Evelyne Coudrier

Rab GTPases and Myosin Motors in Organelle Motility

The actin cytoskeleton is essential to ensure the proper location of, and communication between, intracellular organelles. Some actin‐based myosin motors have been implicated in this process, particularly members of the class V myosins. We discuss here the emerging role of the Ras‐like GTPases of the Rab family as regulators of myosin function in organelle transport. Evidence from yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab GTPases are crucial components of the myosin organelle receptor machinery. Better understood is the case of the melanosome where Rab27a recruits a specific effector called melanophilin, which in turn binds myosin Va. The presence of a linker protein between a Rab and a myosin may represent a general mechanism. We argue that Rabs are ideally suited to perform this role as they are exquisite organelle markers. Furthermore, the molecular switch property of Rabs may enable them to regulate the timing of the myosin association with the target organelle.

Different Microtubule Motors Move Early and Late Endocytic Compartments

Important progress has been made during the past decade in the identification of molecular motors required in the distribution of early and late endosomes and the proper trafficking along the endocytic pathway. There is little direct evidence, however, that these motors drive movement of the endosomes. To evaluate the contributions of kinesin‐1, dynein and kinesin‐2 to the movement of early and late endosomes along microtubules, we made use of a cytosol‐free motility assay using magnetically isolated early and late endosomes as well as biochemical analyses and live‐cell imaging. By making use of specific antibodies, we confirmed that kinesin‐1 and dynein move early endosomes and we found that kinesin‐2 moves both early and late endosomes in the cell‐free assay. Unexpectedly, dynein did not move late endosomes in the cell‐free assay. We provide evidence from disruption of dynein function and latrunculin A treatment, suggesting that dynein regulates late endosome movement indirectly, possibly through a mechanism involving the actin cytoskeleton. These data provide new insights into the complex regulation of endosomes’ motility and suggest that dynein is not the major motor required to move late endosomes toward the minus end of microtubules.

Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes

Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.

Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica

Entamoeba histolytica is the human parasite responsible of amoebiasis, during which highly motile trophozoites invade the intestinal epithelium leading to amoebic colitis, and disseminate via the blood circulation causing liver abscesses. The invasive process, central to the pathogenesis, is known to be driven by parasites motility. To investigate molecules responsible for in vivo motion, we performed a high resolution dynamic imaging analysis using two‐photon laser scanning microscopy. Image analysis of the parasites during invasion of Caco‐2 cell monolayers, an enterocyte‐like model, and hamster liver shows that E. histolytica undergoes non‐Brownian motion. However, studies of movements of parasite strains dominant negative for myosin II, a central component of the cytoskeleton, and for Gal‐GalNAc lectin, a major adhesion molecule, indicate that myosin II is essential for E. histolytica intercellular motility through intestinal cell monolayers and for its motility in liver. In contrast, the Gal‐GalNAc lectin exclusively triggers invasion of the liver. These observations are in agreement with emerging studies that highlight marked differences in the way that cells migrate in vitro in two dimensions versus in vivo in three dimensions. The approach that we have developed should be powerful to identify adhesive complexes required for in vivo cell migration in normal and pathogenic situations and may, thereby, lead to new therapeutic drug, for pathologies based on cell motility and adhesion.

Truncated brush border myosin I affects membrane traffic in polarized epithelial cells.

We investigate, in this study, the potential involvement of an acto‐myosin‐driven mechanism in endocytosis of polarized cells. We observed that depolymerization of actin filaments using latrunculin A decreases the rate of transferrin recycling to the basolateral plasma membrane of Caco‐2 cells, and increases its delivery to the apical plasma membrane. To analyze whether a myosin was involved in endocytosis, we produced, in this polarized cell line, truncated, non‐functional, brush border, myosin I proteins (BBMI) that we have previously demonstrated to have a dominant negative effect on endocytosis of unpolarized cells. These non‐functional proteins affect the rate of transferrin recycling and the rate of transepithelial transport of dipeptidyl‐peptidase IV from the basolateral plasma membrane to the apical plasma membrane. They modify the distribution of internalized endocytic tracers in apical multivesicular endosomes that are accessible to fluid phase tracers internalized from apical and basolateral plasma membrane domains. Altogether, these observations suggest that an acto‐myosin‐driven mechanism is involved in the trafficking of basolaterally internalized molecules to the apical plasma membrane.

Immunolocalization of the 110,000 molecular weight cytoskeletal protein of intestinal microvilli

Abstract The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (Mr 16,000), actin (Mr 43,000), villin (Mr 95,000) and a protein of Mr 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and Mr 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the Mr 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the Mr 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the Mr 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network. Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca2+ and Mg2+, a polypeptide of Mr 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this Mr 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.

Membrane traffic in the secretory pathway

It is generally thought that microtubule-associated motors insure long-range movements of the secretory vesicles from the center of the cell to its periphery, while myosins insure short-range movements at the cell periphery. However, several of the myosins that have been reported during the last decade to be involved in the exocytic pathway are not processive, meaning that they do not have the ability to move cargos along actin polymers. We will review here the possible mechanisms by which these myosins could contribute to the traffic of secretory proteins from the Golgi complex to the plasma membrane.Abstract.It is generally thought that microtubule-associated motors insure long-range movements of the secretory vesicles from the center of the cell to its periphery, while myosins insure short-range movements at the cell periphery. However, several of the myosins that have been reported during the last decade to be involved in the exocytic pathway are not processive, meaning that they do not have the ability to move cargos along actin polymers. We will review here the possible mechanisms by which these myosins could contribute to the traffic of secretory proteins from the Golgi complex to the plasma membrane. (Part of a Multi-author Review)

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

Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour 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, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.

Myosin 1 controls membrane shape by coupling F-Actin to membrane.

Cellular functions are intimately associated with rapid changes in membrane shape. Different mechanisms interfering with the lipid bilayer, such as the insertion of proteins with amphipatic helices or the association of a protein scaffold, trigger membrane bending. By exerting force on membranes, molecular motors can also contribute to membrane remodeling. Previous studies have shown that actin and myosin 1 participate in the invagination of the plasma membrane during endocytosis while kinesins and dynein with microtubules provide the force to elongate membrane buds at recycling endosomes and at the trans-Golgi network (TGN). Using live cell imaging we have recently shown that a myosin 1 (myosin 1b) regulates the actin dependent post-Golgi traffic of cargo and generates force that controls the assembly of F-actin foci and promotes with the actin cytoskeleton the formation of tubules at the TGN. Our data provide evidence that actin and myosin 1 can regulate membrane remodeling of organelles as well as having an unexpected role in the spatial organization of the actin cytoskeleton. Here, we discuss our results together with the role of actin and other myosins that have been implicated in the traffic of cargo.

Myosin 1b functions as an effector of EphB signaling to control cell repulsion

Myosin 1b functions as an effector of EphB2/ephrinB signaling and controls cell morphology and cell repulsion.