2006 International Conference on

Nanotechnology, April 26-28, 2006

Atlanta, GA

Self assembly and organization of nanofibers using biological

molecular motorsPresented by:Jeffrey M. CatchmarkAssistant Professor, College of Engineering and College of Agricultural SciencesOperations Manager, Nanofabrication facility, National Science Foundation National Nanotechnology Infrastructure Network SiteThe Pennsylvania State University

Outline• Motivation for studying nanoscale fiber assembly• Review biological molecular motors and

microtubules• In-vitro organization of microtubules as 2D and 3D

templates for organizing nanofibers coupled to biological motors

• Results on linking biomotors to cellulose• Summary and next steps

Motivation• Why study nano scale fiber assembly?

– Understand the impact nano scale organization has on macro-scale material properties.

– Explore fundamentally new approaches for creating self-assembled fiber materials with engineered properties to address industry needs:

• More efficient fiber utilization.• Improved fiber based composite materials for

biomedical, electronic and optical.

What is biomimetics?

• Implementing nature’s ‘engineering tricks’ to create better materials, devices and systems.

Fiber assembly at it best: the plant cell wall

• The plant cell wall is an excellent example of nanoscale hierarchical assembly which creates an ideal bulk material.

Cellulose

Microtubules and biomotors: the ‘nanoarchitects’ of the plant cell wall

• Microtubule formation is believed to control the orientation of cellulose fibrils in the plant cell wall. Cellulose producingenzyme rosettes glide between membrane bound microtubules creating aligned fibrils*.

Image by Prof. Malcom Brown,

http://www.botany.utexas.edu/facstaff/facpages/mbrown/newstat/stat38.htm

*Pankaj Dhonukshe, et. al, The Plant Cell, Vol. 15, 2666–2679, November 2003, and references therein.

• Biological molecular motors are also thought to be involved in the movement of microtubules or enzyme rosettes*.

*Clive Lloyd and Jordi Chan, Nature Reviews, Molecular Cell Biology, Vol. 5, pp. 13-22, 2004.

Microtubules and biomotors: the ‘nanoarchitects’ of the plant cell wall

Microtubules and Molecular Motors• Microtubules polymerize inside

cells via the ordered assembly of α and β-tubulin proteins, which are linked with GTP (guanosine tri-phosphate). GTP is hydrolyzed to GDP resulting in depolymerization

• There are many families of biomotor proteins which operate on microtubules including the kinesin and dynein motor families.

• Biomotors and microtubules work cooperatively to organize cellular materials (e.g., responsible for the mechanics of cell division), change the shape of cells, etc.

Kozielski et al., Nogales et al., Cooper, 2nd ed.

7 nmPlus end

25 nm

CentrosomeNewt lung cell

Biomotors: what’s under the hood?

129 Lbs

80,000 Lbs

Kinesin motors can carry 10 million times their own weight

Objectives

• Explore the applicability of microtubules and molecular motors used in vitro to assemble organized nanofiber composites.

• Use the nanoscale organizational control to study the impact of nanoscale assembly on macro scale material properties.

Cooper, 2nd ed.

Glass Substrate

Casein protein

Microtubule Kinesin CargoDirection of motion

+ End- End

Gliding assay (biomotor is bound to surface):

Biomotors and microtubules are studied in vitro using motility assays

+ End- End

Bead assay (Biomotor in solution):

Motility Assays

Microtubules labeled with rhodamine

Gliding Motility Assay Bead Assay

Directional motility on kinesin motors patterned on surfaces using electron beam lithography

Fluorescence indicates region of patterned kinesin motors

Linear array of 3 micron diameter patterned motor regions. Have scaled this to 250nm.

Rhodamine labeled microtubules running along patterned motor regions

Microtubules positioned on surfaces can provide a static or dynamic 2-D template for the organization of nanofibers

Self-organizing asters: 3D templates for nanofiber assembly

F. J. Ne´de´lec, T. Surrey, A. C. Maggs & S. Leibler, NATURE, VOL 389, 18, pp. 305-308, 1997

Aster formation at different motor assembly concentrations: a) 25μg/ml, b) 37.5μg/ml, c) 50 μg/mland d) 15 μg/ml.

Multi-head motor assembly containing 4 kinesin linking 2 microtubules.

Microtubule aster fireworks

Rhodamine labeled microtubule asters assembled with 4-kinesin motor assembly (40μg/ml) interacting with kinesin coated surface.

Both 2D and 3D structures can be locked in place by introducing adenylimidodiphosphate (AMP-PMP), a nonhydrolyzable ATP analog which stops motor activity but keeps the motor bound to the microtubule.

Using 2D and 3D microtubule templates: linking biomotors to nanofibers

• We have explored biotinylation schemes as a means of linking biomotors to cellulose nanofibers and carbon nanotubes.

• Biotinylated fibers can be linked to biotinylated kinesin biomotors via a neutravidin bond.

• Once a fiber assembly is formed, the microtubules can be depolymerized to remove the template.

Nanofiber

biotinneutravidin

biotinylated biomotor

Using 2D and 3D microtubule templates: linking biomotors to nanofibers

• For cellulose, we have explored 2 molecules:– NHS – Biotin (N-Hydroxysuccinimido Biotin) (previously

demonstrated by Janolino, IFT, 99)

– NHS-dPEG™12 Biotin (Quanta Biodesign)

• Biotinylation procedure: – Dissolve NHS-biotin or NHS-dPEG-biotin in dimethyl sulfoxide (DMSO)

20mg/ml. Dissolve cellulose in 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, at a concentration of 5mg/ml. Mix biotin solution (20µl) in cellulose mixture (0.8ml). Allow to react for 12h.

• Added biotinylated cellulose with multi-head motor assembly containing 4 kinesin and microtubules.

• Observed motility of microtubules on cellulose particles clearly indicating biotinylation was successful.

Microtubules running on cellulose particles

Microtubules running on cellulose particles

Rhodamine labeled microtubules at a concentration of 100μg/ml

Summary and future work

• Have established 2D and 3D static and dynamic microtubule networks.

• Have successfully linked kinesin biomotors to cellulose and observed microtubule motility on cellulose particles.

• Working on assembly of cellulose nanofibers and carbon nanotubes.

• Intend to apply system to cellulose nanofibers being produced by bacteria.

Acknowledgements• Collaborators:

– Prof. Nicole Brown, School of Forest Resources

– Prof. William Hancock, Bioengineering

• Student:– Vivek Verma, Ph.D. Student,

Engineering Science and Mechanics

Acknowledgements• Facilities

– Penn State Nanofabrication Facility, a National Science Foundation National Nanotechnology Infrastructure Network (NSF NNIN) Site.

• Support– National Science Foundation

Materials Research Science and Engineering Center (NSF MRSEC) Center for Nanoscale Science.

Thank You Presented by:Jeffrey M. CatchmarkAssistant Professor, College of Engineering and College of Agricultural SciencesOperations Manager, Nanofabrication facility, National Science Foundation National Nanotechnology Infrastructure Network SiteThe Pennsylvania State Universityjcatchmark@engr.psu.edu