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. 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 reveal that the TFEB/TMEM55B/JIP4 axis coordinates lysosome movement in response to a variety of stress conditions.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
U.S. National Heart Lung and Blood Inst
Zip Code
Choy, Christopher H; Saffi, Golam; Gray, Matthew A et al. (2018) Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence. J Cell Sci 131:
Willett, Rose; Martina, José A; Zewe, James P et al. (2017) TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes. Nat Commun 8:1580
Ai, Teng; Willett, Rose; Williams, Jessica et al. (2017) N-(1-Benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides: Antiproliferative Activity and Effects on mTORC1 and Autophagy. ACS Med Chem Lett 8:90-95
Shang, Peng; Valapala, Mallika; Grebe, Rhonda et al. (2017) The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling. Aging Cell 16:349-359
Sun, Lu; Hua, Yinan; Vergarajauregui, Silvia et al. (2015) Novel Role of TRPML2 in the Regulation of the Innate Immune Response. J Immunol 195:4922-32
Abe, Ken; Puertollano, Rosa (2011) Role of TRP channels in the regulation of the endosomal pathway. Physiology (Bethesda) 26:14-22
Lelouvier, Benjamin; Puertollano, Rosa (2011) Mucolipin-3 regulates luminal calcium, acidification, and membrane fusion in the endosomal pathway. J Biol Chem 286:9826-32
Puertollano, Rosa; Kiselyov, Kirill (2009) TRPMLs: in sickness and in health. Am J Physiol Renal Physiol 296:F1245-54
Martina, Jose A; Lelouvier, Benjamin; Puertollano, Rosa (2009) The calcium channel mucolipin-3 is a novel regulator of trafficking along the endosomal pathway. Traffic 10:1143-56