Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.

The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking.

Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1-infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1-infected macrophages, leading to strong alterations in phagolysosome biogenesis.

MICAL-like1 in endosomal signaling.

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates tight junction assembly and polarized membrane transport in epithelial cells. Using yeast two-hybrid screen, we identified MICAL-like1 (MICAL-L1), a protein that interacts with GTP-bound Rab13 and shares a similar domain organization with MICAL protein family. MICAL-L1 has a calponin homology, Lin11, Isl-1 & Mec-3 (LIM), proline-rich, and coiled-coil domains. It is associated with late and recycling endosomes. Time-lapse video microscopy shows that GFP-Rab7 and cherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of tight junction proteins, but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in late endosomal compartments. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data show that MICAL-L1 inhibits EGFR degradation, suggesting that MICAL-L1 is involved in sorting/targeting the receptor to the recycling pathway. They provide novel insights into MICAL-L1/Rab protein complex that can regulate EGFR trafficking/signaling.

MICAL-like1 mediates epidermal growth factor receptor endocytosis.

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.

Tight junction: a co-ordinator of cell signalling and membrane trafficking.

Increasing evidence indicates that the tight junction plays a role in membrane transport. Various signalling and trafficking molecules localize to the sites of cell-cell junctions in epithelial cells, including Rab proteins, a family of small GTPases that regulate different steps of vesicular transport along the endocytic and exocytic pathways. We have recently shown that Rab13 controls protein kinase A activity, demonstrating a clear biochemical and functional link between Rab13 and protein kinase A signalling during tight junction assembly in epithelial cells. The present article focuses on how protein kinase A signalling and protein trafficking events could be integrated at tight junctions in epithelial cells.

Assay of Rab13 in regulating epithelial tight junction assembly.

Rab13 is recruited to tight junctions from a cytosolic pool after cell-cell contact formation. Tight junctions are intercellular junctions that separate apical from basolateral domains and are required for the establishment/maintenance of polarized transport in epithelial cells. They form selective barriers regulating the diffusion of ions and solutes between cells. They also maintain the cell surface asymmetry by forming a "fence" that prevents apical/basolateral diffusion of membrane proteins and lipids in the outer leaflet of the plasma membrane. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delays the formation of electrically tight epithelial monolayers, induces the leakage of small nonionic tracers from the apical domain, and disrupts the tight junction fence diffusion barrier. It also alters the tight junction strand structure and delays the localization of the tight junction transmembrane protein, claudin1. In contrast, the inactive Rab13T22N mutant does not disrupt tight junction functions, tight junction strand architecture, or claudin1 localization. Here we describe a set of assays that allows us to investigate the role of Rab13 in modulating tight junction structure and function.

Properties of Rab13 interaction with protein kinase A.

The small GTPase Rab13 localizes to tight junctions in epithelial cells and regulates the recruitment of claudin1 and ZO-1, two proteins required for the assembly of functional tight junctions. Rab13 directly binds to the alpha-catalytic subunit of protein kinase A (PKA alpha cat) and reversibly inhibits PKA-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key actin cytoskeletal remodeling protein. The inhibition of VASP phosphorylation abolishes the targeting of VASP to cell-cell junctions, which in turn leads to a delay in the recruitment of claudin1 and ZO1 into tight junctions. Consequently, tight junctions formed in epithelial cells expressing the GTP-bound Rab13 are structurally disorganized and functionally leaky for small molecules (A. M. Marzesco et al. [2002]. Mol. Biol. Cell13, 1819-1831; K. Kohler et al. [2004]. J. Cell Biol. 165, 175-180). Our data provide the first direct link between activation of small GTPases and the recruitment of cytoskeletal modulators into tight junctions. Here, we describe different procedures we used to demonstrate that Rab13 interacts with PKA and reversibly controls phosphorylation and recruitment of VASP.

[Tight junctions, a platform regulating cell proliferation and polarity]. / Les jonctions serrées: plate-forme de régulation de la prolifération et de la polarité cellulaires.

Tight junctions (TJ) are specialized plasma membrane microdomains that encircle the apical pole of each epithelial and endothelial cell, separating apical from basolateral side. They form an intercellular diffusion barrier (or gate) regulating the passage of ions, water, and various macromolecules through the paracellular spaces, and a fence restricting the apical/basolateral diffusion of membrane proteins and lipids. This latter function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Several TJ proteins are involved in organizing signal transduction at TJ. For example, the interaction of TJ protein ZO-1 (zonula occludens 1) with the transcription factor ZONAB (ZO-1 associated nucleic acid protein) is important in the regulation of expression of the proto-oncogene Erb-2 as well as epithelial proliferation/differentiation. Recent studies indicate that different proteins involved in membrane trafficking are associated with tight junctions. Among these proteins are the small G-proteins of the Rab family that regulate specific membrane transport events in both endocytic and exocytic pathways. Rab proteins may coordinate the recruitment of protein complexes necessary for the establishment-maintenance of cell polarity. Based on the studies reviewed here, tight junctions emerge as a platform used to coordinate and regulate cell polarity, and proliferation/differentiation.

Rab13 regulates PKA signaling during tight junction assembly.

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell-cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells.

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.