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 ActinMyosinMicrotubules Cell crawling - actin [D. Bray, Cell Movements, 2001] Cortex intrinsic dynamics???

Dynamics of the actomyosin cortex in suspension cells Microtubules L929 fibroblast

Microtubules PEG Dynamics of the actomyosin cortex in suspension cells

Microtubules NocodazoleMicrotubules PEG Dynamics of the actomyosin cortex in suspension cells

Microtubules NocodazoleMicrotubules PEG [B. Liu et al., Cell Adhes. Commun. 5: (1998)] [M. Krendel et al., Nat. Cell Biol. 4: (2002)] Myosin II Enhanced contractility RhoRho-kinase MT depolymerization GEF-H1 Myosin II Regulatory Light Chain (RLC)

PEG Microtubules NocodazoleMicrotubules Dynamics of the actomyosin cortex in suspension cells Lymphoblasts: [M. Bornens, M. Paintrand, C. Celati, J. Cell Biol. 109: (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: ] Fragments of L929 fibroblasts Movie:

1. Actin and myosin II during the oscillation 2. The mechanism of the oscillation 3. Spontaneous cortical ruptures and cell motility

1. Actin and myosin II during the oscillation Dynamic characterization of actin during the oscillation Cell fragment 5 µm Movie: [E. Paluch et al., Biophys. J., 89: ]

1. Actin and myosin II during the oscillation Dynamic characterization of actin during the oscillation 5 µm [E. Paluch et al., Biophys. J., 89: ]

1. Actin and myosin II during the oscillation Dynamic characterization of myosin II during the oscillation 5 µm Movie: [E. Paluch et al., Biophys. J., 89: ]

1. Actin and myosin II during the oscillation Two-steps mechanism: 1. The actomyosin shell breaks 2. A bulge is expelled and grows

2. The mechanism of the oscillation 3. Spontaneous cortical ruptures and cell motility 1. Actin and myosin II during the oscillation

A mechanism for cortical symmetry breaking σ σ - The actin gel is under tension because of myosin motors - The integrated tension T is homogenous: Where the gel thinner, σ is higher. [K. Sekimoto et al., Eur. Phys. J. E, 13: (2004)] Actin Myosin 2. The mechanism of the oscillation

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) 2. The mechanism of the oscillation

Actin gel Actin nucleator bead Beads mimicking the motility of Listeria monocytogenes 2. The mechanism of the oscillation

[V. Noireaux et al., Biophys. J., 78: (2000)] The actin gel growing around a bead undergoes stress because of the geometry R Stress 2. The mechanism of the oscillation

Observation of symmetry breaking 2. The mechanism of the oscillation

[J. van der Gucht, E. Paluch, J. Plastino, C. Sykes, PNAS, 22: (2005)] Symmetry breaking can be induced Local photolysis of the actin gel 2. The mechanism of the oscillation

t = 13’t = 15’t = 17’ [J. van der Gucht, E. Paluch, J. Plastino, C. Sykes, PNAS, 22: (2005)] Hole formation is reminiscent of a fracture 2 µm 2. The mechanism of the oscillation

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 2. The mechanism of the oscillation

Bulge growth can be induced pipette: cytochalasin latrunculin… substrate cell medium 2. The mechanism of the oscillation

Local stress application induces bulge growth P = 200 Pa flow of medium pipette cell 2. The mechanism of the oscillation [E. Paluch et al., Biophys. J., 89: ]

Protrusion growth 2. The mechanism of the oscillation

The oscillation is driven by contraction of the actomyosin cortex contraction velocity: 1 to 9 µm/min 2. The mechanism of the oscillation 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: ] Actin Myosin 2. The mechanism of the oscillation

3. Spontaneous cortical ruptures and cell motility 2. The mechanism of the oscillation 1. Actin and myosin II during the oscillation

… + microtubules reduce myosin II activity Actin Myosin Microtubules 3. Spontaneous cortical ruptures and cell motility

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, (2004)] “Migration involves the coordinated two-stroke movement of a perinuclear tubulin ‘cage’, and the centrosome, with the centrosome moving forward before nuclear translocation…” Movie:

And if contractility is enhanced? 3. Spontaneous cortical ruptures and cell motility

[K. Wolf et al., J. Cell Biol., 160: (2003)] I. Contraction waves [E. Sahai, C. Marshall, Nat. Cell Biol., 5: (2003)] II. Multiple blebs And if contractility is enhanced? 3. Spontaneous cortical ruptures and cell motility

And if contractility is enhanced? - 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] 3. Spontaneous cortical ruptures and cell motility

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 Joanny Jacques Prost - GFP constructs: Beat Imhof (University of Geneva) Rex Chisholm (Northwestern University, Chicago) - Deconvolution: Jean-Baptiste Sibarita (Institut Curie)