 3–4 daysarrest and proliferate in secondary organs of the embryo.Measuring a small number of early-arriving metastaticcells (<200) growing and expanding in the secondaryorgan has always been technically difficult. We now usean approach in which unique regions of human DNA,known as Alu repeat sequences, are amplified by poly-merase chain reaction from the total DNA extractedfrom various organs of the tumor-bearing chick embryo.Chicken DNA contains no Alu sequences, so any prod-uct generated by the polymerase chain reaction indi-cates that human tumor cells are present in the chickembryo organ and would have arrived there via theknown sequential steps in metastasis. We can nowdetect as few as 25–50 human tumor cells present inthe entire chick embryo lung, liver, or brain and canmeasure the expansion of these metastatic cells by usingreal-time polymerase chain reaction.

We are using various screening procedures in thismodel system to identify molecules that enhance, orconversely inhibit, the appearance of metastatic humantumor cells in organs of chick embryos. The screeningprocedures include direct inoculation of primary tumorcells that have been transfected with various small inter-fering RNA constructs to silence specific genes that mightcontribute to metastatic dissemination. Inoculating mono-clonal antibodies directly into the tumor-bearing embryoand monitoring the influence of the antibodies on metas-tasis are also part of our screening procedures.

We are also using a more conventional method ofmonitoring human tumor metastasis in specific immun-odeficient mice. However, compared with our chickembryo metastasis assay, this method is less quantitative,requires more time (3–5 weeks), and is more difficultto use for inhibitor screening and molecular intervention.We are using the mouse metastasis assay to take advan-tage of mouse genetics and to confirm the efficacy ofvarious effector molecules and inhibitors that initiallyare identified in the chick embryo metastasis assay.

A N G I O G E N E S I S

One of the most commonly used in vivo assays forangiogenesis is the chick embryo chorioallantoic mem-brane assay. We developed a quantitative variation ofthis assay that enables us to detect and measure thenewly sprouting blood vessels responding to an angio-genic stimulus such as a specific growth factor or agrowing tumor. A highly specific metalloproteinase,MMP-13, has been implicated in the tissue remodel-ing that occurs during the formation of the new bloodvessels. We characterized this specific proteolytic eventand found that specific collagen-cleaving metallopro-teinases are implicated directly in the outgrowth ofnew vessels.

We also found that another metalloproteinase,MMP-9 (gelatinase B), most likely is involved in angio-genic tissue remodeling. The proteolytic activity of thisenzyme also appears to be necessary for a full angio-genic response. Interestingly, these 2 critical enzymesare actively imported into the vascular/stromal tissueby distinct inflammatory cells responding to the angio-genic stimulation. Neutrophil-like heterophils rapidlyand almost immediately import MMP-9 into the tissue,whereas monocyte/macrophages actively deliver MMP-13 1–2 days later, possibly in response to specificsecreted products of the early-arriving heterophils. Thus,normal angiogenesis and tumor angiogenesis are closelylinked to an accompanying host inflammatory responsethat contributes critical functional molecules to theangiogenic process.

We are dissecting out and identifying the specificmolecules and cells that link the inflammatory responseto the angiogenic process and to the progression ofmalignant neoplasms. We are also trying to decipherwhether the relevant functional molecules are derivedfrom host cells or tumor cells.

I N T R A V A S A T I O N

We are also investigating intravasation, the entryof primary tumor cells into the host vasculature, oftenthe vasculature that is newly formed during tumor angio-genesis. Intravasation appears to be the least-studiedprocess in the metastatic cascade but most likely is arate-limiting step in tumor dissemination. We recentlyisolated 2 isogenic variants of a human fibrosarcomacell line that differ 100-fold in their ability to enter thevasculature in vivo and in their ability to metastasize.We are using array technology, proteomics approaches,and intravital microscopy in the cellular and molecularanalysis of these 2 variants. The results should indi-


cate specific molecules that are functionally importantin interactions between tumor cells and the vascula-ture and contribute to intravasation. Using activity-based protein profiling, we found that the proteolyticenzyme urokinase is differentially activated duringintravasation and catalytically contributes to theenhanced entry of tumor cells into the vasculature.

PUBLICATIONSDeryugina, E.I., Zijlstra, A., Partridge, J.J., Kupriyanova, T.A., Madsen, M.A.,Papagiannakopoulos, T., Quigley, J.P. Unexpected effect of matrix metalloproteinasedown-regulation on vascular intravasation and metastasis of human fibrosarcomacells selected in vivo for high rates of dissemination. Cancer Res. 65:10959, 2005.

Lewis, J.D., Destito, G., Zijlstra, A., Gonzalez, M.J., Quigley, J.P., Manchester,M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat.Med. 12:354, 2006.

Madsen, M.A., Deryugina, E.I., Niessen, S., Cravatt, B.F., Quigley, J.P. Activity-based protein profiling implicates urokinase activation as a key step in humanfibrosarcoma intravasation. J. Biol. Chem. 281:15997, 2006.

Zijlstra, A., Seandel, M., Kupriyanova, T.A., Partridge, J.J., Madsen, M.A., Hahn-Dantona, E.A., Quigley, J.P., Deryugina, E.I. Proangiogenic role of neutrophil-likeinflammatory heterophils during neovascularization induced by growth factors andhuman tumor cells. Blood 107:317, 2006.

Regulators of Clathrin-MediatedEndocytosis S.L. Schmid, J. Chappie, S.D. Conner, M. Ishido, M. Leonard,

R. Ramachandran, F. Soulet, B.D. Song, M.C. Surka, D. Yarar

Clathrin-mediated endocytosis is essential for theefficient uptake of nutrients and other macro-molecules into cells and for the regulation of

signaling by cell-surface receptors. The process occursat clathrin-coated pits, which concentrate receptor-ligand complexes, deform the membrane, invaginate,and eventually pinch off, forming clathrin-coated vesicles(CCVs). The major components involved in formation ofCCVs are clathrin, adaptor proteins, and dynamin, anatypical GTPase.

Clathrin self-assembles into a polygonal latticeand serves as a scaffold for the formation of coatedpits. Adaptor protein-2 is a heterotetrameric proteinthat triggers clathrin assembly at the plasma mem-brane and interacts directly with the cytoplasmic tailsof surface receptors to concentrate the receptors intothe assembling coated pit. We view dynamin as themaster regulator of endocytosis.

Previously, we developed a cell-free assay in whichCCVs are reconstituted from sheets of purified plasmamembranes from rat liver. Using this in vitro reconstitu-

tion system and a new biochemical complementationassay, we explored the limiting cytosolic requirementsfor endocytosis of the low-density lipoprotein recep-tor–related protein (LRP) from isolated plasma mem-branes. LRP, also known as a scavenger receptor,binds multiple, distinct ligands and participates inconstitutive endocytosis and signal transduction.

We found that clathrin, adaptor protein-2, anddynamin do not support efficient LRP uptake; additionalfactors present in a 30% ammonium sulfate supernatantfraction of bovine brain cytosol are required. Fraction-ation of the supernatant revealed that multiple andredundant factors are required to support LRP endocy-tosis. Our data suggest that LRP, which has severaldistinct endocytic motifs in its cytoplasmic domain,may use multiple pathways for endocytosis in vitro.The factors we identified, 70-kD heat-shock cognateprotein, synaptojanin, and collapsin response mediatorprotein-2, are all implicated in clathrin-mediated endo-cytosis in vivo, thus validating the assay. However, wefound that these factors were sufficient but not neces-sary for formation of CCVs. Thus, functional redun-dancy and complexity make this assay biochemicallyintractable, and we have chosen, at least for themoment, to abandon it.

We continue to analyze the structure and functionof dynamin. Dynamin is a multidomain protein con-sisting of an N-terminal GTPase domain, a middledomain of previously unknown function, a PH domainthat binds phosphatidylinositol 4,5-biphosphate, aGTPase effector domain (GED) required for dynaminself-assembly that functions as an assembly-dependentGTPase-activating protein for dynamin, and a C-terminalproline-arginine–rich domain. Dynamin self-assembles invitro into spirals on liposomes containing phosphatidyli-nositol 4,5-biphosphate to tubulate the liposomes,resulting in a greater than 100-fold stimulation ofGTPase activity. Self-assembly and assembly-stimu-lated GTPase activity at the necks of deeply invagi-nated coated pits are thought to drive conformationalchanges that mediate membrane fission.

Our collaborators recently solved the crystal struc-ture of the isolated, unoccupied GTPase domain of ratdynamin-1. Unlike the situation in other GTPases, thenormally unstructured switch 1 and switch 2 regionsthat surround the unoccupied GTP binding pocket wereordered. On the basis of the structure, 2 arginines nearthe active site were predicted to be required for catal-ysis, but our enzymatic analysis of lysine and alanine


substitutions of these residues suggested otherwise.Thus, the mechanism of GTP hydrolysis remains obscure.However, the structure revealed the existence of a hydro-phobic groove created by the N- and C-terminal α-helicesof the GTPase domain, which was suggested to be adocking site for the GED.

In a recent study, we identified mutations in theGED that resulted in reduced GTPase activity withoutaffecting self-assembly. Because these mutations mappedto a predicted amphipathic helix at the extreme C termi-nus of GED, we speculated that this GED helix formed a3-helical bundle with the GTPase domain helices. Totest this hypothesis, we have generated a construct con-sisting of the GTPase of dynamin together with a C-ter-minal extension composed of a short sequence ofturn-preferring residues followed by the 20 amino acidC-terminal GED helix. This construct, unlike GTPasedomain or GED constructs is largely soluble whenexpressed in Escherichia coli and has basal GTPaseactivity equivalent to that of full-length dynamin.

These exciting results suggest that we have fullyreconstituted the GED-GTPase interactions. We arecurrently expressing the GTPase-GED peptide constructfor high-resolution x-ray crystallography studies, andmutagenesis is under way to probe the mechanisms ofGED-stimulated GTPase activity.

We have also identified a new class of mutants inthe middle domain that alter the quaternary structureof dynamin. Native dynamin exists as a tetramer, butanalytical ultracentrifugation and gel filtration chromatog-raphy coupled to multiangle light scattering have con-firmed that the middle domain mutants are dimeric.Kinetic studies established that the basal GTPase activityof dynamin requires a highly cooperative, GTP-depen-dent conformational change in dynamin tetramers. Thedimeric dynamin mutants are defective in both activa-tion in the basal state and self-assembly into higherorder structures.

Finally, in following up our earlier discovery thatactin dynamics are required for multiple stages ofclathrin-mediated endocytosis, we found that the pro-tein sorting nexin 9 is a molecular link between dynaminand actin assembly. Sorting nexin 9 binds dynaminthrough the nexin’s SH3 domain and activates neuralWiskott-Aldrich syndrome protein to trigger actin-relatedprotein 2/3–dependent actin assembly into branchedfilaments. Thus, sorting nexin 9 may trigger the burstof actin assembly that accompanies the scission ofcoated vesicles.

PUBLICATIONSLeonard, M., Song, B.D., Ramachandran, R., Schmid, S.L. Robust colorimetricassays for dynamin’s basal and stimulated GTPase activities. Methods Enzymol.404:490, 2005.

Miwako, I., Schmid, S.L. A cell-free biochemical complementation assay revealscomplex and redundant cytosolic requirements for LRP endocytosis. Exp. Cell Res.312:1335, 2006.

Miwako, I., Schmid, S.L. Clathrin-coated vesicle formation from isolated plasmamembranes. Methods Enzymol. 404:503, 2005.

Reubold, T.F., Eschenburg, S., Becker, A., Leonard, M., Schmid, S.L., Vallee,R.B., Kull, F.J., Manstein, D.J. Crystal structure of the GTPase domain of ratdynamin 1. Proc. Natl. Acad. Sci. U. S. A. 102:13093, 2005.

Molecular Biology of OlfactionL. Stowers, I.S. Bharati, P. Chamero, J. Cruz, K. Flanagan,

J. Lin, D. Logan, T. Marton, C. Ramos

Every breath samples the environment for olfac-tory chemical information, determining the qual-ity of food, warning of danger, and confirming

safety. The neurons that mediate olfaction are of 2 types:those that mediate an evocative perception that varieswith each individual’s experience and those that regu-late stereotyped innate social behaviors such as aggres-sion and mating. Neurons that elicit odorant perceptionreside in the olfactory epithelium and relay chemicalinformation through activation of cAMP-responsivechannels. Recently, we showed that behavior-generat-ing neurons are located in the vomeronasal organ andrespond to pheromones through a cascade that ultimatelyactivates C-type transient-receptor potential 2 (TRP2)channels. We are using a molecular genetic approachto characterize the function of these pheromone-respon-sive neurons.

Through electrophysiologic recordings, we haveshown that mutant mice lacking C-type TRP2 channelsdo not depolarize in response to natural sources ofpheromones. Behavioral assays with these animalsrevealed that this pheromone response is necessary forboth intermale aggression and gender recognition. Weare identifying other unique molecular subpopulationsof pheromone-responsive neurons, and through geneticablation, biochemistry, and electrophysiology, we areassigning biological function to each neuron type.

A full characterization of the repertoire of chemo-sensory neurons will be essential in understanding thelogic of olfactory information coding. To this end, weare investigating a novel class of olfactory neurons thatlack both the cAMP and C-type TRP2 signaling com-ponents. Analysis of these neurons by transcriptional


profiling and then molecular genetics and biochemistryis being used to identify their role in olfactory function.

Elucidation of the function of specific neural circuitsthat regulate mammalian behavior has been hinderedbecause the pheromone compounds that signal eachbehavior have not been identified from their complexnatural sources. To obtain these important molecules,we are fractionating natural sources of pheromones andthen using biobehavioral assays to identify the mole-cules that initiate activity. The results will enable us toboth activate specific neural circuits and analyze thenatural production and regulation of the signalingligands. In total, we expect to define the pheromoneresponse pathway of mice and to reveal general princi-ples of neurons that govern complex social behavior.

Molecular Regulation ofVascular System Developmentin MammalsH. Stuhlmann, M.J. Fitch, Z. Zou, S. Chitnis, J.D. Lewis,

A. Durrans, W. LeVine

Establishment of a functional circulatory systemduring development is crucial for the delivery ofnutrients and oxygen to embryos. Defects in the

development of blood vessels result in death before birthor in congenital cardiovascular abnormalities. We exam-ine the molecular and genetic pathways that regulatethe 3 principal processes of vascular development: deter-mination of vascular lineage, vasculogenesis, and angio-genesis. We focus on the mouse model because of theready availability of genetic information on mice andexperimental tools and because of similarities betweenmice and humans. Using an expression-based “genetrap” screen in mouse embryonic stem cells andembryos, we identified 2 novel genes involved in theseprocesses: Vezf1 and Egfl7.A Z I N C F I N G E R G E N E E S S E N T I A L F O R N O R M A L

V A S C U L A R A N D L Y M P H A T I C D E V E L O P M E N T

Vezf1 is the gene for an early zinc finger transcrip-tion factor that controls the development of blood ves-sels and the lymphatic system in mice. Using functionalgenetic studies, we previously showed that Vezf1 playsan essential and dosage-dependent role in the prolifer-ation, remodeling, and integrity of the developing vas-culature. We recently extended these studies by using

in vitro differentiation of embryonic stem cells intoembryoid bodies. Our results indicate that Vezf1 affectsvascular differentiation by regulating cell proliferation,differentiation, and deposition of extracellular matrix.

We are examining the molecular pathways of Vezf1function. In collaborative studies with L. Benjamin, BethIsrael Deaconess Medical Center, Boston, we found thatVEZF1, the protein encoded by Vezf1, interacts withRho GTPases to modulate the function of Rho in theendothelium. Microarray cDNA analysis with RNA fromwild-type embryos and embryos lacking Vezf1 sug-gested that genes for fibrinogen and claudin and sev-eral genes involved in metabolite transport are targetgenes for Vezf1.A N E A R L Y M A R K E R F O R E N D O T H E L I A L C E L L S A N D

T H E I R P R O G E N I T O R S

Expression of a second endothelial gene identifiedin our screen, Egfl7, is restricted to the vascular endo-thelium and endothelial progenitors in the yolk sacmesoderm. Egfl7 is also expressed in multipotent stemcells in embryos, in primordial germ cells, and duringspermatogenesis. In the quiescent vasculature in adults,overall Egfl7 expression is downregulated. During physi-ologic angiogenesis in the uterus during pregnancy andin the regenerating endothelium after vascular injury,expression of Egfl7 is upregulated. EGFL7, the proteinencoded by Egfl7, is partially secreted and acts as achemoattractant on both endothelial cells and embry-onic fibroblasts in in vitro migration assays. EGFL7 isa compact 278 amino acid protein with an amino-ter-minal signal peptide; an EMI domain; and 2 centralepidermal growth factor–like domains, one of whichcontains a putative Notch interaction domain. Impor-tantly, recent collaborative studies with J. Kitajewski,Columbia University, New York City, provide supportfor our hypothesis that EGFL7 binds to Notch recep-tors and acts as an agonist for Notch signaling duringvascular development.D E V E L O P M E N T O F M U L T I V A L E N T V I R A L

N A N O P A R T I C L E S F O R I N V I V O V A S C U L A R

T A R G E T I N G A N D I M A G I N G

In collaboration with M. Manchester, Departmentof Cell Biology, we have developed viral nanoparticlesbased on the cowpea mosaic virus (CPMV) for nonin-vasive imaging and targeting of the mammalian cardio-vascular system. CPMV can be fluorescently labeled tohigh densities with no quenching, resulting in brightparticles that allow high-resolution intravital imagingof the vascular endothelium and blood flow deep inside


mouse or chick embryos for up to 72 hours. Using ahuman fibrosarcoma model for tumor angiogenesis, wefound that fluorescent CPMV can be used to distinguishbetween arterial and venous vessels and to monitor theneovascularization of the tumor microenvironment. Weare extending these studies by conjugating peptides tothe CPMV capsid to target the peptides to the vascula-ture, both during development and in disease models.

PUBLICATIONSLewis, J.D., Destito, G., Gonzalez, M.J., Zijlstra, A., Quigley, J.P., Manchester,M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat.Med. 12:354, 2006.

Kuhnert, F., Stuhlmann, H. Role of Vezf1 during vascular and lymphatic develop-ment. In: Endothelial Biomedicine. Aird, W.C. (Ed.), Cambridge University Press,New York, in press.

Zijlstra, A., Lewis, J.D., DeGryse, B., Stuhlmann, H., Quigley, J.P. Inhibition oftumor cell intravasation and subsequent metastasis through the regulation ofCD151-mediated in vivo tumor cell motility. Cancer Cell, in press.

Ion Channels and Fast Synaptic TransmissionN. Unwin

Ion channels play a central role in the rapid trans-mission of electrical signals throughout the nervoussystem. To determine how these membrane proteins

work, my colleagues and I are using electron micros-copy to analyze the structures of the proteins trappedin different physiologic states. Current studies centeron the nicotinic acetylcholine receptor at the nerve-muscle synapse. We wish to find out how this ion chan-nel achieves its ion selectivity and high transport rateand how it opens and desensitizes in response toacetylcholine released into the synaptic cleft. For ourstudies, we use postsynaptic membranes isolated fromthe (muscle-derived) electric organ of the Torpedo ray,which form tubular crystals of acetylcholine receptors.

The acetylcholine receptor is a member of asuperfamily of transmitter-gated ion channels, whichincludes the receptors for serotonin 5-HT3, γ-aminobu-tyric-acids A and C, and glycine. It has a cation-selec-tive pore, delineated by a ring of 5 similar subunits,that opens upon binding of acetylcholine to the 2ligand-binding (α) subunits at the subunit interfaces.

Recently, we obtained a refined atomic model ofthe acetylcholine receptor in the closed-channel form.We found that the individual subunits in the N-termi-nal ligand-binding domain are organized around 2 sets

of β-sheets packed in a curled β-sandwich, as in therelated soluble pentameric acetylcholine-binding pro-tein. Each of the subunits in the membrane-spanningdomain is made from 4 α-helical segments. The heli-cal segments arrange symmetrically, forming an innerring of helices that shape a water-filled pore and anouter shell of helices that coil around each other andshield the inner ring from the lipids. In the closedchannel, the helices in the inner ring come togethernear the middle of the membrane and make a con-stricting hydrophobic girdle. This girdle, which is about50 Å from the acetylcholine-binding sites, constitutesan energetic barrier to ion permeation and functionsas the gate of the channel.

These details, together with those obtained earlierfrom studies of the receptor trapped in the open-chan-nel form, have enabled us to understand in outline thestructural mechanism by which acetylcholine opensthe pore. In the absence of acetylcholine, the pore isnormally closed. When acetylcholine enters the bind-ing sites, localized rearrangements in the α-subunitsoccur that stabilize an alternative extended conforma-tion of the channel in which the inner sets of β-sheetsare rotated by about 10° about axes perpendicular tothe membrane plane, relative to the orientations of thesheets in the closed channel. These rotations are com-municated through the inner membrane-spanninghelices and open the pore by breaking the hydropho-bic girdle apart.

Improvements in resolution of the 3-dimensionalstructure, in both the closed- and open-channel forms,are now being attempted so that the structural mecha-nism of gating of the channel can be described ingreater detail. The knowledge gained from the refinedstructure of the locations of amino acid residues, inrelation to the ion pathway, is also being used todevelop quantitative explanations of how the highcation selectivity and high conduction rates of thischannel are achieved. These studies are yielding cru-cial insight into the nature of a number of neuromus-cular disorders, including several well-characterizedcongenital myasthenic syndromes. They are also pro-viding important 3-dimensional information about thebinding sites for drugs that affect the brain by modu-lating the function the related γ-aminobutyric acid,serotonin, glycine, and neuronal acetylcholine receptors.

PUBLICATIONSO’Brien, J., Unwin, N. Organization of spines on the dendrites of Purkinje cells.Proc. Natl. Acad. Sci. U. S. A. 103:1575, 2006.


Microscopes and Motility:Systems Integration in Cell MigrationC.M. Waterman-Storer, O. Rodriguez, S.L. Gupton, K. Kita,

R. Littlefield, K. Hu, A. Wheeler, W. Shin, M.L. Gardel,

I.C. Schnieder, A. Parapera, J. Lim

Cell migration is critical to development, theimmune response, and wound healing. In can-cer cells, loss of regulation of cell motility results

in deadly metastasis. The locomotion of vertebrate tis-sue cells is thought to require complex and dynamicinteractions between the microtubule and actin cyto-skeletal polymers, the endomembrane trafficking system,and focal adhesions to the extracellular environment.We develop quantitative light microscopy methods toanalyze the dynamic interactions between these complexmacromolecular systems in living cells to understand howthe systems are spatiotemporally coordinated to drivedirected cell movement. We then use these microscopicassays to analyze cells with specific perturbations ofcytoskeletal, membrane, or adhesive proteins to dis-sect the molecular mechanisms of the regulation ofthe proteins and their contribution to cell morphogene-sis and migration.

We pioneered fluorescent speckle microscopy, apowerful method that allows quantitative analysis of thedynamics of macromolecular assemblies in living cells.Recently, we extended the technology to multispectraltotal internal fluorescence reflection fluorescence micros-copy, allowing analysis of the integration of proteinswithin focal adhesions with the actin cytoskeleton dur-ing cell migration. In collaboration with G. Danuser,Department of Cell Biology, we developed correlationalfluorescent speckle microscopy to measure the couplingof focal adhesion proteins to the actin cytoskeleton.

We found that different classes of focal adhesionstructural and regulatory molecules have different degreesof correlated motions with actin filaments, indicating dif-ferential transmission of actomyosin motion through focaladhesions. Our results suggest that transient interactionsbetween focal adhesion proteins and actin filaments con-stitute a friction clutch between the cytoskeleton and theextracellular environment that is regulated during the mor-phodynamic transitions of cell migration.

On the basis of a mathematical model, researcherspredicted 15 years ago that migration speed would

have a biphasic response to increasing strength of celladhesion, with slow migration occurring at low and highstrengths and fast migration at intermediate strength.The assumption of the model was that migrating cellshave an asymmetry in adhesion strength from the frontpart of the mass of cells to the rear part, with the cellsconnected by symmetric contractile elements, butdynamic organizational states of F-actin and focaladhesions were not considered. This biphasic depen-dence of migration velocity on increasing adhesionstrength has since been supported experimentally, anda front-to-rear gradient in cell adhesion strength hasalso been shown.

We sought to determine if distinct organizationalstates of F-actin, myosin II, and focal adhesions accom-pany adhesion-dependent changes in velocity. Wecharacterized a unique phenotype for optimal migra-tion at intermediate adhesion strength, entailing rapidflow convergence and local depolymerization of F-actin,local activation of myosin II, rapid renewal of the com-ponents of focal adhesions, and intermediate lifetimeand turnover rates of focal adhesions. We recapitulatedthis phenotype and fast migration at a nonoptimal adhe-sion strength by manipulating the activity of myosin II.In contrast to the results with simple models, we foundthat a complex spatiotemporal integration and feed-back between F-actin, myosin II, and focal adhesionsmediates the classically observed biphasic migrationvelocity response to increasing adhesion strength, sothat a specific balance between adhesion and contrac-tion induces maximal migration velocity.

The microtubule and actin cytoskeletons interactin cells to promote a coordinated effort to drive protru-sion of the leading edge of cells during cell migration.The tumor suppressor protein adenomatous polyposiscoli and its binding partner EB1 accumulate on theends of microtubules. The tumor suppressor proteinspecifically collects on microtubules in cell protrusions,suggesting that it may promote cell protrusion. Wesimultaneously visualized dynamics of the suppressorprotein and microtubules in living cells. We found thatthe association of the protein with the ends of micro-tubules correlates with the increased growth stabilityof the microtubules and that this stabilization can occurindependent of the association of the protein with EB1.We also found that the protein and EB1 associate withthe ends of microtubules by distinct mechanisms. Thus,cancer-causing mutations in this tumor suppressorprotein may arise from defects in microtubule stabilityand cell migration.


PUBLICATIONSDanuser, G., Waterman-Storer, C.M. Quantitative fluorescent speckle microscopyof cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35:361, 2006.

deRooij, J., Kerstens, A., Danuser, G., Schwartz, M.A., Waterman-Storer, C.M.Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J.Cell Biol. 171:153, 2005.

Gupton, S.L., Waterman-Storer, C.L. Live-cell fluorescent speckle microscopy(FSM) of actin cytoskeletal dynamics and their perturbation by drug perfusion. In:Cell Biology: A Laboratory Handbook, 3rd ed. Celis, J., et al. (Eds.). AcademicPress, San Diego, 2005, Vol. 3, p. 137.

Gupton, S.L., Waterman-Storer, C.L. Spatiotemporal feedback between actomyosinand focal-adhesion systems optimizes rapid cell migration. Cell 125:1361, 2006.

Kita, K., Wittmann, T., Nathke, I.S., Waterman-Storer, C.L. Adenomatous polypo-sis coli on microtubule plus ends in cell extensions can promote microtubule netgrowth with or without EB1. Mol. Biol. Cell 17:2331, 2006.

Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman-Storer, C.M., Danuser, G.Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithe-lial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J.89:3456, 2005.

Systems Biology and MalariaE.A. Winzeler, C. Kidgell, V. Ramachandran, N. Kato,

K. Henson, J. Young, J. Johnson

Despite the widespread impact of malaria on theworld’s health and economies, relatively littleis known about the function of the majority of

the 5300 genes in the genome of Plasmodium falci-parum, the causative agent of the most severe form ofmalaria in humans. This lack of knowledge retards thedevelopment of drugs and vaccines against the parasite.We use systematic discovery-based approaches to pre-dict the function of uncharacterized Plasmodium genes;our goal is to facilitate the discovery of new treatments.We are using mRNA and protein expression to revealgenetic regulatory networks and to suggest protein-pro-tein interactions. We are also developing new methodsthat can be used in systems biology research.

In addition to our past work on blood-stage parasites,we have characterized the expression program of thesexual stages of malarial parasites. These stages, whichare essential for the mosquito transmission of the dis-ease, are the focus of the development of drugs andvaccines that block transmission. To better understandgenes important to sexual development, we used a full-genome high-density oligonucleotide microarray to profilethe transcriptomes of P falciparum gametocytes. Tointerpret this transcriptional data, we developed andused a novel knowledge-based data-mining algorithmtermed ontology-based pattern identification. With thisalgorithm, published or custom gene classifications

are used to optimize normalization methods and clusterboundaries so that the largest number of any given genetype is found in the smallest cluster size.

This analysis resulted in the identification of a sexualdevelopment cluster containing 246 genes, of whichapproximately 75% were unclassified and which con-tained most known sexual stage genes. These genes hadhighly correlated, gametocyte-specific expression pat-terns. Statistical analysis of the upstream promoterregions of these 246 genes revealed putative cis regula-tory elements. In addition, we extended the ontology-based pattern identification by using current annotationsprovided by the Gene Ontology Consortium to identify380 statistically significant clusters containing genes withexpression patterns characteristic of various biologicalprocesses, cellular components, and molecular functions.

We are also studying genetic diversity by usinghybridization-based approaches to further characterizeparasite genes. By performing a full genome scan ofallelic variability of 14 field and laboratory strains ofP falciparum, we showed that 10% of the genomehas higher than neutral rates of diversity at tens ofthousands of loci. We found that whereas many genesare exceptionally well conserved across parasite iso-lates, paralog genes (i.e., genes related by duplicationwithin a genome that have different functions), genesnear the ends of chromosomes, genes that encode pro-teins that are trafficked to the surface of the infectedred cell, and genes that encode known and potentialdrug targets are exceptionally diverse. These data sug-gest that rates of mitotic recombination are elevatedamong genes with paralogs and that selection pres-sure on those without paralogs is strong.

We also revealed gene amplification events, includ-ing one associated with pfmdr1, the gene for multidrugresistance in P falciparum, and a previously uncharac-terized amplification centered on the gene for GTPcyclohydrolase, the first enzyme in the folate biosynthe-sis pathway. Although GTP cyclohydrolase is not theknown target of any current drugs, downstream mem-bers of the pathway are targeted by several widely usedantimalarial agents. We propose that amplification ofthe GTP cyclohydrolase enzyme in the folate biosynthe-sis pathway may facilitate increased flux through thispathway and increase resistance to antifolate drugs.

These data and recent publications indicating that90% of a small eukaryote’s genetic variation can becaptured in a single microarray hybridization, suggestthat population genomics will be a fruitful approach


for discovering new determinants of drug resistance ina variety of infectious agents.

PUBLICATIONSCarret, C.K., Horrocks, P., Konfortov, B., Winzeler, E., Qureshi, M., Newbold, C.,Ivens, A. Microarray-based comparative genomic analyses of the human malariaparasite Plasmodium falciparum using Affymetrix arrays. Mol. Biochem. Parasitol.144:177, 2005.

Kidgell, C., Winzeler, E.A. Using the genome to dissect the molecular basis of drugresistance. Future Microbiol. 1:185, 2006.

Winzeler, E.A. Applied systems biology and malaria. Nat. Rev. Microbiol. 4:145,2006.

Young, J.A., Fivelman, Q.L., Blair, P.L., de la Vega, P., Le Roch, K.G., Zhou, Y.,Carucci, D.J., Baker, D.A., Winzeler, E.A. The Plasmodium falciparum sexualdevelopment transcriptome: a microarray analysis using ontology-based patternidentification. Mol. Biochem. Parasitol. 143:67, 2005.

Advancing Applications in MassSpectrometry–Based ProteomicsJ.R. Yates III, A.O. Bailey, G.T. Cantin, E. Chen, D. Cociorva,

J. Coppinger, C. Delahunty, M.Q. Dong, J. Hewel,

J.R. Johnson, L. Liao, B.W. Lu, I. McLeod, D. McClatchy,

R. Park, E. Romijn, H. Prieto, C.I. Ruse, J. Venable,

J. Wohlschlegel, C. Wong, T. Xu

Mass spectrometry has emerged as a powerfultechnique for cellular proteomics, comple-menting traditional gene-by-gene approaches

with a comprehensive description of the molecular fac-tors that contribute to a biologically relevant system.We remain at the forefront of this field, developing newstrategies to address more sophisticated scientific ques-tions through proteomics, such as how to measureglobal changes in protein abundance and how to char-acterize complex posttranslational modifications.Q U A N T I T A T I V E P R O T E O M I C A N A L Y S I S O F I N S U L I N

S I G N A L I N G

Quantitative mass spectrometry–based proteomicsrelies on internal isotopic standards. Metabolic label-ing with nitrogen 15 is a preferred method of intro-ducing internal standards. It produces a standard forevery peptide or protein to be characterized. In addi-tion, it is stable, nonradioactive, less error-prone thanchemical labeling, and relatively inexpensive. We haveused metabolic labeling with nitrogen 15 and quanti-tative mass spectrometry to study insulin signaling inthe worm Caenorhabditis elegans.

Insulin regulates a wide range of processes, includ-ing metabolism, development, and aging, but only ahandful of its downstream targets are known. We

hypothesized that a subset of insulin-signaling targets,specifically, daf-2 (the gene for an insulin receptor)and daf-16 (the gene for a transcription factor nega-tively regulated by daf-2) mutants, might be differen-tially expressed in wild-type C elegans and thus couldbe identified by using our quantitative proteomicsapproach. We identified 104 proteins that were pre-sent in higher or lower levels in daf-2 mutants than inwild-type and daf-16 mutant organisms, including theknown targets superoxide dismutase 3 and catalases.Gene ontology analysis revealed that the upregulatedproteins in daf-2 mutants were overrepresented in themetabolism of reactive oxygen species, metabolism ofcarbohydrates, and biosynthesis of amino acids; thedownregulated proteins were enriched in proteinsinvolved in translation and lipid transport.

We confirmed by genetics analysis that some of thepossible insulin-signaling components identified in thestudy play a role in regulating life span and/or formationof dauer larvae, both of which are regulated by C ele-gans insulins. Among the confirmed targets is a proteinphosphatase. Using a green fluorescent protein as alabel, we found that the phosphatase was upregulated inworms in which daf-2 was inactivated via RNA interfer-ence, consistent with the results of mass spectrometry.Further genetic analysis indicated that this phosphataseacted upstream of and/or in parallel to the protein DAF-16 in regulation of aging and dauer formation.

Taken together, our data suggest that this proteinphosphatase is both a downstream target and a regu-lator of insulin signaling. Therefore it may be part of afeedback loop. This study illustrates the effectivenessof combining quantitative mass spectrometry and Celegans genetics. Such an approach can be extendedto other studies beyond insulin signaling.P R O T E I N S U M O Y L A T I O N

The characterization of posttranslational modifica-tions is also an emerging application of mass spectrom-etry–based proteomics. We are developing proteomictools to study the family of small ubiquitin-like modi-fiers (SUMOs). Recently, we focused on Smt3p, thebudding yeast homolog of a human SUMO. Althoughgenetic studies have indicated that Smt3p is requiredfor proper regulation of a variety of different cellularprocesses, including transcription, intracellular trans-port, progression of the cell cycle, and the mainte-nance of genome integrity, the mechanisms by whichit does so remain largely unknown.

Using proteomic approaches, we addressed 2 majorareas in protein sumoylation: the large-scale identifi-


cation of sumoylation targets and the development ofstrategies for mapping SUMO modification sites. Bycombining different affinity chromatography strategieswith our multidimensional protein identification plat-form, we identified 271 new SUMO targets. Thesesubstrates play roles in a diverse set of biological pro-cesses and greatly expand the known scope of SUMOregulation in eukaryotic cells. This research alsorevealed coordinated SUMO modification of multipleproteins in well-defined macromolecular complexes.This intriguing result suggests that sumoylation maytarget protein complexes rather than individual pro-teins. We are also characterizing the mechanism thatunderlies this observation.

Characterization of many of the new substrates iden-tified in our study was limited by difficulties in identifyingSUMO attachment sites in the target of interest. To solvethis problem, we recently developed a method for therapid and efficient identification of SUMO attachmentsites in cellular proteins. In this method, differentSUMO mutants are used in combination with variousprotease digestion strategies, and then mass spec-trometry is used to directly and specifically map thelocations of the modified lysine residues. We showed theusefulness of this method by identifying SUMO modifi-cation sites in an assortment of model SUMO sub-strates and in complex mixtures. The development ofa method for identifying SUMO attachment sites willbe a powerful tool for the characterization of the newsubstrates identified in our initial global analysis.

PUBLICATIONSCantin, G.T., Venable, J.D., Cociorva, D., Yates, J.R. III. Quantitative phosphopro-teomic analysis of the tumor necrosis factor pathway. J. Proteome Res. 5:127, 2006.

Chen, E.I., Hewel, J., Felding-Habermann, B., Yates, J.R. III. Large scale proteinprofiling by combination of protein fractionation and multidimensional protein iden-tification technology (MudPIT). Mol. Cell. Proteomics 5:53, 2006.

Sadygov, R., Wohlschlegel, J., Park, S.K., Xu, T., Yates, J.R. III. Central limit theoremas an approximation for intensity-based scoring function. Anal. Chem. 78:89, 2006..

Venable, J.D., Xu, T., Cociorva, D., Yates, J.R. III. Cross-correlation algorithm forcalculation of peptide molecular weight from tandem mass spectra. Anal. Chem.78:1921, 2006.

Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Improved identifica-tion of SUMO attachment sites using C-terminal SUMO mutants and tailored pro-tease digestion strategies. J. Proteome Res. 5:761, 2006.

Macromolecular AssembliesVisualized by ElectronCryomicroscopy and ImageAnalysis: Membrane Proteinsand VirusesM. Yeager, R. Abagyan,**** B.D. Adair, K. Baker, K. Altieri,

A. Cheng, M.J. Daniels, K.A. Dryden, B. Ganser, J. Harless,

Y. Hua, M. Matho, F.A. Palida, M.A. Arnaout,*

A.R. Bellamy,** N. Ben-Tal,*** M.J. Buchmeier,****

F.V. Chisari,**** K. Coombs,***** H.B. Greenberg,†

J.E. Johnson,**** S. Matsui,† L.H. Philipson,††

T.D. Pollard,††† A. Rein,†††† A. Schneemann,****

J.A. Tainer,**** J.A. Taylor,** V.M. Unger†††

* Harvard Medical School, Boston, Massachusetts

** University of Auckland, Auckland, New Zealand

*** Tel-Aviv University, Tel-Aviv, Israel

**** Scripps Research

***** University of Manitoba, Winnipeg, Manitoba

† Stanford University, Stanford, California

†† University of Chicago, Chicago, Illinois

††† Yale University, New Haven, Connecticut

†††† National Cancer Institute, Frederick, Maryland

The ultimate goal of our studies is to gain a deeperunderstanding of the molecular basis of impor-tant human diseases, such as sudden death, heart

attacks, and HIV infection, that cause substantial mor-tality and suffering. The structural details revealed byour research may provide clues for the design of moreeffective and safer medicines.

At the basic science level, we are intrigued by ques-tions at the interface between cell biology and struc-tural biology: How do membrane proteins fold? Howdo membrane channels open and close? How are sig-nals transmitted across a cellular membrane when anextracellular ligand binds to a membrane receptor?How do viruses attach to and enter host cells, repli-cate, and assemble infectious particles? To exploresuch problems, we use high-resolution electron cry-omicroscopy and computer image processing. Withthis approach, we can examine the molecular archi-tecture of supramolecular assemblies such as mem-brane proteins and viruses.

In electron cryomicroscopy, biological specimensare quick frozen in a physiologic state to preserve


their native structure and functional properties. A spe-cial advantage of this method is that we can capturedynamic states of functioning macromolecular assem-blies, such as open and closed states of membranechannels and viruses actively transcribing RNA. Three-dimensional density maps are obtained by digital imageprocessing of the high-resolution electron micrographs.The rich detail in the density maps exemplifies the powerof this approach to reveal the structural organizationof complex biological systems that can be related tothe functional properties of such assemblies.

Ongoing research projects include the structureanalysis of (1) membrane proteins involved in cell-to-cell communication (gap junctions), water transport(aquaporins), ion transport (potassium channels),transmembrane signaling (integrins), and viral recogni-tion (rotavirus NSP4); (2) viruses responsible for sig-nificant human diseases (retroviruses, hepatitis B virus[HBV], rotavirus, astrovirus); and (3) viruses used asmodel systems to understand mechanisms of patho-genesis (arenaviruses, reoviruses, nodaviruses, tetra-viruses and sobemoviruses). The following sectionshighlight selected projects that exemplify the themesof our research program.G A P J U N C T I O N M E M B R A N E C H A N N E L S

Gap junction channels connect the cytoplasms ofadjacent cells by means of an intercellular conduitformed by the end-to-end docking of 2 hexamerichemichannels called connexons. Gap junctions play anessential functional role by mediating metabolic andelectrical communication within tissues. For instance,in the heart, gap junction channels organize the pat-tern of current flow to allow a coordinated contractionof the muscle.

We expressed a recombinant cardiac gap junctionprotein, termed connexin 43, and produced 2-dimen-sional crystals suitable for electron cryocrystallography.Our previous findings indicated that each hexamericconnexon is formed by 24 closely packed α-helices.We have now extended this analysis to 5.7-Å in-planeand 19.8-Å vertical resolution, a step that enables us toidentify the positions and tilt angles for the 24 α-heliceswithin each hemichannel (Fig. 1). The 4 hydrophobicsegments in connexin sequences were assigned to theα-helices in the map on the basis of biochemical andphylogenetic data. Evolutionary conservation and ananalysis of compensatory mutations in connexin evolu-tion were used to identify the packing interfaces betweenthe helices. The final model, which specifies the coordi-

nates of Cα atoms in the transmembrane domain, pro-vides a structural basis for understanding the differentphysiologic effects of almost 30 mutations and poly-morphisms in terms of structural deformations at theinterfaces between helices, revealing an intimate con-nection between molecular structure and disease.I N T E G R I N S

Integrins are a large family of heterodimeric trans-membrane receptor proteins that modulate importantbiological processes such as development, cell adhe-sion, angiogenesis, wound healing, and neoplastictransformation. The ectodomain of the integrin αvβ3crystallizes in a bent, genuflexed conformation, whichis considered to be inactive (i.e., unable to bind physi-ologic ligands in solution) unless it is fully extendedby activating stimuli. To assess whether the bent inte-grin can bind physiologic ligands, we collaboratedwith M.A. Arnaout, Harvard Medical School, Boston,Massachusetts, to generate a stable, soluble complexof the manganese-bound αvβ3 ectodomain with a frag-


F i g . 1 . Intercellular gap junction channels have a diameter of

about 65 Å and are formed by the end-to-end docking of 2 hemi-

channels, each composed of a hexamer of connexin subunits. A Cαmodel (ribbons) for the membrane-spanning α-helices of the hemi-

channels was derived by combining the information from a compu-

tational analysis of connexin sequences, the results of more than a

decade of biochemical studies, and the constraints provided by a

3-dimensional map derived by using electron cryocrystallography.

Although individually, none of these approaches provided high-reso-

lution information, their sum yielded an atomic model that predicts

how connexin mutations (spheres), which result in diseases such

as nonsyndromic deafness and Charcot-Marie-Tooth disease, may

interfere with formation of functional channels by disrupting helix-

helix packing.

ment of fibronectin containing type III domains 7–10and the EDB domain. Electron microscopy and single-particle image analysis were used to determine the 3-dimensional structure of this complex (Fig. 2).

Most αvβ3 particles, whether unliganded or boundto fibronectin, had compact, triangular shapes. A differ-ence map comparing ligand-free and fibronectin-boundintegrin revealed density that could accommodate thefibronectin type III domain 10 containing arginine–glycine–aspartic acid in proximity to the ligand-bindingsite of β3, with domain 9 just adjacent to the synergysite binding region of αv.

This study suggests that the ectodomain of αvβ3 hasa bent conformation that can stably bind a physiologicligand in solution. These results are relevant for under-standing how binding of ligands to the extracellulardomain leads to conformational changes that transmitsignals across the plasma membranes of cells, culminat-ing in changes in gene transcription in the nucleus.H E P A T I T I S B V I R U S

HBV currently infects more than 350 million peo-ple, of which 1 million will die every year. The infec-tious virion is an enveloped capsid containing the viralpolymerase and the double-stranded DNA genome. Thestructure of the capsid assembled in vitro from expressed

core protein has been studied intensively. However, lit-tle is known about the structure and assembly of nativecapsids present in infected cells, and even less is knownabout the structure of mature virions. We used electroncryomicroscopy and image analysis to examine HBVvirions (also called Dane particles) isolated from theserum of a patient with hepatitis B and capsids posi-tive and negative for HBV DNA isolated from the liversof transgenic mice (Fig. 3).

Both types of capsids assembled as icosahedralparticles indistinguishable from previous image recon-structions of capsids. Likewise, the virions containedcapsids with either T = 3 or T = 4 icosahedral sym-metry. Projections extending from the lipid envelope


F i g . 2 . The 3-dimensional density map (grayscale transparency)

of the integrin αvβ3 in a complex with fibronectin was determined

by using electron microscopy and image analysis. The x-ray structures

of the αv and β3 proteins have been docked into the electron micros-

copy density envelope. Additional density (lower right) can accom-

modate fibronectin domain 10 adjacent to the ligand-binding site

as well as domain 9 at the synergy site. The complex is shown adja-

cent to the white box, which represents the 30-Å-thick hydrophobic

part of the cellular membrane across which signals are transmitted.

F i g . 3 . A model of the HBV virion (diameter ~450 Å) based on

electron cryomicroscopy and image analysis. A, The double-stranded

DNA genome is encapsidated by an icosahedral capsid shell com-

posed of 120 spikes. The surface is studded with glycoproteins

spaced about 60 Å apart that bind to membrane receptors on liver

cells. B, In the close-up view, the x-ray crystal structure of a

recombinant capsid has been docked into the electron cryomi-

croscopy density map of the virion capsid. The core spikes are in

close apposition but do not penetrate the envelope.

were attributed to surface glycoproteins. The packingof the projections was unexpectedly nonicosahedral butconformed to an ordered lattice. These structural fea-tures distinguish HBV from other enveloped viruses.

PUBLICATIONSDaniels, M.J., Wood, M.R., Yeager, M. In vivo functional assay of a recombinantaquaporin in Pichia pastoris. Appl. Environ. Microbiol. 72:1507, 2006.

Daniels, M.J., Yeager, M. Phosphorylation of aquaporin PvTIP3;1 defined by massspectrometry and molecular modeling. Biochemistry 44:14443, 2005.

Dryden, K.A., Wieland, S.F., Whitten-Bauer, C., Chisari, F.V., Yeager, M. Nativehepatitis B virions and capsids visualized by electron cryomicroscopy. Mol. Cell23:843, 2006.

Greig, S.L., Berriman, J.A., O’Brien, J.A., Taylor, J.A., Bellamy, A.R., Yeager, M.,Mitra, A.K. Structural determinants of rotavirus subgroup specificity mapped bycryo-electron microscopy. J. Mol. Biol. 356:209, 2006.

Wiedenheft, B., Mosolf, J., Willits, D., Yeager, M., Dryden, K.A., Young, M., Douglas,T. An archaeal antioxidant: characterization of a Dps-like protein from Sulfolobussolfataricus. Proc. Natl. Acad. Sci. U. S. A. 102:10551, 2005.


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