Lysosomes provide a niche for molecular digestion and are a convergence point for endocytic trafficking, phagosome maturation and autophagy. Typically, lysosomes are small, globular organelles that appear punctate under the fluorescence microscope. However, activating agents like phorbol esters transform macrophage lysosomes into tubular lysosomes (TLs), which have been implicated in retention of pinocytic uptake and phagosome maturation. Moreover, dendritic cells exposed to lipopolysaccharides (LPSs) convert their punctate class II major histocompatibility complex compartment, a lysosome-related organelle, into a tubular network that is thought to be involved in antigen presentation. Other than a requirement for microtubules and kinesin, little is known about the molecular mechanisms that drive lysosome tubulation. Here, we show that macrophage cell lines readily form TLs after LPS exposure, with a requirement for the Rab7 GTPase and its effectors RILP (Rab7-interacting lysosomal protein) and FYCO1 (coiled-coil domain-containing protein 1), which respectively modulate the dynein and kinesin microtubule motor proteins. We also show that Arl8B, a recently identified lysosomal GTPase, and its effector SKIP, are also important for TL biogenesis. Finally, we reveal that TLs are significantly more motile than punctate lysosomes within the same LPS-treated cells. Therefore, we identify the first molecular regulators of lysosome tubulation and we show that TLs represent a more dynamic lysosome population.
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"These structures were induced by FLCN/FNIP2 expression as we did not observe them in control cells stained for Rab7. Although we did not observe striking FLCN/FNIP2 induced perinuclear clustering in these conditions, morphologically and dynamically similar tubules have been observed in macrophages following LPS stimulation and require the activity of several lysosome associated small GTPases, effector and motor proteins  and so are consistent with our hypothesis that FLCN/FNIP have an intrinsic capacity to impact upon lysosome membrane dynamics . "
"Among these, microtubules appear to provide a scaffold along which the plus end-directed motor protein kinesin-1 and the minus end-directed motor dynein act in concert to 'stretch' lysosomes, though this likely requires alterations in membrane trafficking as well. These motors link to lysosomes through the small GTPases Rab7 and Arl8b and their effectors; Rab7 recruits RILP and FYCO1 to respectively link to dynein and kinesin-1, while Arl8b links to kinesin-1 through its effector SKIP (Cantalupo et al., 2001; Jordens et al., 2001; Pankiv et al., 2010; Rosa-Ferreira and Munro, 2011; Mrakovic et al., 2012). However, it remains unclear how LPS-dependent signaling interfaces with the microtubule-dependent motors to modulate lysosome morphology and identity. "
[Show abstract][Hide abstract]ABSTRACT: Macrophages and dendritic cells exposed to lipopolysaccharides (LPS) convert their lysosomes from small punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid-phase and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex-II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation, independently of IRAK1/4 and TBK. Notably, we found that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation and that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we showed that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but had no impact in retrograde movement upon acetate removal. Overall, our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.
Full-text · Article · Nov 2015 · Molecular Biology of the Cell
[Show abstract][Hide abstract]ABSTRACT: The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knockout mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knockout mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.
No preview · Article · Mar 2013 · Journal of Biological Chemistry