Rab35 GTPase et initiation de la polarité apico-basal dans un modèle cellulaire 3D

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), I 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.L'établissement de la polarité apico-basale dans les tissus épithéliaux est étroitement lié à la division cellulaire, mais les mécanismes moléculaires sous-jacents n'ont pas encore été établis. A l'aide d'un modèle de culture en 3 dimensions de cellules rénales (MDCK), j'ai montré que lors du développement d'un cyst, la GTPase Rab35 joue un rôle majeur dans l'établissement de la polarité et le positionnement du lumen pendant la première division cellulaire. Au niveau moléculaire, Rab35 permet de coupler l'initiation de la polarité apico-basale avec la cytocinèse via l'attachement au sillon de clivage de vésicules intracellulaires contenant des déterminants clé de l'établissement de la polarité. Ces vésicules transportent notamment les protéines aPKC, Cdc42, Crumbs3 ainsi que le facteur d'ouverture de la lumière Podocalyxin. De plus, l'attachement de ces vésicules au sillon de clivage dépend de l'interaction directe entre Rab35 et la queue cytoplasmique de Podocalyxin. Par conséquence, l'inactivation de Rab35 entraine une inversion complète de la polarité apico-basale des kystes 3D. J'ai mis en évidence un nouveau mécanisme de ciblage des vésicules intracellulaire au site de clivage dépendant de la protéine Rab35 impliqué à la fois dans l'initiation de la polarité apico-basale et dans l'ouverture de la lumière au centre du cyst

Rab35 GTPase et initiation de la polarité apico-basal dans un modèle cellulaire 3D

L'établissement de la polarité apico-basale dans les tissus épithéliaux est étroitement lié à la division cellulaire, mais les mécanismes moléculaires sous-jacents n'ont pas encore été établis. A l'aide d'un modèle de culture en 3 dimensions de cellules rénales (MDCK), j'ai montré que lors du développement d'un cyst, la GTPase Rab35 joue un rôle majeur dans l'établissement de la polarité et le positionnement du lumen pendant la première division cellulaire. Au niveau moléculaire, Rab35 permet de coupler l'initiation de la polarité apico-basale avec la cytocinèse via l'attachement au sillon de clivage de vésicules intracellulaires contenant des déterminants clé de l'établissement de la polarité. Ces vésicules transportent notamment les protéines aPKC, Cdc42, Crumbs3 ainsi que le facteur d'ouverture de la lumière Podocalyxin. De plus, l'attachement de ces vésicules au sillon de clivage dépend de l'interaction directe entre Rab35 et la queue cytoplasmique de Podocalyxin. Par conséquence, l'inactivation de Rab35 entraine une inversion complète de la polarité apico-basale des kystes 3D. J'ai mis en évidence un nouveau mécanisme de ciblage des vésicules intracellulaire au site de clivage dépendant de la protéine Rab35 impliqué à la fois dans l'initiation de la polarité apico-basale et dans l'ouverture de la lumière au centre du cyst.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), I 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

Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening.

International audienceEstablishment 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

Rab35-regulated lipid turnover by myotubularins represses mTORC1 activity and controls myelin growth

International audienceInherited 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

Aurora A depletion reveals centrosome-independent polarization mechanism in Caenorhabditis elegans

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

Systemic and Cardiac Depletion of M2 Macrophage through CSF-1R Signaling Inhibition Alters Cardiac Function Post Myocardial Infarction

<div><p>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 Gr1^lo and F4/80^hi 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.</p></div

Oxidation of F-actin controls the terminal steps of cytokinesis

International audienceCytokinetic 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

Effect of CD206+ M2 macrophage depletion on collagen deposition and cell infiltration within the infarct 2 weeks post MI in MAFIA mice.

<p>A. Representative Sirius Red staining on histological sections from MAFIA mice 2 weeks post MI. B. Quantification of collagen staining as a percentage of LV from MAFIA mice 2 weeks post MI (Scale bar: 1 mm, n = 6 animals per group, ≥8 images per animal). C. Representative hematoxilin and eosin staining on 20x infarct or remote histological section from MAFIA mice 2 weeks post MI showing an increase in inflammatory infiltrates in animals treated with GW2580. D. Quantification of nuclei number per mm² in MAFIA remote and infarct zone in MAFIA mice 2 weeks post MI. (n = 4–6 animals per group). E. Representative images of immunofluorescent staining of CD206+ M2 macrophages, Gr1+ M1 macrophages and Ly6G+ neutrophils within the infarct zone from MAFIA mice 2 weeks post MI. Quantification of Ly6G+ neutrophil, Gr1+ M1 macrophage and CD206+ M2 macrophage infiltration within the infarct zone from MAFIA mice 2 weeks post MI (n = 4 animals per group, 10 images/animal). * <i>p</i><0.05, ** <i>p</i><0.01.</p

Depletion of the circulating monocyte and Gr1^lo and F4/80^hi populations following 1 week GW2580 treatment.

<p>A. Identification of total monocyte population in mouse blood using MAFIA-GFP. B Following one week of GW2580 treatment, no difference in total circulating monocytes was observed. FACS quantification of Gr1^hi (M1) and Gr1^lo (M2; C,D) and F4/80^hi (E,F) in total monocytes following 1 week GW2580 treatment (n = 6 and n = 4 animals per group, respectively).</p

CD206+ M2 macrophage depletion reduces LV ejection fraction but not infarct size 2 weeks post MI in MAFIA mice.

<p>A. mRNA expression relative to GAPDH of M1 markers IL6, IL1B, and (b) M2 markers Arg1 and CD206 respectively within the heart 2 weeks post MI (n = 4–6 animals per group). C. Determination of ejection fraction based on pressure-volume loop measurements vehicle or GW2580-treated MAFIA mice 2 weeks post MI. (* <i>p</i><0.05. n = 11–13 animals per group). D. Upper panels: representative MAFIA heart sections after TTC staining. Middle panels: representative MAFIA heart section under fluorescent microscope allowing the detection of coloured microspheres distributed in the perfused area. Lower panels: Infarct areas are represented as white, non-perfused area-at risk (AAR) of infarction (dark red) and perfused areas (blue) are highlighted. The area at risk corresponds to the non-perfused area. E. AAR/LV ratio quantification in MAFIA mice 2 weeks post MI using coloured microspheres. F. IZ/LV ratio quantification in MAFIA mice 2weeks post MI using TTC staining and coloured microspheres (* <i>p</i><0.05. n = 11–13 animals per group).</p