Sélection de travaux publiés

Midbody remnant engulfment after cytokinesis abscission in mammalian cells.

Crowell EF, Gaffuri AL, Gayraud-Morel B, Tajbakhsh S and Echard A

Journal of Cell Science: 127 jcs.154732 (2014)

 

The midbody remnant (MBR) that is generated after cytokinesis abscission has recently attracted a lot of attention, since it may have crucial consequences for cell differentiation and tumorigenesis in mammalian cells. In those cells, it has been reported that the MBR is either released into the extracellular medium, or retracted into one of the two daughter cells where it can be degraded by autophagy. Here, we describe a major alternative pathway in a variety of human and mouse immortalized/cancer and primary stem cells. Using correlative light/scanningEM microscopy and quantitative assays, we found that sequential abscissions on both sides of the midbody generate free MBRs, which are tightly associated to the cell surface through a Ca++/Mg++-dependent receptor. Surprisingly, MBRs move over the cell surface for several hours, before being eventually engulfed by an actin-dependent phagocytosis-like mechanism. Mathematical modelling combined to experiments further demonstrates that lysosomal activities fully account for clearance of MBRs after engulfment. This study changes our vision of how MBRs are inherited and degraded in mammalian cells, and suggests a mechanism by which MBRs might signal over long distances between cells.

                  

SLK-dependent activation of ERM controls LGN/NuMA localization and spindle orientation.

Machicoane M, de Frutos C, Fink J, Rocancourt M, Lombardi Y, Garel S, Piel M and Echard A

Journal of Cell Biology: 205, 791-799 (2014)

 

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane-actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN-NuMA force generator complexes and hence for proper spindle orientation.

 

An ARF6/Rab35 GTPase Cascade for Endocytic Recycling and Successful Cytokinesis. 
Chesneau L, Dambournet D, Machicoane M, Kouranti I, Fukuda M, Goud B, Echard A.
Current Biology: 22, 147-53 (2012)
 

Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo. Membrane trafficking is crucial for animal cell cytokinesis, and several endocytic pathways regulated by distinct GTPases (Rab11, Rab21, Rab35, ARF6, RalA/B) contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis. Here, we report that the ARF6 GTPase negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis.

 

Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.
Dambournet D., Machicoane M., Chesneau L. Rocancourt M., Formstecher E., Salomon R., Goud B. and Echard A.
Nature Cell Biology: 13, 981-88 (2011)
 

Abscission is currently the least understood step of cytokinesis. It consists in the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, which presumably implies membrane trafficking, lipid and cytoskeleton remodelling. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages. Here, we report that the PI(4,5)P2 5-phosphatase OCRL, which is mutated in Lowe syndrome patients, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PI(4,5)P2 and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patients and can be corrected by addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PI(4,5)P2 hydrolysis is important for normal cytokinesis abscission in order to locally remodel the F-actin cytoskeleton in the intercellular bridge. It also reveals an unexpected role of the phosphatase OCRL in cell division and sheds new light on the pleiotropic phenotypes associated with the Lowe disease.

 

Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex.
Miserey-Lenkei S, Chalancon G, Bardin S, Formstecher E, Goud B, Echard A.
Nature Cell Biology: 12, 645-54 (2010)
 

Trafficking between membrane compartments is a characteristic of eukaryotic cells and relies on transport carriers that bud and fission from a donor membrane, before being transported and fusing with the correct acceptor compartment. Rab GTPases ensure specificity and directionality of trafficking steps by regulating the movement of transport carriers along cytoskeletal tracks, and the recruitment of tethering factors required for the docking and fusion processes. Here we show that Rab6, a Golgi-associated Rab, forms a complex with myosin II, contributes to its localization at the Golgi complex and, unexpectedly, controls the fission of Rab6 vesicles. Inhibition of either Rab6 or myosin II function impairs both the fission of Rab6 transport carriers from Golgi membranes and the trafficking of anterograde and retrograde cargo from the Golgi. These effects are consistent with myosin II being an effector of Rab6 in these processes. Our results provide evidence that the actomyosin system is required in vesicle biogenesis at the Golgi, and uncover a function for Rab GTPases in vesicle fission.

 

Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells.
Carreno S., Kouranti I., Glusman E.S., Fuller M.T. and Echard A. / Payre F.
Journal of Cell Biology:  180, 739-46 (2008)
 

Cell division requires cell shape changes involving the localized reorganization of cortical actin, which must be tightly linked with chromosome segregation operated by the mitotic spindle. How this multistep process is coordinated remains poorly understood. In this study, we show that the actin/membrane linker moesin, the single ERM (ezrin, radixin, and moesin) protein in Drosophila melanogaster, is required to maintain cortical stability during mitosis. Mitosis onset is characterized by a burst of moesin activation mediated by a Slik kinase-dependent phosphorylation. Activated moesin homogenously localizes at the cortex in prometaphase and is progressively restricted at the equator in later stages. Lack of moesin or inhibition of its activation destabilized the cortex throughout mitosis, resulting in severe cortical deformations and abnormal distribution of actomyosin regulators. Inhibiting moesin activation also impaired microtubule organization and precluded stable positioning of the mitotic spindle. We propose that the spatiotemporal control of moesin activation at the mitotic cortex provides localized cues to coordinate cortical contractility and microtubule interactions during cell division.

 

Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.
Kouranti I., Sachse M., Arouche N., Goud B. and Echard A.
Current Biology : 16 : 1719-25 (2006)
 

Cytokinesis is the final step of cell division and leads to the physical separation of the daughter cells. After the ingression of a cleavage membrane furrow that pinches the mother cell, future daughter cells spend much of the cytokinesis phase connected by an intercellular bridge. Rab proteins are major regulators of intracellular transport in eukaryotes, and here, we report an essential role for human Rab35 in both the stability of the bridge and its final abscission. We find that Rab35, whose function in membrane traffic was unknown, is localized to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway. Consistent with a key requirement for Rab35-regulated recycling during cell division, inhibition of Rab35 function leads to the accumulation of endocytic markers on numerous cytoplasmic vacuoles in cells that failed cytokinesis. Moreover, Rab35 is involved in the intercellular bridge localization of two molecules essential for the postfurrowing steps of cytokinesis: the phosphatidylinositol 4,5-bis phosphate (PIP2) lipid and the septin SEPT2. We propose that the Rab35-regulated pathway plays an essential role during the terminal steps of cytokinesis by controlling septin and PIP2 subcellular distribution during cell division.

 

Terminal cytokinesis events uncovered after an RNAi screen.
Echard A., Hickson G., Foley E. and O’Farrell P.H.
Current Biology : 14, 1685-93 (2004) 
 

Much of our understanding of animal cell cytokinesis centers on the regulation of the equatorial acto-myosin contractile ring that drives the rapid ingression of a deep cleavage furrow. However, the central part of the mitotic spindle collapses to a dense structure that impedes the furrow and keeps the daughter cells connected via an intercellular bridge. Factors involved in the formation, maintenance, and resolution of this bridge are largely unknown. Using a library of 7,216 double-stranded RNAs (dsRNAs) representing the conserved genes of Drosophila, we performed an RNA interference (RNAi) screen for cytokinesis genes in Schneider's S2 cells. We identified both familiar and novel genes whose inactivation induced a multi-nucleate phenotype. Using live video microscopy, we show that three genes: anillin, citron-kinase (CG10522), and soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (alpha-SNAP), are essential for the terminal (post-furrowing) events of cytokinesis. anillin RNAi caused gradual disruption of the intercellular bridge after furrowing; citron-kinase RNAi destabilized the bridge at a later stage; alpha-SNAP RNAi caused sister cells to fuse many hours later and by a different mechanism. We have shown that the stability of the intercellular bridge is essential for successful cytokinesis and have defined genes contributing to this stability.
 

Mis à jour le 01/09/2014

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