Development of live-cell single molecule localization microscopy to study lipid droplet biology and fatty acid trafficking at super-resolution
2022-06
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Development of live-cell single molecule localization microscopy to study lipid droplet biology and fatty acid trafficking at super-resolution
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2022-06
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Abstract
Quantitative Single-Molecule Localization Microscopy (SMLM) is evolving into a powerful biophysical technique to study biological processes below the optical
diffraction limit. However, due to the motion of the imaged structures during the
long data acquisition times, most studies have been limited to fixed cells. In
addition, it has been a challenge to label hydrophobic environments such as lipid
droplets (LDs) due to the properties of available fluorophores. LDs are highly
dynamic and actively regulated lipid and energy storage organelle of a cell that is
essential to maintain lipid and energy homeostasis in the cell during metabolic
changes and to buffer the fatty acid levels to protect the cell from lipotoxicity. Cells
tightly control the number, size, and spatial distribution of LDs by regulating the
density of functional enzymes on the LD surface.
In this thesis, I present the application of quantitative SMLM to lipid droplet biology
to reveal how yeast cells dynamically regulate the density of fatty acid activating
enzymes on their lipid droplets. This density regulation enables cells to respond to
the energy and lipid demands during metabolic changes. I develop a novel
mechanism of conventional BODIPY conjugates that enables quantitative SMLM
imaging of fatty acid and neutral lipid distributions with two colors in living yeast
and mammalian cells. The obtained high resolution and single-molecule sensitivity
of these measurements allowed us to gain new biological insights into fatty acid
trafficking and metabolism. Finally, I will present the development of motion-corrected photo-activated localization microscopy (mcPALM) for imaging moving
structures with super-resolution and show the potential for new insights into the
structure and dynamics of endosomes and telomeres in living yeast and
mammalian cells
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University of Minnesota Ph.D. dissertation. June 2022. Major: Physics. Advisor: Elias Puchner. 1 computer file (PDF); viii, 161 pages.
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Adhikari, Santosh. (2022). Development of live-cell single molecule localization microscopy to study lipid droplet biology and fatty acid trafficking at super-resolution. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/253430.
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