PROJECT SUMMARY A hallmark of eukaryotic cells is the ability to compartmentalize essential reactions into membrane-bound and membrane-less organelles. Membrane-bound organelles form collaborative networks through transport vesicles and inter-organellar interaction domains to traffic cargo and distribute lipids throughout the cell. Conversely, membrane-less organelles mobilize the aqueous portion of cells into condensed structures called condensates. The majority of membrane-less condensates enrich for RNAs and various proteins, which control RNA processing and function at nearly every step of the mRNA life cycle. We and others have recently identified a new class of inter-organelle interactions between membrane-bound organelles, such as the endoplasmic reticulum, and membrane-less RNA-sequestering condensates, such as stress granules. Thus, the long-term goal of my research program is to define how and why these two types of organelles interact to gain a more seamless understanding of how the cytoplasm is organized in health and disease. The objective of this proposal is to identify the factors and utilities of interaction domains between the endoplasmic reticulum and two well- studied RNA-sequestering condensates. Our central hypothesis is that the endoplasmic reticulum plays crucial roles in RNA condensate formation, maintenance, and disassembly through the formation of membrane-to- condensate interactions. Although in vitro and in vivo studies on RNA and protein condensation have revealed the some molecular and biophysical principles of condensates, the contribution of membranes to condensate mechanisms are poorly understood. In the first Project, my lab will elucidate how condensate components are recruited to the endoplasmic reticulum to stimulate RNA condensate formation and inhibition of mRNA translation. We will use biochemical and live-cell imaging approaches to identify the key membrane factors that control this new class of interaction domains. These studies will allow us to identify the molecular mechanisms behind our previous discovery of a surprising dependence of membrane-less condensate abundance on endoplasmic reticulum morphology. In the second Project, we will dissect endoplasmic reticulum-dependent mechanisms of stress granule disassembly. Specifically, we will identify new disassembly factors by taking advantage of our newly developed ability to uncouple stress granule fission from dissolution. Additionally, we will test whether increasing the rate of endoplasmic reticulum-mediated stress granule fission can drive the disassembly of disease-associated aggregates. Collectively, this work will reveal how a new cellular niche between membrane-bound and membrane-less organelles drives the dynamic compartmentalization and quality control of RNA processes. Therefore, we anticipate that the proposed work will have important implications for both basic science and translational medicine targeting neurodegeneration, cancer, and RNA v...