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Cortical actomyosin gel breakage triggers shape oscillations in cells and cell fragments Ewa Paluch Institut Curie/CNRS, Paris 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] Cell crawling - actin Cortex nucleus intrinsic dynamics??? [D. Bray, Cell Movements, 2001] Actin Myosin Microtubules Dynamics of the actomyosin cortex in suspension cells Microtubules L929 fibroblast Dynamics of the actomyosin cortex in suspension cells Microtubules PEG Dynamics of the actomyosin cortex in suspension cells Microtubules Nocodazole PEG Microtubules MT depolymerization GEF-H1 Rho Rho-kinase Myosin II Regulatory Light Chain (RLC) Myosin II Enhanced contractility [B. Liu et al., Cell Adhes. Commun. 5:249-255 (1998)] [M. Krendel et al., Nat. Cell Biol. 4:294-301 (2002)] Microtubules Nocodazole PEG Microtubules Dynamics of the actomyosin cortex in suspension cells Microtubules Nocodazole Microtubules PEG Lymphoblasts: [M. Bornens, M. Paintrand, C. Celati, J. Cell Biol. 109:1071-1083 (1989)] Fragments of L929 fibroblasts Movie: http://www.biophysj.org/content/vol0/issue2005/images/data/biophysj.105.060590/DC1/Paluch-Movie1.mov Cytoplast Centrifugation after microfilaments and microtubules depolymerization Fragments L929 fibroblasts Nucleus [E. Paluch, M. Piel, J. Prost, M. Bornens, C. Sykes, Biophys. J., 89:724-733] 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 Movie: http://www.biophysj.org/content/vol0/issue2005/images/data/biophysj.105.060590/DC1/Paluch-Movie3.mov Cell fragment 5 µm [E. Paluch et al., Biophys. J., 89:724-733] 1. Actin and myosin II during the oscillation Dynamic characterization of actin during the oscillation 5 µm [E. Paluch et al., Biophys. J., 89:724-733] 1. Actin and myosin II during the oscillation Dynamic characterization of myosin II during the oscillation Movie: http://www.biophysj.org/content/vol0/issue2005/images/data/biophysj.105.060590/DC1/Paluch-Movie4.mov 5 µm [E. Paluch et al., Biophys. J., 89:724-733] 1. Actin and myosin II during the oscillation Two-steps mechanism: 1. The actomyosin shell breaks 2. A bulge is expelled and grows 1. Actin and myosin II during the oscillation 2. The mechanism of the oscillation 3. Spontaneous cortical ruptures and cell motility 2. The mechanism of 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: rext T de rint Where the gel thinner, σ is higher. Actin Myosin [K. Sekimoto et al., Eur. Phys. J. E, 13:247-259 (2004)] 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 Beads mimicking the motility of Listeria monocytogenes Actin nucleator bead Actin gel 2. The mechanism of the oscillation The actin gel growing around a bead undergoes stress because of the geometry Stress R [V. Noireaux et al., Biophys. J., 78:1643-1654 (2000)] 2. The mechanism of the oscillation Observation of symmetry breaking 2. The mechanism of the oscillation Symmetry breaking can be induced Local photolysis of the actin gel [J. van der Gucht, E. Paluch, J. Plastino, C. Sykes, PNAS, 22:7847-7852 (2005)] 2. The mechanism of the oscillation Hole formation is reminiscent of a fracture 2 µm t = 13’ t = 15’ t = 17’ [J. van der Gucht, E. Paluch, J. Plastino, C. Sykes, PNAS, 22:7847-7852 (2005)] 2. The mechanism of the oscillation Cell cortex: stress due to MYOSIN motors The actin shell around a bead: stress due to GEOMETRY bead The rupture mechanism seems comparable. The controlled bead system can help understading cell cortex ruptures (role of various actin-binding proteins, etc). 2. The mechanism of the oscillation Bulge growth can be induced medium cell substrate pipette: cytochalasin latrunculin… 2. The mechanism of the oscillation Local stress application induces bulge growth pipette cell flow of medium P = 200 Pa [E. Paluch et al., Biophys. J., 89:724-733] 2. The mechanism of the oscillation Protrusion growth 2. The mechanism of the oscillation Is actomyosin shrinkage due to depolymerization or contraction? contraction velocity: 1 to 9 µm/min The oscillation is driven by contraction of the actomyosin cortex 2. The mechanism of the oscillation A mechanism for the oscillation Actin Myosin [E. Paluch, M. Piel, J. Prost, M. Bornens, C. Sykes, Biophys. J., 89:724-733] 1. Actin and myosin II during the oscillation 2. The mechanism of the oscillation 3. Spontaneous cortical ruptures and cell motility 3. Spontaneous cortical ruptures and cell motility … + 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? “Migration involves the coordinated two-stroke movement of a perinuclear tubulin ‘cage’, and the centrosome, with the centrosome moving forward before nuclear translocation…” QuickTime™ and a YUV420 codec decompressor are needed to see this picture. [D. J. Solecki et al., Nat. Neurosc., 7, 1195-1203 (2004)] Movie: http://www.nature.com/neuro/journal/v7/n11/extref/nn1332-S8.mpg 3. Spontaneous cortical ruptures and cell motility And if contractility is enhanced? 3. Spontaneous cortical ruptures and cell motility And if contractility is enhanced? I. Contraction waves [K. Wolf et al., J. Cell Biol., 160:267-277 (2003)] II. Multiple blebs [E. Sahai, C. Marshall, Nat. Cell Biol., 5:711-719 (2003)] 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] 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 Physics group Cécile Sykes Jasper van der Gucht Biology group Julie Plastino Theorists Michel Bornens Matthieu Piel Jean-François Joanny Jacques Prost - GFP constructs: Beat Imhof (University of Geneva) Rex Chisholm (Northwestern University, Chicago) - Deconvolution: Jean-Baptiste Sibarita (Institut Curie)