Intellectual merit. The aim of this project is to decipher mechanisms of lysosome biogenesis. Lysosomes are the main membrane-bound, degradative organelles of eukaryotic cells. In addition to degrading molecules taken up from the extracellular medium by endocytosis, lysosomes regulate multiple essential cellular processes, including normal turnover of components of cellular organelles, degradation of cellular material in response to starvation, degradation of signaling receptors as needed to regulate developmental pathways, fusion with the plasma membrane to initiate wound healing, and participation in some cell death pathways. Given this central importance of lysosomes, it is striking that little is known about basic principles of how lysosomes are formed. Studies have shown that lysosomal biogenesis proceeds by budding of a membrane extension from the endosomal compartment followed by vesicle scission to release a newly formed lysosome. Preliminary studies for this project identified the TRPML1 protein as an initial regulator of lysosome biogenesis. The studies showed that this protein, which has also been shown to be a channel protein that is dysfunctional in lysosomal storage disorders, is required for the membrane extension and scission steps. Based on protein interactor analyses and drug inhibition studies, a model of lysosome biogenesis was proposed in which TRPML1 recruits proteins following budding that initiate actin fiber formation at the sites of lysosome biogenesis. TRPML1 channel activity then promotes actin polymerization/force generation that leads to membrane extension and eventually scission. This project tests this model and will establish the first detailed mechanistic view of lysosomal biogenesis.
Broader impacts. These studies will transform current views of membrane transport by establishing the first molecular model of lysosomal biogenesis that likely will lead to new understanding of both lysosomal and other transport processes. In addition, this research program will rely heavily on direct undergraduate participation to conduct the experiments for which the PI will provide a mentorship role. This faculty member will continue training efforts, which in the past nine years have involved twenty undergraduate students, including thirteen from under-represented groups. As in the past, the majority of these students will likely continue in graduate programs, will present the results of this research at conferences, and will be co-authors on peer-reviewed publications. The research program will be used as a platform for the scientific development of the next generations of scientists and health professionals from this institution.
Intellectual Merit: Lysosomes are the major membrane-bound degradative organelles in eukaryotic cells. Lysosomes degrade material internalized from the extracellular space in addition to autophagic material, thus providing nutrients and metabolites that are essential for cell growth and function. In addition to this metabolic function, lysosomes function in healing damage to the plasma membrane and participate in some pathways of cell death. Despite this essential function of lysosomes, nothing is known about the origin of these organelles, how they are formed. Our studies in live mouse cells showed that lysosomes form from parent organelles (endosomes) by budding. These buds, the nascent lysosomes, extend directionally away from parent organelles while remaining connected to the parent organelles by a membrane bridge. At an average of 16.8 +/- 4.7 seconds after the nascent lysosome buds off the parent organelle, the membrane bridge ruptures releasing lysosomes; this rupture occurs at an average distance of 0.45 +/- 0.26 μm between the nascent lysosomes and the parent organelles. This result is the first qualitative and quantitative demonstration of lysosome formation. We also showed that the cation channel protein TRPML1 is required for the directional movement of nascent lysosomes away from parent compartments and for the scission of the membrane connecting nascent lysosomes to parent compartments. TRPML1 is the protein that is defective in the lysosomal storage disorder Mucolipidosis type IV. Thus TRPML1 is the first known regulator of lysosome formation that does not function in nascent lysosome formation but rather regulates the scission of the membrane connecting nascent lysosomes to parent compartments. Our future studies will focus on identifying additional regulators of lysosomes formation; indeed, as part of this study, we identified several candidate proteins that physically associate with TRPML1. Our studies will lead to the first molecular model of lysosome formation. Broader Impacts: Our studies are transforming the field of lysosome biology by providing a molecular mechanism for lysosome formation. This will impact many fields of biology, including studies on endocytosis, autophagy, cell signaling, wound healing, and cell death. In addition, our studies will allow a more in depth analysis of the defects in ~50 human diseases referred to as lysosomal storage disorders. Undergraduate students perform most of the studies in my laboratory. I use my research as a vehicle to train undergraduate students and to retain them in research and teaching careers. In the past eleven years as a faculty member, I have had one research specialist, seven rotation graduate students, one graduate student, two postdoctoral scientists, and 31 undergraduate student researchers who remain in my laboratory for an average of two years, including summers when funds are available. I directly mentor all of my undergraduate students such that I personally supervise their research and assist them in preparing for their future careers. The 31 undergraduate students include fifteen female and five minority students (three males, two female). The 31 undergraduate students (only counting students who stayed in my laboratory for more than one year) include 16 female and 6 minority students (four males, two females). Of the 31 undergraduate student researchers, 7 are currently in my laboratory. Of the 24 students who have graduated, one is working as a research specialist, one is pursuing a career as a social worker/counselor, and the remaining 22 (91%; 12 female, 5 minority) are in or have completed graduate/medical studies in science or in health-related fields. Thus, the majority of my students continue their graduate studies in science or in health-related fields.
