Microtubule Assembly Microtubule Assembly Dynamics at the Dynamics at the

NanoscaleNanoscale

Henry T. Schek, IIIHenry T. Schek, IIIEuropean Molecular Biology LaboratoryEuropean Molecular Biology Laboratory

Melissa K. GardnerMelissa K. Gardner David OddeDavid Odde

University of Minnesota University of Minnesota

Jun ChengJun ChengAlan J. HuntAlan J. Hunt

University of MichiganUniversity of Michigan

METHODS, MEASUREMENTS, AND IMPLICATIONS FOR UNDERSTANDING MICROTUBULE DYNAMIC INSTABILITY

BME Department

Microtubules

Dynamic Instability

From Fygenson et al, Phys Rev E, 1994

BME Department

Microtubule Polymerization in Cells

• Works with actin to guide an axonal growth cone

• Kinetochore attachments

• Push on mitotic chromosomes arms

Why Study Polymerization Under Load?

• Quantify forces

• Study microtubule dynamics with nanometer resolution

• Improve models of dynamic instability

BME Department

Optical Tweezers

Cellular & Molecular Biomechanics Lab

Optical Tweezers Features• Extremely stable (<1 nm drift/min)• Multiple Independently Maneuverable Traps• Sub-Nanometer Detection and Manipulation• Forces from Less than one pN to greater than 100 pN• Servo-control for Force Clamp or Position Clamp

Experimental Strategy

Tightly Focused Trapping Laser

Trapped Particle

Attached Microtubule Seed

Barrier on cover glass

Barrier Design

• Vertex• Undercut• Laser footprint • LOR, SU-8

• No interference• No image

degradation• Constrain MT• Short MT

Barrier Fabrication

Primary structure and undercut fabrication are independent

Barrier Results

Scale bar=14 m Scale bar=2 m

What an Experiment Looks Like

Stationary Trap Results

• Microtubule growth is highly variable, and exhibits pauses over a broad range of forces, even in a single microtubule• F-V relation is complex

Stationary trap underestimates filament displacement

Force Clamping

Force Clamp-MT length change

1.6 1.4 1.3 1.1 0.9 0.7Clamped Force (pN)

Large Growth Rate VariabilityM

icro

tubu

le L

engt

hC

hang

e (n

m)

Close to zero = 3% (speed < 1 nm/s)

Growth= 55%

Shortening=41%

Growth rates{

Variability on Longer ScalesM

icro

tubu

le L

engt

hC

hang

e (n

m)

Nano-Shortening Events

Mic

rotu

bule

Len

gth

Cha

nge

(nm

)

9/minute, do not lead to rapid shortening

Summary so far

• Forces greater than 1 pN - plenty to influence chromosome movements*

• Large growth rate variability

• Frequent shortening events > 20 nm

• Persistent velocities at longer times scales

*Joglekar & Hunt. Biophys. J. (2002) Marshall et al. Curr. Biol. (2001)

GTP-Cap Hypothesis

• Grows with GTP-cap

• Occasionally loses cap

• Rapid Shortening results

1) Bayley et al., FEBS Lett., 1989; Bayley et al., J. Cell Sci., 1990; O'Brien et al., Biochemistry, 1987; Panda et al., Biochemistry, 2002; Stewart et al., Biochemistry, 1990

2) Vandecandelaere et al., Biochemistry, 1999; Voter et al., Cell Motil. Cyto., 1991

Trouble for the GTP cap?

It has been widely argued that the GTP-cap is at most one layer thick1, or slightly larger2 (e.g. lateral cap

hypothesis)

A microtubule that shortens sufficiently to lose more than one layer of tubulin subunits (~ 8 nm) will transition to rapid shortening.

This is contradicted by our results.

If the GTP-cap is one layer thick,

then:

D

Can these results be explained by a mechanochemical model?

VanBuren, Cassimeris & Odde, Biophys. J. 2005

This…

Or this?

No

Yes

Ave. = –9.7 nm

Ave. = –6.4 nm < -9.7 nm (p < 10-6)

*Nature, 2006

In contrast to the conclusions of Kerssemakers et al*, no evidence of oligomer addition

Resolution is sufficient to detect addition of individual subunits.

Step-like events when data is processed in a manner similar to

Kerssemakers et al

What’s a step?

+ XMAP215

Time

Dis

pla

cem

ent

Growth rate depends more on the evolving tip structure than force.

*

*S.E. < 0.1 nm/sec

Conclusions• At the nanoscale, microtubule growth is

highly variable• Frequent shortening events: as large as

80 nm, > 30 nm (2 layers) @ 8/min, > 40 nm @ 1/min ]

• Oligomer addition occurs rarely, if ever• Average growth rate is weakly dependent

on force, strongly dependent on tip structure

• Shortening excursions are smaller at higher forces

Conclusions II

• Finding consistent with a physically simple mechanochemical model, which explains:– Unexpected growth-phase shortening– Smaller shortening excursions at higher force– Weak force dependence of average growth rate

• Reject other models for microtubule polymerization– Small cap induced hydrolysis models (i.e. “lateral

cap”)– “Coupled hydrolysis” models (Flyvbjerg et al, Phys.

Rev. Lett., 1994; Phys. Rev. E, 1996)

Acknowledgements

• NSF

• Whitaker Foundation (to H. Schek)

• Burroughs Wellcome Fund