Pom1 regulates the assembly of Cdr2-Mid1 cortical nodes for robust spatial control of cytokinesis

Rincon SA, Bhatia P, Bicho C, Guzman-Vendrell M, Fraisier V, Borek WE, de Lima Alves F, Dingli F, Loew D, Rappsilber J, Sawin KE, Martin SG and Paoletti A J Cell Biol. 2014 Jul 7;206(1):61-77. doi: 10.1083/jcb.201311097. Epub 2014 Jun 30 Learn more Hide

Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning.

Catch-bond behavior facilitates membrane tubulation by non-processive myosin 1b

Ayako Yamada, Alexandre Mamane, Jonathan Lee-Tin-Wah, Aurélie Di Cicco, Coline Prévost Daniel Lévy, Jean-François Joanny, Evelyne Coudrier and Patricia Bassereau. Nat Commun. 2014 Apr 7;5:3624. doi: 10.1038/ncomms4624. Learn more Hide

natureCom2014Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a
catch-bond behavior in response to load. In vivo, myosin 1b is required to form membrane
tubules at both endosomes and the trans-Golgi network. To establish the link between these
two fundamental properties, we investigate the capacity of myosin 1b to extract membrane
tubes along bundled actin filaments in a minimal reconstituted system. We show for the first
time that single-headed non-processive myosin 1b can extract membrane tubes at low density,
which is biologically relevant. In contrast to kinesins we did not observe motor accumulation
at the tip, suggesting that the underlying mechanism for tube formation is different. Our
theoretical model emphasizes the importance of the catch-bond properties to facilitate tube
extraction upon increasing membrane tension

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells

Reffay M, Parrini MC, Cochet-Escartin O, Ladoux B, Buguin A, Coscoy S, Amblard F, Camonis J, Silberzan P. Nat Cell Biol. 2014 Feb Learn more Hide

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Border forces and friction control epithelial closure dynamics.

Cochet-Escartin O., Ranft J., Silberzan P., Marcq P. Biophysical Journal 2014 Learn more Hide

We study the closure dynamics of a large number of well-controlled circular apertures within an epithelial monolayer, where the collective cell migration responsible for epithelization is triggered by the removal of a spatial constraint rather than by scratching. Based on experimental observations, we propose a physical model that takes into account border forces, friction with the substrate, and tissue rheology. Border protrusive activity drives epithelization despite the presence of a contractile actomyosin cable at the periphery of the wound. The closure dynamics is quantified by an epithelization coefficient, defined as the ratio of protrusive stress to tissue-substrate friction, that allows classification of different phenotypes. The same analysis demonstrates a distinct signature for human cells bearing the oncogenic RasV12 mutation, demonstrating the potential of the approach to quantitatively characterize metastatic transformations.

ESCRT machinery is required for plasma membrane repair.

Jimenez AJ, Maiuri P, Lafaurie-Janvore J, Divoux S, Piel M, Perez F. Science. 2014 Feb 28;343(6174):1247136. doi: 10.1126/science.1247136. Epub 2014 Jan 30. Learn more Hide

Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.

Membrane shape modulates transmembrane protein distribution.

Aimon S, Callan-Jones A, Berthaud A, Pinot M, Toombes GE, Bassereau P. Dev Cell. 2014 Jan 27;28(2):212-8. doi: 10.1016/j.devcel.2013.12.012. Learn more Hide

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins.

Structural basis of myosin V Rab GTPase-dependent cargo recognition.

Pylypenko O, Attanda W, Gauquelin C, Lahmani M, Coulibaly D, Baron B, Hoos S, Titus MA, England P, Houdusse AM. Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20443-8. doi: 10.1073/pnas.1314329110. Epub 2013 Nov 18. Learn more Hide

Specific recognition of the cargo that molecular motors transport or tether to cytoskeleton tracks allows them to perform precise cellular functions at particular times and positions in cells. However, very little is known about how evolution has favored conservation of functions for some isoforms, while also allowing for the generation of new recognition sites and specialized cellular functions. Here we present several crystal structures of the myosin Va or the myosin Vb globular tail domain (GTD) that gives insights into how the motor is linked to the recycling membrane compartments via Rab11 or to the melanosome membrane via recognition of the melanophilin adaptor that binds to Rab27a. The structures illustrate how the Rab11-binding site has been conserved during evolution and how divergence at another site of the GTD allows more specific interactions such as the specific recognition of melanophilin by the myosin Va isoform. With atomic structural insights, these structures also show how either the partner or the GTD structural plasticity upon association is critical for selective recruitment of the motor.

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip.

Bornschlögl T, Romero S, Vestergaard CL, Joanny JF, Van Nhieu GT, Bassereau P. Proc Natl Acad Sci U S A. 2013 Nov 6 Learn more Hide

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range.

Identification and characterisation of multiple novel Rab-myosin Va interactions

Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B. Mol Biol Cell. 2013 Sep 4 Learn more Hide

Myosin Va is a widely expressed actin based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A’, 6B, 11B, 14, 25, 39B), which includes interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Interestingly, myosin Va only interacts with a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively-spliced exon and the globular tail). While the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14- positive endosomes.