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

Hotaka Kobayashi,Mitsunori Fukuda,Norihiko Ohbayashi,Kan Etoh

doi:10.1242/bio.20148771

Abstract

ABSTRACTSmall 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.

Highlights

Membrane trafficking, which is a process that transports proteins and lipids from one organelle to another organelle by means of vesicular carriers, is fundamental to various cellular processes, including cell division, signal transduction, and neuronal functions (Maxfield and McGraw, 2004; Grant and Donaldson, 2009; Sorkin and von Zastrow, 2009; Scita and Di Fiore, 2010)
The results showed that MICAL-L1 interacts with constitutively active mutants (Rab8A-Q67L, Rab8B-Q67L, Rab10-Q68L, Rab13-Q67L, Rab15-Q67L, and Rab36-Q116L), but not with their constitutively inactive mutants (Rab8A-T22N, Rab8B-T22N, Rab10-T23N, Rab13T22N, Rab15-T22N, and Rab36-T71N), the same as it recognized active Rab35 (Fig. 1A), suggesting that MICAL-L1 may function as an effector molecule for these other Rabs
Because Rab effectors generally localize to specific intracellular compartments through interaction with their partner Rabs (Stenmark, 2009), we investigated whether these MICALL1-binding Rabs localize at Arf6-positive recycling endosomes in nerve growth factor (NGF)-stimulated PC12 cells, the same as MICAL-L1 does

Summary

Introduction

Membrane trafficking, which is a process that transports proteins and lipids from one organelle to another organelle by means of vesicular carriers, is fundamental to various cellular processes, including cell division, signal transduction, and neuronal functions (Maxfield and McGraw, 2004; Grant and Donaldson, 2009; Sorkin and von Zastrow, 2009; Scita and Di Fiore, 2010). Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan. Because each Rab isoform has a distinct effector-binding property (Fukuda et al, 2008; Kanno et al, 2010), the output of each Rab-signaling is unique and isoform-specific

Methods

Results

Conclusion