Self-Regulation of Cytoplasmic Dynein through its Unconventional Force Response
1
migration in eukaryotes. The building block for MT polymerization is the aß- tubulin heterodimer, which is present as a tightly regulated soluble pool in the cytoplasm. Despite the importance of aß-tubulin heterodimer in the regu- lation of MT dynamics, it remains unclear how nascent and folded a and ß- tubulin are assembled and activated into a single heterodimer configuration that universally dictates dynamic MT polymerization. It remains unknown how five conserved tubulin cofactors (TBC-A,B,C,D and E) and a dedicated Arl2 G-protein promote aß-tubulin biogenesis, activation and degradation, and how such activities impact MT function. In contrast to a long-standing hypothesis in which individual tubulin cofactors bind sequentially to a and ß-tubulin monomers and assemble aß-tubulin dimers through dynamic inter- actions, we show based on biochemical and structural studies that multiple tubulin cofactors and Arl2 form multi-subunit platforms for aß-tubulin dimer assembly, activation and degradation. We show that multi-subunit tubulin cofactor and Arl2 platform are soluble aß-tubulin regulators that are powered by GTP hydrolysis cycles. We have determined tubulin cofactor platform structure, conformational changes upon tubulin dimer binding, and the mech- anism of GTP hydrolysis activation. Surprisingly, we further show, using reconstitution of these complexes with soluble tubulin dimer and dynamic MTs, that they enhance aß-tubulin polymerizing state at MT plus ends in a manner dependent on Arl2 and tubulin GTP hydrolysis. Our data surprisingly suggest tubulin cofactors are potent regulators of soluble tubulin dimer state, and promote soluble tubulin activation required for all MT dynamics. Our model explains long-standing cell biology and genetics data about the roles of tubulin cofactors in regulating the soluble aß-tubulin pool and MT homeostasis. 2238-Plat Structural Basis for Nucleotide Exchange and Power Stroke Generation by the Kinesin Molecular Motor Zhiguo Shang 1 , Roseanne Csencsits 2 , Chen Xu 3 , Jared C. Cochran 4 , Charles Vaughn Sindelar 1 . 1 Yale University, New Haven, CT, USA, 2 Lawrence Berkeley National Laboratory, Berkeley, CA, USA, 3 Brandeis University, Waltham, MA, USA, 4 Indiana University, Bloomington, IN, USA. Kinesin molecular motors use energy derived from ATP to step along microtu- bules, driving many essential processes in eukaryotic cells, including mitosis, vesicle transport and cytoskeletal remodeling. Several conformational states of kinesin have been identified by X-ray crystallography, but the structural tran- sitions used by kinesin to generate force and movement along intact microtu- bules have remained unclear. We used recent improvements in cryo-EM methodology and instrumentation to capture the conformation of microtubule-attached kinesin at the beginning (no-nucleotide) and end (ATP analog-bound) of the force generation process at 5-6A ˚ resolution. We derived all-atom models for these two maps from a crystal structure of the tubulin- kinesin complex, using explicitly solvated molecular dynamics simulations combined with restraints derived from the maps. This analysis revealed that, contrary to existing models, kinesin’s central beta sheet serves as the primary transducer in the motor’s force-generation mechanism, twisting to drive ADP release and subsequently untwisting upon ATP binding to trigger a power stroke. We identified conserved residues on the motor domain, supported by additional structural and biochemical analysis of site-directed mutations, which serve as allosteric latches during the motor’s microtubule-attached phase. These latches regulate the sheet-twisting motion and couple key properties of motor function to each other, including nucleotide binding, hydrolysis, and generation of a power stroke. These findings reveal how interactions with the microtubule can fundamentally alter kinesin’s energetic landscape in order to initiate productive motility. 2239-Plat Bimodality in a System of Active and Passive Kinesin-1 Motors Lara Scharrel 1 , Rui Ma 2,3 , Frank Ju ¨licher 4 , Stefan Diez 1,5 . 1 B CUBE, TU Dresden, Dresden, Germany, 2 Max Planck Institute for the Physics of Complex Systems, Dresden, Germany, 3 Institute for Advanced Study, Tsinghua University, Bejing, China, 4 Max Planck Institute for the Physics of Complex System, Dresden, Germany, 5 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Long-range directional transport in cells is facilitated by microtubule-based mo- tor proteins. One example is transport in a nerve cell, where small groups of mo- tor proteins, such as kinesin and dynein, work together to perform the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer and other neurodegenerative diseases. In particular two diseases, Hereditary Spastic Paraplegia (HSP) and Charcot-Marie-Tooth type 2A neuropathy (CMT2A) are connected to mutations of kinesin family members in the motor domain that affect their ATPase activity. However, it is not known how in detail multi-motor based cargo transport is impacted if the mo- tor function of a fraction of motors is inhibited. In order to mimic hindered multi- motor transport in-vitro, we performed gliding motility assays with varying frac- tions of active kinesin-1 and passivated kinesin-1 (rigor mutants). We found that hindered gliding manifests in three motility regimes: gliding at the velocity of single motors, simultaneous gliding and stopping (bistable movement), and stop- ping. Notably, an abrupt transition from gliding to stopping occurred at a certain threshold fraction. Furthermore, we developed a theoretical description based on single motor parameters. Our model explains the bimodal microtubule move- ment as well as the sharp transition from gliding to stopping. Our results demon- strate that hindered transport is acting in a bimodal "either-or-fashion": depending on the fraction of passive motors, transport by a multi-motor system is either performed close to full speed or not at all. 2240-Plat A Comparative Study of the Major Biochemical States of Kinesin-MT Complex using Computational Techniques and All-Atom Structural Models Srirupa Chakraborty, Wenjun Zheng. Biophysics, University at Buffalo, Buffalo, NY, USA. Here we apply computational molecular dynamics simulation techniques to do a comprehensive comparative study of the three major biochemical states of a kinesin - microtubule (MT) complex. These states are namely, the nucleotide- free APO state, the ADP-bound state and the ATP-bound state. We built atom- istic structure models for these key MT-binding states with different nucleotide content using available crystal structures, homology modeling and flexible fitting of high-quality cryo-electron-microscopy (EM) maps. We next explore how MT binding modulates active site dynamics of kinesin, predict some of the structural changes and pin-point some of the key residues that control the transition states. We further study the binding free-energy between kinesin head and MT in the three states and also identify a list of interactions (hydrogen bonds and salt bridges) between kinesin and MT and also between kinesin and ligand (ADP). We further perform steered molecular dynamics to mimic the in- tramolecular strain and identify some residues in force regulation of binding af- finity. This study helps us identify promising targets for future mutational and functional studies of the kinesin-MT complex. 2241-Plat Self-Regulation of Cytoplasmic Dynein through its Unconventional Force Response Takayuki Torisawa 1 , Ken’ya Furuta 2 , Akane Furuta 2 , Muneyoshi Ichikawa 1 , Kei Saito 1 , Kazuhiro Oiwa 2,3 , Hiroaki Kojima 2 , Yoko Yano Toyoshima 1 . 1 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan, 2 Advanced ICT Research Institute, National Institute of Information and Communications Technology, Hyogo, Japan, 3 Graduate School of Life Science, University of Hyogo, Hyogo, Japan. Cytoplasmic dynein is essential for a wide range of cellular activities, including intracellular transport, cell division, and cell migration in most of eukaryotic organisms. To achieve these various functions, dynein activity must be tightly controlled. However, the motility of mammalian cytoplasmic dynein has been controversial in previous studies, which makes it unclear how dynein activity is regulated and tuned for a specific function. Here, using electron microscopy and DNA nanostructure-based motility assays, we investigated how the control of dynein activity is achieved. We showed that single dynein molecules diffused along microtubules in an autoinhibited state, in which two motor heads were stacked together. This state was released when multiple dynein molecules worked together on a single cargo or when dynein was pulled by an optical tweezer, suggesting that individual dynein molecules in the team were acti- vated through destabilization of the stacked conformation by mechanical strain generated between dynein molecules. We confirmed this force-dependent acti- vation mechanism by observing the movement of a chimeric dynein fused with a inactive kinesin, which we assume acts as a load. This mechanism would function at a fundamental level of dynein regulation that does not require an external regulator, thereby serving as a stable basis for the higher-level regula- tion of dynein-driven transport. 2242-Plat Dynein’s C-Terminal Domain Plays a Novel Role in Regulating Force Gen- eration Peter Ho ¨o ¨k 1 , Matthew P. Nicholas 2 , Sibylle Brenner 2 , Caitlin Lazar 1 , Sarah J. Weil 1 , Richard B. Vallee 1 , Arne Gennerich 2 . 1 Columbia University College of Physicians and Surgeons, New York, NY, USA, 2 Albert Einstein College of Medicine, Bronx, NY, USA. Cytoplasmic dynein is a homodimeric, minus-end-directed microtubule motor protein involved in a wide range of both low and high force requiring functions Tuesday, February 18, 2014 443a
Transcript of Self-Regulation of Cytoplasmic Dynein through its Unconventional Force Response
Tuesday, February 18, 2014 443a
migration in eukaryotes. The building block for MT polymerization is the aß-tubulin heterodimer, which is present as a tightly regulated soluble pool inthe cytoplasm. Despite the importance of aß-tubulin heterodimer in the regu-lation of MT dynamics, it remains unclear how nascent and folded a and ß-tubulin are assembled and activated into a single heterodimer configurationthat universally dictates dynamic MT polymerization. It remains unknownhow five conserved tubulin cofactors (TBC-A,B,C,D and E) and a dedicatedArl2 G-protein promote aß-tubulin biogenesis, activation and degradation,and how such activities impact MT function. In contrast to a long-standinghypothesis in which individual tubulin cofactors bind sequentially to a andß-tubulin monomers and assemble aß-tubulin dimers through dynamic inter-actions, we show based on biochemical and structural studies that multipletubulin cofactors and Arl2 form multi-subunit platforms for aß-tubulin dimerassembly, activation and degradation. We show that multi-subunit tubulincofactor and Arl2 platform are soluble aß-tubulin regulators that are poweredby GTP hydrolysis cycles. We have determined tubulin cofactor platformstructure, conformational changes upon tubulin dimer binding, and the mech-anism of GTP hydrolysis activation. Surprisingly, we further show, usingreconstitution of these complexes with soluble tubulin dimer and dynamicMTs, that they enhance aß-tubulin polymerizing state at MT plus ends in amanner dependent on Arl2 and tubulin GTP hydrolysis. Our data surprisinglysuggest tubulin cofactors are potent regulators of soluble tubulin dimer state,and promote soluble tubulin activation required for all MT dynamics. Ourmodel explains long-standing cell biology and genetics data about the rolesof tubulin cofactors in regulating the soluble aß-tubulin pool and MThomeostasis.
2238-PlatStructural Basis for Nucleotide Exchange and Power Stroke Generation bythe Kinesin Molecular MotorZhiguo Shang1, Roseanne Csencsits2, Chen Xu3, Jared C. Cochran4,Charles Vaughn Sindelar1.1Yale University, New Haven, CT, USA, 2Lawrence Berkeley NationalLaboratory, Berkeley, CA, USA, 3Brandeis University, Waltham, MA, USA,4Indiana University, Bloomington, IN, USA.Kinesin molecular motors use energy derived from ATP to step along microtu-bules, driving many essential processes in eukaryotic cells, including mitosis,vesicle transport and cytoskeletal remodeling. Several conformational statesof kinesin have been identified by X-ray crystallography, but the structural tran-sitions used by kinesin to generate force and movement along intact microtu-bules have remained unclear. We used recent improvements in cryo-EMmethodology and instrumentation to capture the conformation ofmicrotubule-attached kinesin at the beginning (no-nucleotide) and end (ATPanalog-bound) of the force generation process at 5-6A resolution. We derivedall-atom models for these two maps from a crystal structure of the tubulin-kinesin complex, using explicitly solvated molecular dynamics simulationscombined with restraints derived from the maps. This analysis revealed that,contrary to existing models, kinesin’s central beta sheet serves as the primarytransducer in the motor’s force-generation mechanism, twisting to drive ADPrelease and subsequently untwisting upon ATP binding to trigger a powerstroke. We identified conserved residues on the motor domain, supported byadditional structural and biochemical analysis of site-directed mutations, whichserve as allosteric latches during the motor’s microtubule-attached phase.These latches regulate the sheet-twisting motion and couple key properties ofmotor function to each other, including nucleotide binding, hydrolysis, andgeneration of a power stroke. These findings reveal how interactions with themicrotubule can fundamentally alter kinesin’s energetic landscape in order toinitiate productive motility.
2239-PlatBimodality in a System of Active and Passive Kinesin-1 MotorsLara Scharrel1, Rui Ma2,3, Frank Julicher4, Stefan Diez1,5.1B CUBE, TU Dresden, Dresden, Germany, 2Max Planck Institute for thePhysics of Complex Systems, Dresden, Germany, 3Institute for AdvancedStudy, Tsinghua University, Bejing, China, 4Max Planck Institute for thePhysics of Complex System, Dresden, Germany, 5Max Planck Institute ofMolecular Cell Biology and Genetics, Dresden, Germany.Long-range directional transport in cells is facilitated bymicrotubule-basedmo-tor proteins. One example is transport in a nerve cell, where small groups of mo-tor proteins, such as kinesin and dynein, work together to perform the supply andclearance of cellular material along the axon. Defects in axonal transport havebeen linked to Alzheimer and other neurodegenerative diseases. In particulartwo diseases, Hereditary Spastic Paraplegia (HSP) and Charcot-Marie-Toothtype 2A neuropathy (CMT2A) are connected to mutations of kinesin familymembers in the motor domain that affect their ATPase activity. However, it is
not knownhow in detailmulti-motor based cargo transport is impacted if themo-tor function of a fraction ofmotors is inhibited. In order tomimic hinderedmulti-motor transport in-vitro, we performed glidingmotility assayswith varying frac-tions of active kinesin-1 and passivated kinesin-1 (rigor mutants).We found thathindered gliding manifests in three motility regimes: gliding at the velocity ofsinglemotors, simultaneous gliding and stopping (bistablemovement), and stop-ping. Notably, an abrupt transition from gliding to stopping occurred at a certainthreshold fraction. Furthermore, we developed a theoretical description based onsingle motor parameters. Our model explains the bimodal microtubule move-ment as well as the sharp transition from gliding to stopping. Our results demon-strate that hindered transport is acting in a bimodal "either-or-fashion":depending on the fraction of passive motors, transport by a multi-motor systemis either performed close to full speed or not at all.
2240-PlatA Comparative Study of the Major Biochemical States of Kinesin-MTComplex using Computational Techniques and All-Atom StructuralModelsSrirupa Chakraborty, Wenjun Zheng.Biophysics, University at Buffalo, Buffalo, NY, USA.Here we apply computational molecular dynamics simulation techniques to doa comprehensive comparative study of the three major biochemical states of akinesin - microtubule (MT) complex. These states are namely, the nucleotide-free APO state, the ADP-bound state and the ATP-bound state. We built atom-istic structure models for these key MT-binding states with different nucleotidecontent using available crystal structures, homology modeling and flexiblefitting of high-quality cryo-electron-microscopy (EM) maps. We next explorehow MT binding modulates active site dynamics of kinesin, predict some ofthe structural changes and pin-point some of the key residues that control thetransition states. We further study the binding free-energy between kinesinhead and MT in the three states and also identify a list of interactions (hydrogenbonds and salt bridges) between kinesin and MT and also between kinesin andligand (ADP). We further perform steered molecular dynamics to mimic the in-tramolecular strain and identify some residues in force regulation of binding af-finity. This study helps us identify promising targets for future mutational andfunctional studies of the kinesin-MT complex.
2241-PlatSelf-Regulation of Cytoplasmic Dynein through its Unconventional ForceResponseTakayuki Torisawa1, Ken’ya Furuta2, Akane Furuta2,Muneyoshi Ichikawa1, Kei Saito1, Kazuhiro Oiwa2,3, Hiroaki Kojima2,Yoko Yano Toyoshima1.1Department of Life Sciences, Graduate School of Arts and Sciences, TheUniversity of Tokyo, Tokyo, Japan, 2Advanced ICT Research Institute,National Institute of Information and Communications Technology, Hyogo,Japan, 3Graduate School of Life Science, University of Hyogo, Hyogo, Japan.Cytoplasmic dynein is essential for a wide range of cellular activities, includingintracellular transport, cell division, and cell migration in most of eukaryoticorganisms. To achieve these various functions, dynein activity must be tightlycontrolled. However, the motility of mammalian cytoplasmic dynein has beencontroversial in previous studies, which makes it unclear how dynein activity isregulated and tuned for a specific function. Here, using electron microscopyand DNA nanostructure-based motility assays, we investigated how the controlof dynein activity is achieved. We showed that single dynein moleculesdiffused along microtubules in an autoinhibited state, in which two motor headswere stacked together. This state was released when multiple dynein moleculesworked together on a single cargo or when dynein was pulled by an opticaltweezer, suggesting that individual dynein molecules in the team were acti-vated through destabilization of the stacked conformation by mechanical straingenerated between dynein molecules. We confirmed this force-dependent acti-vation mechanism by observing the movement of a chimeric dynein fused witha inactive kinesin, which we assume acts as a load. This mechanism wouldfunction at a fundamental level of dynein regulation that does not require anexternal regulator, thereby serving as a stable basis for the higher-level regula-tion of dynein-driven transport.
2242-PlatDynein’s C-Terminal Domain Plays a Novel Role in Regulating Force Gen-erationPeter Hook1, Matthew P. Nicholas2, Sibylle Brenner2, Caitlin Lazar1,Sarah J. Weil1, Richard B. Vallee1, Arne Gennerich2.1Columbia University College of Physicians and Surgeons, New York, NY,USA, 2Albert Einstein College of Medicine, Bronx, NY, USA.Cytoplasmic dynein is a homodimeric, minus-end-directed microtubule motorprotein involved in a wide range of both low and high force requiring functions
![Crystal clear insights into how the dynein motor moves · 2013. 4. 10. · 2010)]. In dynein, four of the AAA+ domains bind nucleotides. The size of the dynein motor domain, the presence](https://static.fdocuments.net/doc/165x107/60ed0c0f1235ef420447d9e4/crystal-clear-insights-into-how-the-dynein-motor-moves-2013-4-10-2010-in.jpg)
