All Publications

  • LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade. Life science alliance Dhekne, H. S., Yanatori, I., Vides, E. G., Sobu, Y., Diez, F., Tonelli, F., Pfeffer, S. R. 2021; 4 (5)


    Activating mutations in LRRK2 kinase causes Parkinson's disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va's role in ciliogenesis.

    View details for DOI 10.26508/lsa.202101050

    View details for PubMedID 33727250

  • Rab29 Fast Exchange Mutants: Characterization of a Challenging Rab GTPase. Methods in molecular biology (Clifton, N.J.) Gomez, R. C., Vides, E. G., Pfeffer, S. R. 2021; 2293: 19-25


    Rab29 has been implicated in multiple membrane trafficking processes with no described effectors or regulating proteins. Its fast nucleotide exchange rate and inability to bind GDI in cytosol make it a unique and poorly understood Rab. Because the conventional, "GTP-locked" Rab mutation does not have the desired effect in Rab29, we present here the use of a fluorescence-based assay to characterize novel Rab29 mutants (I64T and V156G) that display faster nucleotide exchange rates, allowing for GEF-independent Rab29 activation.

    View details for DOI 10.1007/978-1-0716-1346-7_2

    View details for PubMedID 34453707

  • Endogenous Rab29 does not impact basal or stimulated LRRK2 pathway activity. The Biochemical journal Kalogeropulou, A. F., Freemantle, J. B., Lis, P., Vides, E. G., Polinski, N. K., Alessi, D. R. 2020


    Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinson's disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinson's, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans Golgi network. Here we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on assessment of Rab10 and Rab12 phosphorylation, in wildtype LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that stimulation of Rab10 and Rab12 phosphorylation induced by agents that stress the endolysosomal system (nigericin, monensin, chloroquine, and LLOMe) was not blocked in Rab29 deficient mouse embryonic fibroblasts. From the agents tested, nigericin induced the greatest increase in pRab10 and pRab12 phosphorylation (5-9 fold). Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes.

    View details for DOI 10.1042/BCJ20200458

    View details for PubMedID 33135724