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. The Va phenotype results from a punctual mutation (A419P) in the pore region of MCOLN3 that locks the channel in an open conformation causing massive entry of calcium inside cells and inducing cell death by apoptosis. Overexpression of wild-type 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. To understand better the physiological role of MCOLN3, we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3 and measure several endosomal parameters including luminal calcium, pH, and endosomal fusion. We found impairment of MCOLN3 activity caused a significant accumulation of luminal calcium at endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating calcium homeostasis at the endosomal pathway and confirm 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 first 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. Overall, our study reveals interesting differences in the regulation and distribution of the members of the MCOLN family and identifies a novel role for MCOLN2 in the innate immune response. The endolysosomal pathway, and in particular MCOLN1 and MCOLN3, play also an important role regulating the rate of autophagosome degradation. Recent evidence suggests that the elimination of damaged cellular components mediated by autophagy contributes to the progression of certain tumors. In collaboration with the laboratory of Dr. Liqiang Chen, we have characterized two novel anticancer compounds that showed antiproliferative activity in pancreatic ductal adenocarcinoma cells by disrupting autophagy flux and reducing mTORC1 activity.
