Rab7 and Arl8b mediate vesicle transport and fusion with lysosomes. Marwaha et al. show that the Rab7 effector PLEKHM1 competes with PLEKHM2/SKIP for binding to Arl8b and that Arl8 mediates recruitment of the HOPS complex to PLEKHM1-positive vesicles for fusion, suggesting that Arl8b and its effectors orchestrate lysosomal transport and fusion.

Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3'-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the DQ-Red BSA trafficking assay that can be used to study endocytic trafficking in various cell types.

As a sedentary epithelium turns motile during wound healing, morphogenesis, and metastasis, the Golgi apparatus moves from an apical position, above the nucleus, to a basal position. This apical-to-basal repositioning of Golgi is critical for epithelial cell migration. Yet the molecular mechanism underlying it remains elusive, although microtubules are believed to play a role. Using live-cell and super-resolution imaging, we show that at the onset of collective migration of epithelial cells, Golgi stacks get dispersed to create an unpolarized transitional structure, and surprisingly, this dispersal process depends not on microtubules but on actin cytoskeleton. Golgi–actin interaction involves Arp2/3-driven actin projections emanating from the actin cortex, and a Golgi-localized actin elongation factor, MENA. While in sedentary epithelial cells, actin projections intermittently interact with the apically located Golgi, and the frequency of this event increases before the dispersion of Golgi stacks, at the onset of cell migration. Preventing Golgi–actin interaction with MENA-mutants eliminates Golgi dispersion and reduces the persistence of cell migration. Taken together, we show a process of actin-driven Golgi dispersion that is mechanistically different from the well-known Golgi apparatus fragmentation during mitosis and is essential for collective migration of epithelial cells.

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.

Collective cell migration during morphogenesis, cancer metastasis, and wound healing depends on the emergence of leader cells at the migration front. However, the cellular changes that enable only a few cells to become the leader cells, remain elusive. Here we show that the leader cells in Drosophila embryo and mammalian epithelial monolayer emerge through a mechanosensitive relocalization of lysosomes. Before the leader cells display their characteristic lamellipodial protrusions, lysosomes accumulate at their leading periphery. Promoting this lysosome accumulation augments the leader cell emergence, while inhibiting it suppresses the latter. Moreover, experiments modulating cellular forces by chemical inhibition, optogenetics, and micropatterning show that peripheral accumulation of lysosomes depends on the actomyosin contractility. Mechanistically, peripheral lysosomes associate with the inactive form of small RhoGTPase Rac1 and remove it from the vicinity of plasma membrane. Removal of inactive Rac1 molecules leads to an increased Rac1-activity at the leading periphery, triggering actin polymerization and lamellipodium formation. Taken together, lysosome appears as a unique intracellular platform that links mechanical and biochemical signals and thereby controls the emergence of leader cells. We, therefore, discover a previously unknown function of lysosome in collective cell migration, significantly expanding its scope in cell and developmental biology.