An ARF6/Rab35 GTPase Cascade for Endocytic Recycling and Successful Cytokinesis

Abstract

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.

Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells

“…In our preliminary experiments, the Rab35 SR (T76S/T81A) mutant was unable to rescue the multinucleation phenotype of HeLaS3 cells induced by Rab35 siRNA (15). 3 Because Arf6 is also known to be required for cytokinesis (16), there is a strong possibility that centaurin-␤2-mediated functional cross-talk between Rab35 and Arf6 occurs in cytokinesis. Investigation of the possible functional involvement of centaurin-␤2 in cytokinesis is now underway in our laboratory.…”

“…Rab35 is one such Rab protein and has been shown to bind various candidate effector molecules, including MICAL-1 (7), MICAL-L1 (7,9,10), MICAL-cl (7), OCRL (7,11), RUSC2 (12), Fascin1 (13), and centaurin-␤2 (8,14), and to be involved in various cellular events, including cytokinesis (11,15,16), cell migration (17,18), phagocytosis (19,20), immunological synapse formation (21), myelination (22), and neurite outgrowth (10,14,23,24), most likely through regulation of endocytic recycling (25). The pleiotropic roles of Rab35 in membrane trafficking may be attributable to the presence of multiple Rab35 effectors, but the involvement of individual Rab35 effectors in the above cellular events has remained largely unknown.…”

RAB-10-GTPase–mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate

“…In HeLa cells, Chesneau et al (38) showed that Arf6-GTP can interact with Rab35 GAP EPI64B, negatively regulating Rab35 activity during cytokinesis. Together with our results, these observations provide compelling evidence that Rab-to-Arf and Arf-to-Rab regulatory loops represent a general mechanism for the coordinate regulation of Rabs and Arfs during membrane trafficking.…”

Interactions between Rab and Arf GTPases regulate endosomal phosphatidylinositol-4,5-bisphosphate during endocytic recycling

“…In cultured human cells, Arf6(GTP) can interact with the Rab35 GAP EPI64B, negatively regulating Rab35 activity. 32 This work indicates that Arf6-to-Rab35 regulation is required for endocytic pathways important for cytokinesis. Taken together, the discoveries of Rab-to-Arf and Arf-to-Rab regulatory cascades suggest that signaling between these classes of small GTPases represent an evolutionally conserved mechanism that coordinates remodeling capability.…”

“…15,[30][31][32] Our studies provide an example of a Rab-toArf regulatory cascade, with RAB-10 and ARF-6 acting in sequential steps to regulate endosomal PI(4,5)P2 levels, thus regulating endosomal recruitment of PI(4,5)P2-binding proteins implicated in membrane bending and membrane fission. In addition to the Rab-to-Arf regulatory loops, an Arf-to-Rab regulatory cascade has also been recently reported.…”