Regulation of the Endo/Lysosomal pathway

National Heart, Lung, And Blood Institute

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Abstract

Mucolipins (or TRPMLs) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3). MCOLN1 is the best-characterized member of the family due to the fact that mutations in this protein are associated with a human disease known as mucolipidosis type IV (MLIV). We and others have shown that the primary role of MCOLN1 in cells is to mediate calcium efflux from late endosomes and lysosomes, thus promoting organelle fusion and regulating endosomal trafficking. Gain-of-function mutation in MCOLN3 causes the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction, and coat color dilution. We showed that overexpression of MCOLN3 produces severe alterations of the endosomal pathway, including enlargement and clustering of endosomes, delayed EGF receptor degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. Inhibition of MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3 caused a significant accumulation of luminal calcium at endosomes, leading to severe defects in endosomal acidification and increased endosomal fusion. Our findings revealed a prominent role for MCOLN1 and MCOLN3 in regulating calcium homeostasis at the endosomal pathway and confirmed the importance of luminal calcium for proper acidification and membrane trafficking. The cellular function of MCOLN2 is far less characterized. To address MCOLN2 function in a physiologically relevant cell type, we analyzed MCOLN2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of MCOLN2 at the transcriptional level. While MCOLN2 expression was negligible in resting macrophages, its mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, MCOLN1 and MCOLN3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous MCOLN2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with MCOLN1 and MCOLN3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of MCOLN2, we generated a MCOLN2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in MCOLN2-knockout mice. Furthermore, MCOLN2-knockout mice displayed impaired recruitment of peripheral macrophages in response to intra peritoneal (IP) injections of LPS and live bacteria, suggesting a potential defect in the immune response. These observations were further expanded in a collaboration with the laboratory of Dr. Christian Grimm from the Ludwig-Maximilians-Universität in Germany, confirming treatment with ML2-SA1, a novel MCOLN2-specific agonist, increased CCL2 secretion in LPS-stimulated macrophages and promoted migration. MCOLN2 functions as an hypotonicity/mechanosensitive endolysosomal cation channel that enhances fast recycling and secretion pathways in activated macrophages. Furthermore, a recent collaborative study with the group of Dr. Denis Ko (Duke University) showed a key function of MCOLN2 in nutritional immunity against Salmonella enterica serovar Typhi (S. Typhi). By performing cellular genome-wide association analysis of intracellular replication by S. Typhi in nearly a thousand cell lines from around the world, as well as extensive follow-up using intracellular S. Typhi transcriptomics and manipulation of magnesium availability, we demonstrated that MCOLN2 restricts S. Typhi intracellular replication through magnesium deprivation. Overall, these studies reveal interesting differences in the regulation and distribution of the members of the MCOLN family and corroborates the critical role of MCOLN2 in the innate immune response. Recent evidence suggests that lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Moreover, alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. We have identified a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. These data revealed that the TFEB/TMEM55B/JIP4 axis coordinates lysosome movement in response to nutrient deprivation. Our follow-up studies revealed an important function of TMEM55B in response to oxidative stress. We found that TMEM55B mediates NEDD4-dependent PLEKHM1 ubiquitination, causing PLEKHM1 proteasomal degradation and halting autophagosome/lysosome fusion. TMEM55B also promotes recruitment of components of the ESCRT machinery to lysosomal membranes to stimulate lysosomal repair. Finally, TMEM55B sequesters the FLCN/FNIP complex to facilitate translocation of the transcription factor TFE3 to the nucleus, allowing expression of transcriptional programs that enable cellular adaptation to stress. Knockout of tmem55 genes in zebrafish embryos increases their susceptibility to oxidative stress, causing early death of tmem55-KO larvae in response to arsenite toxicity. This work identifies a novel role for TMEM55B as a molecular sensor that coordinates autophagosome degradation, lysosomal repair, and activation of stress responses. More recently we are addressing how lysosomal positioning may influence the formation and stability of lysosome-mitochondria contact sites and how these interactions affect mitochondria quality control pathways and cellular homeostasis both in normal and pathological conditions.