[Abstract unavailable

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.

Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.

Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as 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 phosphatidylinositol-4,5-bisphosphate (PtdIns(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 PtdIns(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 the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P2 hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease.

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

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.

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gai/ LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGNindependent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.

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.