Rab35 promotes the recruitment of Rab8, Rab13 and Rab36 to recycling endosomes through MICAL-L1 during neurite outgrowth

Abstract

Small GTPase Rab35 is an important molecular switch for endocytic recycling that regulates various cellular processes, including cytokinesis, cell migration, and neurite outgrowth. We previously showed that active Rab35 promotes nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells by recruiting MICAL-L1, a multiple Rab-binding protein, to Arf6-positive recycling endosomes. However, the physiological significance of the multiple Rab-binding ability of MICAL-L1 during neurite outgrowth remained completely unknown. Here we report that Rab35 and MICAL-L1 promote the recruitment of Rab8, Rab13, and Rab36 to Arf6-positive recycling endosomes during neurite outgrowth. We found that Rab35 functions as a master Rab that determines the intracellular localization of MICAL-L1, which in turn functions as a scaffold for Rab8, Rab13, and Rab36. We further showed by functional ablation experiments that each of these downstream Rabs regulates neurite outgrowth in a non-redundant manner downstream of Rab35 and MICAL-L1, e.g. by showing that knockdown of Rab36 inhibited recruitment of Rab36-specific effector JIP4 to Arf6-positive recycling endosomes, and caused inhibition of neurite outgrowth without affecting accumulation of Rab8 and Rab13 in the same Arf6-positive area. Our findings suggest the existence of a novel mechanism that recruits multiple Rab proteins at the Arf6-positive compartment by MICAL-L1.

Intercellular transfer of transferrin receptor by a contact‐, Rab8‐dependent mechanism involving tunneling nanotubes

“…pmCherry and eGFP‐f were purchased from Clontech Laboratories (Palo Ato, CA, USA), and GFP‐EEA1 wild‐type (WT) was a gift from S. Corvera (University of Massachusetts Medical School, Worcester, MA, USA) (Addgene plasmid 42307; http://www.addgene.org). pEGFPC1 Rab8a WT, Rab8a S22N, and Rab8a Q70L were kindly provided by M. Fukuda (Tohoku University, Sendai, Japan) (24). The pEGFP‐EPS15 EH29 mutant was a kind gift from A. Benmerah (Imagine Institute, Paris, France).…”

Recycling endosomes

“…MICAL-L1 recruits both Arf6 and EHD1 to recycling membranes and promotes tubulation [7,9,10 • ,11]. The association of MICAL-L1 with tubular endosomes is regulated by Rab35 [8,12]. These studies suggest that regulation of the morphology of the endosomal recycling membranes may reflect alterations in trafficking patterns.…”

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

“…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.…”