Dlg1 controls planar spindle orientation in the neuroepithelium through direct interaction with LGN.

Oriented cell divisions are necessary for the development of epithelial structures. Mitotic spindle orientation requires the precise localization of force generators at the cell cortex via the evolutionarily conserved LGN complex. However, polarity cues acting upstream of this complex in vivo in the vertebrate epithelia remain unknown. In this paper, we show that Dlg1 is localized at the basolateral cell cortex during mitosis and is necessary for planar spindle orientation in the chick neuroepithelium. Live imaging revealed that Dlg1 is required for directed spindle movements during metaphase. Mechanistically, we show that direct interaction between Dlg1 and LGN promotes cortical localization of the LGN complex. Furthermore, in human cells dividing on adhesive micropatterns, homogenously localized Dlg1 recruited LGN to the mitotic cortex and was also necessary for proper spindle orientation. We propose that Dlg1 acts primarily to recruit LGN to the cortex and that Dlg1 localization may additionally provide instructive cues for spindle orientation.

SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation.

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane-actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN-NuMA force generator complexes and hence for proper spindle orientation.

An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis.

Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo. Membrane trafficking is crucial for animal cell cytokinesis, and several endocytic pathways regulated by distinct GTPases (Rab11, Rab21, Rab35, ARF6, RalA/B) contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis. Here, we report that the ARF6 GTPase negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis.

Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.

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.