Unraveling TRPML1 regulation and its role in lysosomal morphology and function

The National Institute of Child Health and Human Development awarded the University of Pennsylvania $100,228 on November 1, 2026, under the Child Health and Human Development Extramural Research program (CFDA 93.865) to support research into TRPML1 regulation and its role in lysosomal morphology and function.

The research addresses knowledge gaps in lysosomal biology, focusing on how the transient receptor potential mucolipin 1 (TRPML1) ion channel regulates lysosomal function and morphology. TRPML1 mediates calcium and cation flux from the lysosomal lumen to the cytosol and plays roles in diseases including Alzheimer's disease, Parkinson's disease, Duchenne muscular dystrophy, and mucolipidosis type IV. The project examines TRPML1 modulation through small molecule modulators PRU-12 and MK6-83, with prior evidence linking PRU-12 to reduced migration and invasion in triple-negative breast cancer cells and MK6-83 to restoration of activity in TRPML1 mutants associated with mucolipidosis type IV.

The award period runs through June 30, 2028. Work is performed in Philadelphia, Pennsylvania.

Abstract

This proposal seeks to address critical knowledge gaps in lysosomal biology. Lysosomes are dynamic organelles essential for metabolic regulation and degradative recycling. Their activity and signaling mechanisms play pivotal roles in maintaining endo-lysosomal flux and cellular homeostasis. As the cell's second-largest calcium store, lysosomes rely on calcium flux for effective signaling and function. Although typically depicted as vesicular, lysosomes can form tubular structures or projections, yet the biological mechanisms and relevance of lysosomal tubulation remain understudied. A growing body of work is revealing that tubular lysosomes exist as heterogeneous populations with diverse morphology, acidity, organellar interactions, and potentially disparate roles. The transient receptor potential mucolipin 1 (TRPML1) ION channel is a lysosomal membrane protein that is critical for regulating lysosomal function and morphology. As a nonselective cation-permeable channel, TRPML1 mediates ION flux, including calcium, from the lysosomal lumen to the cytosol. Emerging evidence links TRPML1 activity to lysosomal tubulation and to diseases characterized by disrupted endo-lysosomal homeostasis, such as alzheimer's and parkinson's disease, duchenne muscular dystrophy, and mucolipidosis type IV (mliv). Despite its significance, the mechanisms by which TRPML1 influences lysosomal tubulation are not fully understood. This proposal focuses on understanding TRPML1 modulation and its role in lysosomal tubulation. The small molecules PRU-12 and MK6-83 were identified as specific TRPML1 modulators. Previous studies demonstrated that the TRPML1 inhibitor PRU-12 reduces migration and invasion of triple-negative breast cancer cells, while the TRPML1 agonist MK6-83 restores activity in two mliv-inducing TRPML1 mutants. Cholesterol is structurally similar to PRU-12, indicating IT may bind TRPML1 in a similar manner. However, the molecular binding mechanisms of these ligands remain unknown. In aim 1, i will characterize the structural interactions between TRPML1 and these modulators using cryo-electron microscopy (cryo-EM) and mutagenesis. This work will provide insights into TRPML1's structural regulation and could aid the development of effective and specific pharmacological modulators. In addition, TRPML1 influences lysosomal tubulation via calcium signaling, which alters the recruitment of motor trafficking complexes that can stretch lysosomal membranes. However, the subpopulations of lysosomal tubules that exist and their dependence on TRPML1 activity remain unclear. In aim 2, i will investigate the influence of TRPML1 in lysosomal tubulation using live- cell imaging, cryo-focused ION beam scanning electron microscopy, and cryo-electron tomography. In summary, this project will advance understanding of TRPML1 structural modulation, elucidate its role in lysosomal morphology, and characterize mechanisms underlying lysosomal tubulation.