Arf-like GTPase─Arl8b: a key regulator of lysosome/lysosome-related organelle motility and exocytosis

Lysosomes are fundamental organelles that serve as the major site of degradation of macromolecules delivered via endocytic, autophagic and phagocytic membrane-trafficking pathways. However, the function of lysosomes is not restricted to protein degradation. In certain cell types lysosomes acquire more specialized secretory functions, collectively termed as Lysosome-related organelles (LROs)/secretory lysosomes, and undergo regulated exocytosis in response to an external stimulus. Examples of LROs include melanosomes in melanocytes, lytic granules in natural killer (NK) cells and cytolytic T lymphocytes, and granules in mast cells, platelets, basophils and neutrophils. Abnormalities in lysosomes/LROs secretion have been observed in many human genetic diseases like Chediak-Higashi Syndrome, Hermansky-Pudlak Syndrome, Griscelli Syndrome, Familial Haemophagocytic Lymphohistiocytosis, platelet deficiency and lung fibrosis. However, the molecular mechanism(s) controlling lysosomal motility and exocytosis are still not known. Specifically, what proteins mediate the motility of lysosomes/LROs on microtubule network; what enables lysosomes to take on the dual role of secretory granule and degradative organelle; and to what extent are these processes similar and different between different cell types. To identify molecular mediators of lysosome exocytosis, we carried out a shRNA screening using a preassembled library of shRNAs targeting all known endocytic regulatory genes and identified a novel member of the Ras superfamily of small GTPases, Arf-like GTPase (Arl)-8b, as an essential regulator of lysosome exocytosis. We found that knockdown of Arl8b expression led to clustering of lysosomes at the cell center, while its over expression led to peripheral distribution of lysosomes. Based on these observations, we hypothesized that Arl8b regulates motility and exocytosis of lysosomes/LROs which is required to mediate various physiological events including target-cell killing by NK cells and repair of damaged plasma membrane (PM) during cell injury. We found endogenous Arl8b co-localized with perforin and granzyme containing lytic granules in both ex vivo NK cells and immortalized NK cell lines. Moreover, Arl8b-deficient NK cells clearly demonstrated a significant loss of cytolytic activity compared with control cells. To gain insight into the mechanism of Arl8b action, we assessed which step during lytic granule exocytosis is impaired upon Arl8b knockdown. It was previously reported that the microtubule organizing centre (MTOC) translocates towards the immunological synapse (IS) as it matures. This reorientation is essential for the movement and polarization of the granules to the site of release. We examined the role of Arl8b in these processes using Arl8b-deficient cells. Interestingly, compared to the control knockdown, in NK cells lacking Arl8b, lytic granules and MTOC failed to polarize at the IS and remained almost at the opposite pole to that of the IS. In addition to lytic granules, we also analyzed Arl8b role in regulating the motility and exocytosis of conventional lysosomes during the process of PM repair. Similar to our findings with lytic granules, conventional lysosomes also showed significantly reduced exocytosis upon treatment with ionomycin, an ionophore commonly used to induce PM repair response. Taken together, our findings suggest Arl8b to be a critical factor required for motility and exocytosis of lysosomes and lysosomes-related organelles.