Organelle morphology and positioning orchestrate physiological and disease-associated processes.

In cells, organelles are distributed nonrandomly to regulate cells' physiological and disease-associated processes. Based on their morphology, position within the cell, and contacts with other organelles, they exert different biological functions. Endo-lysosomes are critical cell metabolism and nutrient-sensing regulators modulating cell growth and cellular adaptation in response to nutrient availability. Their spatial distribution is intimately linked to their function. In this review, we will discuss the role of endolysosomes under physiological conditions and in the context of cancer progression, with a special focus on their morphology, the molecular mechanisms determining their subcellular position, and the contacts they form with other organelles. We aim to highlight the relationship between cell architecture and cell function and its impact on maintaining organismal homeostasis.

Collective heterogeneity of mitochondrial potential in contact inhibition of proliferation.

In the epithelium, cell density and cell proliferation are closely connected to each other through contact inhibition of proliferation (CIP). Depending on cell density, CIP proceeds through three distinct stages: the free-growing stage at low density, the pre-epithelial transition stage at medium density, and the post-epithelial transition stage at high density. Previous studies have elucidated how cell morphology, motion, and mechanics vary in these stages. However, it remains unknown whether cellular metabolism also has a density-dependent behavior. By measuring the mitochondrial membrane potential at different cell densities, here we reveal a heterogeneous landscape of metabolism in the epithelium, which appears qualitatively distinct in three stages of CIP and did not follow the trend of other CIP-associated parameters, which increases or decreases monotonically with increasing cell density. Importantly, epithelial cells established a collective metabolic heterogeneity exclusively in the pre-epithelial transition stage, where the multicellular clusters of high- and low-potential cells emerged. However, in the post-epithelial transition stage, the metabolic potential field became relatively homogeneous. Next, to study the underlying dynamics, we constructed a system biology model, which predicted the role of cell proliferation in metabolic potential toward establishing collective heterogeneity. Further experiments indeed revealed that the metabolic pattern spatially correlated with the proliferation capacity of cells, as measured by the nuclear localization of a pro-proliferation protein, YAP. Finally, experiments perturbing the actomyosin contractility revealed that, while metabolic heterogeneity was maintained in the absence of actomyosin contractility, its ab initio emergence depended on the latter. Taken together, our results revealed a density-dependent collective heterogeneity in the metabolic field of a pre-epithelial transition-stage epithelial monolayer, which may have significant implications for epithelial form and function.

RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes.

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.

Actin-driven Golgi apparatus dispersal during collective migration of epithelial cells.

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.

DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes.

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) etc., 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.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion.