Title: Metabolic Imaging and Applications in Protein and Lipid Homeostasis
Description: Metabolic activity is an important functional parameter of a living cell. Microscopic techniques are demanded to resolve the heterogeneity of metabolic activity from cell to cell and among subcellular compartments. Towards this, work in the Min lab has been dedicated to developing a prevailing metabolic imaging platform that couples chemical imaging by stimulated Raman scattering (SRS) microscopy with small vibrational tags on precursor molecules. This thesis describes efforts along metabolic imaging by SRS microscopy, with focus on visualizing protein and lipid homeostasis. Chapter 1 describes the design principle of metabolic imaging, including selection of vibrational tags and setup of SRS microscopy, and an overview of successful demonstrations of metabolic imaging in protein and lipid metabolism. Chapter 2 describes adoption of such principle to visualize protein turnover with 13C-phenylalanine metabolic labeling under steady-state condition and various perturbations. The rest of this thesis (Chapters 3-6) switches focus to fatty acid metabolism and cellular lipid homeostasis.

As the minimal tagging in vibrational imaging preserves the physicochemical property of lipid molecules to the largest extent, it motivated me to revisit fatty acid metabolism from a biophysical perspective. Bearing the question in mind whether the non-equilibrium metabolic activity could drive phase separation in biological membranes, I thus look into the principle of membrane organization and its implication in biological membranes in Chapter 3. Then in Chapter 4, I describe the discovery and characterization of previously unknown phase separation in endoplasmic reticulum (ER) membrane caused by lipid synthesis. In this case, metabolic imaging by SRS enables identification of solid-like domains formed by saturated fatty acid (SFA) metabolites. This observation further raises the question whether phase separation bears any functional roles in the adverse effects of SFAs (or lipotoxicity). Towards this, Chapter 5 introduces the background of lipotoxicity including its definition and models.

Then I review proposed mechanisms for lipotoxicity, which point to the central role of ER in mediating the stress transduction. In Chapter 6, I present our findings that suggest the association of the observed solid-like domains with ER structural remodeling and local autophagic arrest. Together, these efforts demonstrate the valuable capability of SRS imaging to reveal metabolic heterogeneity and how this aids in the investigation of metabolic stress.