Regulation of Endoplasmic Reticulum (ER) Quality Control Across Cell types with Unique ER morphologies. - PROJECT SUMMARY The endoplasmic reticulum (ER) performs many vital cellular functions, including protein and lipid synthesis and calcium storage, and importantly, the ER is a cell homeostasis sensor and signaling network. For example, when protein synthesis gets overloaded in the ER, the ER signals the integrated stress response. The ER also forms contacts with other organelles to signal and transport materials like calcium and lipids – ER health is therefore at the foundation of cellular health and disease prevention. In order to perform ER localized protein synthesis and signaling to other organelles, the ER network dynamically rearranges to form structures for dedicated functions. For instance, secretory cells are filled with expansive ribosome-studded sheets for protein synthesis, whereas cells which are constantly signaling, like neurons, are jam-packed with bundled tubules. So, what defines these functions and shapes? Changing ER protein levels alters both shape and function. I previously showed that ER-phagy, a process where ER membrane and attached proteins are selectively degraded, is required to set up the ER network as cells differentiate. Specifically, by deleting key autophagy and selective ER-phagy machineries in stem cells, I showed ER accumulation in axons and alterations in the ER proteome when they converted to neurons. These observations raise a key question: Are there similarities and/or differences in how these machineries function or are regulated in cell types with different functional outputs? In addition, we are missing key details about ER-phagy site formation and regulation, both under basal conditions in different non-dividing cell types and in post mitotic cells when stressors like antioxidant damage or protein misfolding occur. Thus, I will combine quantitative methods including ER protein turnover assays and mass spectrometry, with top-of-the-line imaging techniques including live-cell imaging and cryo-electron tomography, and functional assays to establish how ER clearance controls (i) the ER proteome landscape, (ii) ER structural rearrangements into tubules and sheets, and (iii) ER functions vital to cell-type establishment and homeostasis. In essence, the ultimate goal of my research program is to provide a blueprint detailing connections between the underlying ER proteome and how its regulation controls cellular function as cells specialize into unique types or respond to stress conditions. With extensive experience studying both ER-phagy and ER dynamics, I am well suited to achieve the goals of the proposed study, which lies at the intersection of the fields of organelle biogenesis and quality control. Defining the mechanisms driving the clearance of regions of the ER network and the specificity of these ER proteins in homeostasis and disease is essential to the field. In conclusion, the proposed work will lay the foundation for my laboratory, accelerating our research to the forefront of our field and deepening our understanding of key processes relevant to the areas of cell and developmental biology.
