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Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Using 3D cultures of renal MDCK cells (cysts), we found that the Rab35 GTPase plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of cyst development. At the molecular level, Rab35 physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site. These vesicles transport aPKC, Cdc42, Crumbs3 and the lumen-promoting factor Podocalyxin, and are tethered through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Consequently, Rab35 inactivation leads to complete inversion of apico-basal polarity in 3D cysts. This novel and unconventional mode of Rab-dependent vesicle targeting provides a simple mechanism for triggering both initiation of apico-basal polarity and lumen opening at the centre of cysts. Establishment and maintenance of apico-basal polarity in epithelial organs needs to be tightly coupled with cell division. Here the authors show that the Rab35 GTPase tethers intracellular vesicles containing key apical determinants at the cleavage site, connecting cytokinesis to apico-basal polarity.

How living systems break symmetry in an organized manner is a fundamental question in biology. In wild-type Caenorhabditis elegans zygotes, symmetry breaking during anterior-posterior axis specification is guided by centrosomes, resulting in anterior-directed cortical flows and a single posterior PAR-2 domain. We uncover that C. elegans zygotes depleted of the Aurora A kinase AIR-1 or lacking centrosomes entirely usually establish two posterior PAR-2 domains, one at each pole. We demonstrate that AIR-1 prevents symmetry breaking early in the cell cycle, whereas centrosomal AIR-1 instructs polarity initiation thereafter. Using triangular microfabricated chambers, we establish that bipolarity of air-1(RNAi) embryos occurs effectively in a cell-shape and curvature-dependent manner. Furthermore, we develop an integrated physical description of symmetry breaking, wherein local PAR-2-dependent weakening of the actin cortex, together with mutual inhibition of anterior and posterior PAR proteins, provides a mechanism for spontaneous symmetry breaking without centrosomes.

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division. Cytokinetic abscission relies on the local constriction after cytoskeleton disassembly, but it is not known how the actin filaments are disassembled. Here, the authors show that the redox enzyme MICAL1 is recruited by Rab35 and induces oxidation-mediated depolymerization of actin, which is required to recruit ESCRT-III and complete abscission.

Inherited peripheral neuropathies (IPNs) represent a broad group of disorders including Charcot-Marie-Tooth (CMT) neuropathies characterized by defects primarily arising in myelin, axons, or both. The molecular mechanisms by which mutations in nearly 100 identified IPN/CMT genes lead to neuropathies are poorly understood. Here we show that the Ras-related GTPase Rab35 controls myelin growth via complex formation with the myotubularin-related phosphatidylinositol (PI) 3-phosphatases MTMR13 and MTMR2, encoded by genes responsible for CMT-types 4B2 and B1 in humans, and found that it downregulates lipid-mediated mTORC1 activation, a pathway known to crucially regulate myelin biogenesis. Targeted disruption of Rab35 leads to hyperactivation of mTORC1 signaling caused by elevated levels of PI 3-phosphates and to focal hypermyelination in vivo. Pharmacological inhibition of phosphatidylinositol 3,5-bisphosphate synthesis or mTORC1 signaling ameliorates this phenotype. These findings reveal a crucial role for Rab35-regulated lipid turnover by myotubularins to repress mTORC1 activity and to control myelin growth. Charcot-Marie-Tooth (CMT) is an inherited peripheral neuropathy. Here, the authors show that Rab35 forms a complex with genes implicated in CMT, MTMR13 and MTMR2, which regulates myelin growth by controlling mTORC1 signaling through lipid turnover.

Mutant p53s (mutp53) increase cancer invasiveness by upregulating Rab-coupling protein (RCP) and diacylglycerol kinase-α (DGKα)-dependent endosomal recycling. Here we report that mutp53-expressing tumour cells produce exosomes that mediate intercellular transfer of mutp53’s invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53’s ability to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment in distant organs in a way that can support invasive growth. Some p53 mutants promote invasive migration of cancer cells and metastasis of tumours in vivo. However the key mechanistic details behind these phenomena remain unclear. Here the authors propose a non-cell autonomous mechanism involving fibroblasts, whereby mutant p53-expressing cancer cells activate an exosome-mediated mechanism that influences integrin recycling in fibroblasts, thus influencing extracellular matrix remodelling to favour cancer cell invasion and migration.

The heart hosts tissue resident macrophages which are capable of modulating cardiac inflammation and function by multiple mechanisms. At present, the consequences of phenotypic diversity in macrophages in the heart are incompletely understood. The contribution of cardiac M2-polarized macrophages to the resolution of inflammation and repair response following myocardial infarction remains to be fully defined. In this study, the role of M2 macrophages was investigated utilising a specific CSF-1 receptor signalling inhibition strategy to achieve their depletion. In mice, oral administration of GW2580, a CSF-1R kinase inhibitor, induced significant decreases in Gr1lo and F4/80hi monocyte populations in the circulation and the spleen. GW2580 administration also induced a significant depletion of M2 macrophages in the heart after 1 week treatment as well as a reduction of cardiac arginase1 and CD206 gene expression indicative of M2 macrophage activity. In a murine myocardial infarction model, reduced M2 macrophage content was associated with increased M1-related gene expression (IL-6 and IL-1β), and decreased M2-related gene expression (Arginase1 and CD206) in the heart of GW2580-treated animals versus vehicle-treated controls. M2 depletion was also associated with a loss in left ventricular contractile function, infarct enlargement, decreased collagen staining and increased inflammatory cell infiltration into the infarct zone, specifically neutrophils and M1 macrophages. Taken together, these data indicate that CSF-1R signalling is critical for maintaining cardiac tissue resident M2-polarized macrophage population, which is required for the resolution of inflammation post myocardial infarction and, in turn, for preservation of ventricular function.

Background: Previously, we established the importance of the CX3CL1/CX3CR1 axis in the promotion of myeloid cell differentiation into neointimal smooth muscle-like cells (SMLC). Methods: In this study, acute (24 h) endothelial coverage and CX3CL1 expression as well as chronic (2 weeks) vascular remodeling was examined with respect to whether myeloid CX3CR1 + SMLC number in the neointima differed between carotid and femoral artery wire injury. Results and Conclusion: Twenty-four hours after injury, CX3CL1 expression was significantly elevated in injured carotid compared to femoral arteries. In mice with CX3CR1 promoter-driven expression of green fluorescent protein, neointima formation was significantly greater (p < 0.05) 2 weeks after injury in femoral versus carotid arteries as determined by the intima/media ratio. Although the percentage of F4/80/CX3CR1 + cell integration was similar in both models, the carotid lesion had greater proportions of cells coexpressing CX3CR1 and both α-smooth muscle actin and calponin (p < 0.05). Wire injury of carotid arteries was associated with greater CX3CL1 expression in the acute phase followed by greater CX3CR1 coexpressing SMLC content in later lesions as well as less neointima formation than in femoral arteries. This may, in part, explain the variability in lesion composition after carotid versus femoral wire injury.

The heart hosts tissue resident macrophages which are capable of modulating cardiac inflammation and function by multiple mechanisms. At present, the consequences of phenotypic diversity in macrophages in the heart are incompletely understood. The contribution of cardiac M2-polarized macrophages to the resolution of inflammation and repair response following myocardial infarction remains to be fully defined. In this study, the role of M2 macrophages was investigated utilising a specific CSF-1 receptor signalling inhibition strategy to achieve their depletion. In mice, oral administration of GW2580, a CSF-1R kinase inhibitor, induced significant decreases in Gr1lo and F4/80hi monocyte populations in the circulation and the spleen. GW2580 administration also induced a significant depletion of M2 macrophages in the heart after 1 week treatment as well as a reduction of cardiac arginase1 and CD206 gene expression indicative of M2 macrophage activity. In a murine myocardial infarction model, reduced M2 macrophage content was associated with increased M1-related gene expression (IL-6 and IL-1[Beta]), and decreased M2-related gene expression (Arginase1 and CD206) in the heart of GW2580-treated animals versus vehicle-treated controls. M2 depletion was also associated with a loss in left ventricular contractile function, infarct enlargement, decreased collagen staining and increased inflammatory cell infiltration into the infarct zone, specifically neutrophils and M1 macrophages. Taken together, these data indicate that CSF-1R signalling is critical for maintaining cardiac tissue resident M2-polarized macrophage population, which is required for the resolution of inflammation post myocardial infarction and, in turn, for preservation of ventricular function.