PROJECT SUMMARY Lysosomes are essential membrane-bound organelles that play a vital role in cellular degradation and signaling. Damage to lysosomes can be triggered by numerous physiological and pathological conditions, significantly threatening cellular function and survival. Consequently, lysosomal damage is associated with an array of human diseases including cancer, infectious and neurodegenerative diseases, as well as normal aging. Therefore, studying cellular responses to lysosomal damage holds substantial clinical importance. My postdoctoral research provided novel evidence about the molecular aspects of these responses. Additionally, a recent first-author publication, before becoming a Tenure-track Assistant Professor (1/2023), provided the first evidence that lysosomal damage induces the formation of stress granules (SGs). SGs are stress-induced membrane-less organelles believed to protect cells during stress by regulating protein synthesis and prioritizing cellular functions. Although dysfunctional SGs are significantly linked to pathogenic processes of various human diseases, there is a significant knowledge gap regarding the regulation and precise function of SG formation during stress, especially in the context of lysosomal damage. Importantly, recognizing lysosomal damage as a critical internal physiological trigger for SGs highlights the need to better understand the nature of SG formation in disease contexts. Additionally, this novel connection between damaged lysosomes and SGs emphasizes the interplay between membrane-bound and membrane-less organelles, a largely unexplored field of research. Recent preliminary data from my laboratory illustrates that SG formation safeguards lysosomal quality, thereby promoting cell survival. Notably, we also recently published the first evidence that SG-localized proteins can associate with damaged lysosomes, serving additional (largely unknown) functions beyond SG formation. Building on the established platform of seminal work on lysosomal damage, the proposed investigations will focus on two primary goals: (1) Establishing the precise function of SG formation in controlling lysosomal quality, and (2) Determining how lysosomal damage induces SG formation. These goals will be achieved by investigating the function of SGs as organelles and the independent novel activities of SG proteins in controlling lysosomal quality, and defining the molecular mechanisms that transmit signals from lysosomal damage to initiate SG formation, respectively. To complete the two primary goals, the multidisciplinary and diverse team will employ state-of-the-art approaches including high-content microscopy, lysosome immunoprecipitation, proteomic analysis and RNAi screening. Collectively, the investigations will reveal novel insight into how cells respond to lysosomal damage, the precise nature of SG formation, and inter-organelle interactions under stress conditions. These findings have the potential to facilitate in...