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
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