I. Deciphering Lysosome Biogensis
Lysosomes are dynamic structures that undergo cycles of fission and fusion with each other and with late endosomes. Lysosome biogenesis, also referred to as lysosome re-formation, occurs when nascent lysosomes “bud” off hybrid organelles (fusions of lysosomes and late endosomes). The nascent lysosomes extend away from the hybrid organelles they bud from while remaining connected to them by membranes and a shared lumen; fission of the connecting membranes releases the nascent lysosome forming a primary lysosome; this process requires the release of intra-organellar Ca2+. We identified C. elegans CUP-5 as the first protein required for lysosome biogenesis. CUP-5 is the Caenorhabditis elegans orthologue of human TRPML1, a cation channel protein whose loss/perturbation results in the lysosomal storage disorder Mucolipidosis type IV. While C. elegans is the ideal system for sophisticated genetic studies and in vivo analyses of cell biological processes, elucidating mechanisms that regulate lysosome biogenesis requires live imaging. We initiated studies in RAW264.7 cells because they have elaborate lysosomal transport thus facilitating the analysis of lysosome biogenesis. We showed that TRPML1 localizes to nascent lysososomes and lysosomes and that RNAi knockdown of TRPML1 levels results in several lysosomal defects. This provided the foundation for live imaging studies that we are doing to get a dynamic view of lysosome biogenesis and the functions of TRPML1 and other proteins in this process. We have taken two approaches to identify other proteins that function in lysosome biogenesis. First, we identified several proteins that physcially associate with mouse TRPML1. Among the interactors are Rac2, Cdc42, and Phosphatidylinositol 4-phosphate 5-kinase type I, known regulators of actin dynamics. Interestingly, RAB-2, a C. elegans homologue of mammalian Rac2, was shown by another group to be required for lysosome biogenesis. Second, we identified three new lysosome biogenesis genes, cup-15, cup-16, and cup-17 using mutagenesis screens in C. elegans. We will clone and define how these three proteins function with CUP-5 and RAB-2 to regulate lysosome biogenesis using parallel studies in C. elegans and mammalian cells.
Figure I. a.
II. Understanding Basis of Lysosomal Defects and Cell Death in Mucolipidosis type IV
C. elegans cup-5 provides an excellent in vivo model of Mucolipidosis type IV. cup-5 mutants show the same defects as human Mucolipidosis type IV patients, and all of these defects are rescued by expression of the human MCOLN1 gene. We had shown that C. elegans cup-5 mutants show general lysosomal dysfunction in most cells and tissue-specific death, primarily of developing intestinal cells in embryos; 100% of cup-5 mutant embryos fail to hatch and die. We identified the first extrageneic suppressor of cup-5 mutant lethality; mrp-4 mutations almost fully suppress the lethality and the lysosomal defects of developing intestinal cells. MRP-4 is an endosomal ATP-Binding Cassette Transporter whose levels are elevated in cup-5 mutants. We have now identified additional strong extragenic suppressors of cup-5 mutant lethality (>85% rescue). Our studies will lead to two major innovations. First, our studies are deciphering the pathway that leads to tissue degeneration in the absence of CUP-5/TRPML1. Thus we are identifying promising leads for the development of therapies for the treatment of Mucolipidosis type IV and potentially of other diseases with lysosomal pathologies. Second, we are identifying the biochemical links between CUP-5 and downstream efectors that are defining novel pathways of endosomal/lysosomal regulation