Rab7b regulates dendritic cell migration by linking lysosomes to the actomyosin cytoskeleton.

Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MCOLN1), enabling the local activation of myosin II at the cell rear. Taken together, our findings identify Rab7b as the missing physical link between lysosomes and the actomyosin cytoskeleton, allowing control of immune cell migration through lysosomal signaling. This article has an associated First Person interview with the first author of the paper.

Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity.

Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.

TBC1D5 controls the GTPase cycle of Rab7b.

Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various in vitro and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.This article has an associated First Person interview with the first author of the paper.

Rab and Arf proteins at the crossroad between membrane transport and cytoskeleton dynamics.

The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.

Rab7b modulates autophagic flux by interacting with Atg4B.

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.

L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes.

L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.

Spatiotemporal Resolution of Rab9 and CI-MPR Dynamics in the Endocytic Pathway.

Rab9 is a small GTPase that localizes to the trans-Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose-6-phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation-independent (CI-MPR) away from the Golgi yet, has no effect on the retrograde transport of CI-MPR. We also show that CI-MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. CI-MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI-MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI-MPR to the endosomal pathway, entering the maturing endosome at the early-to-late transition.