Mechanics of the Lamellar Actomyosin

Cytoskeleton

Margaret Gardel

University of ChicagoPhysics Department, James Franck Institute, Institute for Biophysical Dynamics

http://squishycell.uchicago.edu/

Mechanics of Cell Adhesion and Migration

(B. Fischer, NHLBI)

Paxillin Traction StressEndothelial cell in collagen gel

U2OS cell on FN-coated PAA gel

MG, Ann. Cell Dev Bio, 2010

Distinct Actin Structure and Dynamics Drive Migration

MG, Ann. Cell Dev Bio, 2010

Actin Paxillin ECM

Extracellular matrix

Plasma Membrane

Force transmission in Cell Adhesion and Migration

Actin filaments, myosin motors

Cross-linking proteins

Focal

Adhesions

Actin Cytoskeleton

Force Generation

& Mechanics

Cell-ECM

Adhesion

ECM

mechanics

Force transmission in Cell Adhesion and Migration

Actin Cytoskeleton

Force Generation

& Mechanics

Cell-ECM

Adhesion

ECM

mechanics

Gardel JCB 2008, Aratyn-Schaus & Gardel, CB, 2010, Stricker et. al., BJ 2011

Extracellular matrix

Plasma Membrane

Force transmission in Cell Adhesion and Migration

Actin filaments, myosin motors

Cross-linking proteins

Focal

Adhesions

Actin Cytoskeleton

Force Generation

& Mechanics

Cell-ECM

Adhesion

ECM

mechanics

Quantitative predictions of how actomyosin cytoskeleton

transmits forces

Diverse Organizations of Lamellar Actomyosin Cytoskeleton

Non-motile Motile

Stiff ECM (2D) Soft ECM (3D?)

High Contractility Weak Contractility

+ 25 mM Blebbistatin

30 min

Dynamically regulate contractile phenotype

Control15 min after Removing

BLEB

Actin

Paxillin

Myosin Light Chain

Blebbistatin Washout Drives Self-Assembly of Lamellar

Networks and Bundles

GFP-actin mApple-Paxillin

Traction

High Resolution Traction Force Microscopy

GFP-actin Cy5 beads

10-20 mm thick polyacrylamide gel

40 nm far red latex spheres

Displacement Field, U

Traction Stress Field, F

Actin Paxillin Traction Stress

140 Pa

100 Pa

60 Pa

20 Pa

Assembly of Contractile Lamella and Traction Forces

Rapid and Slow Phases of Tension Build-Up

Fast Time Scale: Rapid Actin Dynamics

0 sec 5 sec 15 sec

Inverse Force-Velocity Relationship for Lamellar Actin Networks

J. Howard, Mechanics of Motor Proteins

In vitro

trc v

F= 200 nN

Cell

vu 108 nm/s

Fstall 200 nN

In Vitro

100 nm/s

~5 pN

In vitro

J. Howard, Mechanics of Motor Proteins

40000 motors working in parallel

Lamellar mechanics mimics myosin II mechanochemistry

20 mm

GFP-Myosin mApple-Actin

F= 200 nN

0.5 mm

400 mm

~500 pN/mm

~5 pN stall force/motor

~100 motors/mm working in parallel

~ 2 puncta/mm

8 motors per minifilament

6 minifilaments per puncta

Lamellar mechanics mimics myosin II mechanochemistry

Long time scale: Stress Fiber Formation

00:30 30:00

Order Parameter to Measure Extent of Bundling

Aratyn-Schaus Y, Oakes P & Gardel, MBoC, 2011

Myosin II drives stress fiber formation

GFP-myosin light chain

+ 25 mM Blebbistatin

Myosin band spacing decreases as tension builds

0 min

25 minAratyn-Schaus Y, Oakes P & Gardel, MBoC, 2011

a-actinin bands form at increased tension

Control Bleb. Washout Actin a-actinin

0 min

35 min

Lamellar Architecture regulates Force Transmission

Dynamics dictate Force

Rapid (30 s) force build up

“Active” Fluid

(Mogilner, J.F. Joanny, K. Kruse)

)

Myosin-Driven Tension

Structure dictates Force

Slow (10 min) force build up

Solid-like behavior

Aratyn-Schaus Y, Oakes P & Gardel, MBoC, 2011

Contractile Lamellar Networks Insensitive to Substrate Stiffness

Prestress increases the Maxwell Relaxation time of Actomyosin

Networks

Norstrom, M & Gardel, Soft Matter, 2011

Pellegrin & Mellor, JCS 2007

Dorsal Stress Fibers thought to link adhesions to

Lamellar Actin

Stress fiber elongation occurs via mDia1 driven actin

polymerization and myosin-dependent retrograde flow

Hotulainen and Lappalainen, JCB 2006

Myosin II – generates force and retrograde

movement, cross-links F-actin

mDia 1 – actin filament nucleator

a-actinin – actin crosslinking protein

Elongation rate = 5 nm/s

What are the consequences of inhibiting stress fiber assembly?

Formin and a-actinin required for stress fiber assembly

Eliminating stress fibers results in rapid lamellar actin flow

Actin Flow Vectors

Large Traction Forces Generated in the Absence of Stress Fibers

SMIF H2

Tension build up at adhesions occurs in the absence of stress

fiber assembly

Traction Forces

Retrograde Flow

FA lifetime

FA Morphology

FA Composition

ECM Remodeling

Lamellar Network Dorsal Stress Fibers

1-12 nN 1-12 nN

10 nm/s 5 nm/s

30 min 50 min

small large

Inhibiting Stress fiber assembly impairs FA compositional maturation

Reduced pY397 FAK/pY 31 Pxn Impaired formation of fibrillar adhesions

Tension is insufficient to mediate compositional maturation of adhesions

Inhibiting Stress Fiber Assembly Abolishes ECM remodeling

Traction Forces

Retrograde Flow

FA lifetime

FA maturation

FN remodeling

Lamellar Network Dorsal Stress Fibers

1-12 nN 1-12 nN

10 nm/s 5 nm/s

NONO

YES

YES

30 min 50 min

– Disordered Actomyosin networks generate large forces over rapid time

scales

• Rate of Force build up is insensitive to substrate stiffness

– Force-velocity relationship of contractile Lamellar Networks mimics

myosin-II mechanochemistry

• In Lamellar Networks, Contractile Elements add in parallel!

– Bundles of actin (dorsal stress fibers) linking adhesion plaques to

lamellar actomyosin are not needed for efficient force transmission

• Important for adhesion maturation and fibronectin remodeling

– Large Adhesions and Dorsal Stress Fibers do not reflect the extent of

cellular tension (e.g. platelets!)

• Be careful in intuiting mechanics from structure

Thoresen et. al., BJ, 2011

Reconstituted actomyosin bundles have contractile

elements in series

Summary

Hartwig

Funding: Burroughs Wellcome Career Award, NIH Director’s Pioneer Award, Packard Foundation

Cell Biology

Yvonne Beckham (Mol. Biology)

Venkat Maruthamuthu (Chem E)

Patrick Oakes (Physics)

Jonathan Stricker (Physics)

Steve Winter (MD/PhD)

Chris Harland (Physics) – E. Munro

Yvonne Aratyn-Schaus (Cell Bio)

In Vitro

Tobias Falzone (Biophysics) – D. Kovar

Melanie Norstrom (Biochem)

Todd Thoresen (Biochem)

Sam Stam (Biophysics) – W. Zhang

Patrick McCall (Physics)

Michael Murrell (BioE) – D. Kovar

Simulation/Theory

Martin Lenz (Physics) – A. Dinner

Moshe Naoz (Physics) – E. Munro

Tae Yoon Kim (Mech E) – E. Munro

Colloids

Tom Caswell – S. Nagel

Chicago Cytoskeleton Group:

Michael Glotzer

Moe Gupta

Dave Kovar

Ed Munro

Ron Rock

M. Gardel