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Access Type
WSU Access
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Physics and Astronomy
First Advisor
Christopher V. Kelly
Abstract
The diffusion and reorganization of phospholipids and membrane-associated proteins are fundamental for cellular function. Fluorescence cross-correlation spectroscopy (FCCS) measures the diffusion and molecular interactions at nanomolar concentration in biological systems. We have developed a novel, economical method to simultaneously monitor diffusion and oligomerization with the use of super-continuum laser and spectral deconvolution upon a single detector. This method was applied to perform four-color FCCS, as demonstrated with polystyrene nanoparticles and lipid vesicles. Up to four individually customizable excitation channels were selected from the broad-spectrum fiber laser to excite the diffusers within a diffraction-limited spot. We have measured the induced aggregation of nanobeads in solution upon the addition of phosphate buffer saline. Because of the adaptability of investigating spectrally overlapping fluorophores simultaneously we applied FCCS to examine molecular mechanism of lipolysis. This process of fatty acid release, thought to be controlled by interactions of three key LD associated proteins namely alpha-beta hydrolase domain-containing protein 5 (ABHD5), adipose triglyceride lipase (ATGL), and perilipin 5 (PLIN5). The molecular mechanism by which interactions of ABHD5 with ATGL activates essential colipase activity that controls lipolysis is key for triglyceride metabolism. PLIN5 interacts with ABHD5 in the basal state to prevent ABHD5 from activating ATGL. Upon stimulation via lipolysis stimulating ligands, ABHD5 is released and is allowed to interact with ATGL activating lipolysis. Understanding intracellular lipolysis to compliment cell-based analysis, controlled and stable synthetic system of artificial lipid droplet (aLD) have been studied via FCCS that facilitate altering phospholipids, neutral lipids, and protein compositions by maintaining biological functions. These studies will provide further understanding of how lipolysis is regulated via protein interactions with the lipid droplet membrane.
Recommended Citation
Gandhi, Sonali A., "Four-Color Fluorescence Cross-Correlation Spectroscopy To Reveal The Molecular Mechanism Of Lipolysis" (2023). Wayne State University Dissertations. 3796.
https://digitalcommons.wayne.edu/oa_dissertations/3796