Recycling endosomes are stations that sort endocytic cargoes to their appropriate destinations. Tubular endosomes have been characterized as a recycling endosomal compartment for clathrinindependent cargoes. However, the molecular mechanism by which tubular endosome formation is regulated is poorly understood. In this study, we identified Rab10 as a novel protein localized at tubular endosomes by using a comprehensive localization screen of EGFPtagged Rab small GTPases. Knockout of Rab10 completely abolished tubular endosomal structures in HeLaM cells. We also identified kinesin motors KIF13A and KIF13B as novel Rab10interacting proteins by means of in silico screening. The results of this study demonstrated that both the Rab10-binding homology domain and the motor domain of KIF13A are required for Rab10-positive tubular endosome formation. Our findings provide insight into the mechanism by which the Rab10-KIF13A (or KIF13B) complex regulates tubular endosome formation. This article has an associated First Person interview with the first author of the paper.
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.
In lymphocytes, the kinase Mst1 is required for the proper organization of integrins in the plasma membrane at the leading edge of migrating cells, which is critical for lymphocyte trafficking. We found a functional link between the small G protein Rab13 and Mst1 in lymphocyte adhesion and migration. In response to stimulation of T lymphocytes with chemokine, Mst1 promoted phosphorylation of the guanine nucleotide exchange factor DENND1C (differentially expressed in normal and neoplastic cells domain 1C), which activated Rab13. Active Rab13 associated with Mst1 to facilitate the delivery of the integrin LFA-1 (lymphocyte function-associated antigen 1) to the leading edge of lymphocytes. Delivery of LFA-1 involved the recruitment of myosin Va along actin filaments, which extended as a result of the localization of the actin regulatory protein VASP to the cell periphery through phosphorylation of VASP at Ser(157) by Mst1. Inhibition of Rab13 function reduced the adhesion and migration of lymphocytes on ICAM-1 (intercellular adhesion molecule-1), the ligand for LFA-1, and inhibited the formation of a ring-like arrangement of LFA-1 at the contact sites between T cells and antigen-presenting cells. The lymphoid tissues of Rab13-deficient mice had reduced numbers of lymphocytes because of the defective trafficking capability of these cells. These results suggest that Rab13 acts with Mst1 to regulate the spatial distribution of LFA-1 and the motility and trafficking of lymphocytes.
Background: Centaurin-2 specifically recognizes Rab35 but not the other 59 Rabs. Results: The T76S/T81A mutation in the switch II region of Rab35 specifically impaired its centaurin-2 binding activity. Conclusion: Thr-76 and Thr-81 are key residues of Rab35 for the specific recognition by centaurin-2. Significance: Rab35(T76S/T81A) should be a useful tool for evaluating the functional significance of the Rab35/centaurin-2 interaction in Rab35-dependent cellular events.
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