Ewa Paluch Institut Curie/CNRS, Paris Cortical actomyosin gel breakage triggers shape oscillations...

Ewa Paluch

Institut Curie/CNRS, Paris

Cortical actomyosin gel breakage triggers shape oscillations in cells and cell fragments

Present address:Max-Planck-Institute-CBG, Dresden

Cell crawling

[M. Abercrombie, Proc. R. Soc. Lond. B, 1980]

[B. Alberts et al., Molecular Biology of the Cell, 2002]

nucleus

Actin Myosin Microtubules

Cell crawling - actin

[D. Bray, Cell Movements, 2001]

Cortex intrinsic dynamics???

Dynamics of the actomyosin cortex in suspension cells

Microtubules

L929 fibroblast

Microtubules

PEG


Microtubules

Nocodazole Microtubules

PEG


Microtubules


PEG

[B. Liu et al., Cell Adhes. Commun. 5:249-255 (1998)][M. Krendel et al., Nat. Cell Biol. 4:294-301 (2002)]

Myosin IIEnhanced contractility

Rho Rho-kinase

MT depolymerization

GEF-H1Myosin II Regulatory

Light Chain (RLC)

PEG

Microtubules



Lymphoblasts: [M. Bornens, M. Paintrand, C. Celati, J. Cell Biol. 109:1071-1083 (1989)]

L929 fibroblasts

Centrifugation after microfilaments and

microtubules depolymerization

Cytoplast

Fragments

Nucleus

[E. Paluch, M. Piel, J. Prost, M. Bornens, C. Sykes, Biophys. J., 89:724-733]

Fragments of L929 fibroblastsMovie: http://www.biophysj.org/content/vol0/issue2005/images/data/biophysj.105.060590/DC1/Paluch-Movie1.mov

1. Actin and myosin II during the oscillation

2. The mechanism of the oscillation

3. Spontaneous cortical ruptures and cell motility


Dynamic characterization of actin during the oscillation

Cell fragment

5 µm

Movie: http://www.biophysj.org/content/vol0/issue2005/images/data/biophysj.105.060590/DC1/Paluch-Movie3.mov

[E. Paluch et al., Biophys. J., 89:724-733]


Dynamic characterization of actin during the oscillation

5 µm



Dynamic characterization of myosin II during the oscillation

5 µm

Movie: http://www.biophysj.org/content/vol0/issue2005/images/data/biophysj.105.060590/DC1/Paluch-Movie4.mov



Two-steps mechanism:

1. The actomyosin shell breaks

2. A bulge is expelled and grows

A mechanism for cortical symmetry breaking

σ

σ

- The actin gel is under tension because of myosin motors- The integrated tension T is homogenous:

∫=ext

int

r

r

deT σ

Where the gel thinner, σ is higher.

[K. Sekimoto et al., Eur. Phys. J. E, 13:247-259 (2004)]

Actin

Myosin


Beads mimicking the motility of Listeria monocytogenes

Polystyrene beads coated with actin nucleator VCA

Actin(10% fluorescent)

Minimal mixture of proteins :

Arp2/3, gelsolin, cofilin (+ profilin) (+ cross-linkers)


Actin gel

Actin nucleator

bead

Beads mimicking the motility of Listeria monocytogenes


[V. Noireaux et al., Biophys. J., 78:1643-1654 (2000)]

The actin gel growing around a bead undergoes stress because of the geometry

R

Stress


Observation of symmetry breaking


[J. van der Gucht, E. Paluch, J. Plastino, C. Sykes, PNAS, 22:7847-7852 (2005)]

Symmetry breaking can be induced

Local photolysis of the actin gel


t = 13’ t = 15’ t = 17’

[J. van der Gucht, E. Paluch, J. Plastino, C. Sykes, PNAS, 22:7847-7852 (2005)]

Hole formation is reminiscent of a fracture

2 µm


The rupture mechanism seems comparable.

The controlled bead system can help understading cell cortex ruptures (role of various actin-binding proteins, etc).

The actin shell around a bead: stress due to GEOMETRY

Cell cortex: stress due to MYOSIN motors

bead


Bulge growth can be induced

pipette: cytochalasin latrunculin…

substrate

cellmedium


Local stress application induces bulge growth

P = 200 Pa

flow of medium

pipette

cell



Protrusion growth


The oscillation is driven by contraction of the actomyosin cortex

contraction velocity:

1 to 9 µm/min


Is actomyosin shrinkage due to depolymerization or contraction?

A mechanism for the oscillation

[E. Paluch, M. Piel, J. Prost, M. Bornens, C. Sykes, Biophys. J., 89:724-733]

Actin

Myosin


… + microtubules

reduce myosin II activity

Actin

Myosin

Microtubules


The oscillation and motility?

[E. Paluch et al., Trends Cell Biol., in press]

The oscillation and motility?

[D. J. Solecki et al., Nat. Neurosc., 7, 1195-1203 (2004)]

“Migration involves the coordinated two-strokemovement of a perinuclear tubulin ‘cage’, andthe centrosome, with the centrosome movingforward before nuclear translocation…”

QuickTime™ and aYUV420 codec decompressor

are needed to see this picture.

Movie: http://www.nature.com/neuro/journal/v7/n11/extref/nn1332-S8.mpg

And if contractility is enhanced?


[K. Wolf et al., J. Cell Biol., 160:267-277 (2003)]

I. Contraction waves

[E. Sahai, C. Marshall, Nat. Cell Biol., 5:711-719 (2003)]

II. Multiple blebs




- Blebs result from cortical contractility

- Dual behavior comparable to e.g. growth of dry zones in a thin water film

revue: [E. Paluch, C. Sykes, J. Prost, M. Bornens, Dynamic modes of the cortical actomyosin

gel during cell locomotion and division,Trends in Cell Biol., in press]


Summary

• Cortical oscillation is a general phenomenon resulting from elastic gel properties of the actomyosin cortex

• Bleb formation reveals the level of cortical contractility

• Spontaneous cortical ruptures (and blebs) can be used by cells or remain a side-product of cortex contractions

• Cortex breakage in cells // symmetry breaking of gels around beads

Biology group

Michel Bornens

Matthieu Piel

Physics group

Cécile Sykes

Jasper van der Gucht

Julie Plastino

Theorists

Jean-François JoannyJacques Prost

- GFP constructs: Beat Imhof (University of Geneva) Rex Chisholm (Northwestern University, Chicago)

- Deconvolution: Jean-Baptiste Sibarita (Institut Curie)

Ewa Paluch Institut Curie/CNRS, Paris Cortical actomyosin gel breakage triggers shape oscillations...

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