Actin is an abundant and essential cytoskeletal protein that plays a central role in eukaryotic cell structure and motility. The intracellular environment where actin assembly occurs is crowded with various organelles, proteins, and macromolecules that limit the accessible volume for biomolecular reactions. Macromolecular crowding induces excluded volume effects influencing the activity of biological molecules as well as the shape and conformation of proteins. Crowding agents, such as polysaccharides or inert polymeric molecules, are used to mimic the conditions present in intracellular spaces and provide a better understanding of interactions inside the cell. Macromolecular crowding has been shown to affect actin filament assembly, however, how crowder size impacts actin assembly dynamics and kinetics is not well understood. In this thesis, we investigate how the excluded volume effects caused by crowding influences actin filament assembly kinetics by using synthetic polymeric crowder, polyethylene glycol (PEG), of various molecular weights. Using total internal reflection fluorescence (TIRF) microscopy, we directly visualized the assembly of individual actin filaments in various sizes of PEG crowded conditions. We quantified actin filament growth rates that depend on the size of crowder. Bulk fluorescence intensity was monitored to evaluate the effect of crowder size on actin assembly kinetics. These results demonstrate that the size of macromolecular crowding agents can modulate actin filament assembly kinetics, possibly by controlling the volume fractions. This work provides a foundation for a mechanism of how the dynamic cytoskeletal assembly occurs in living cells.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2020-1670 |
| Date | 01 January 2021 |
| Creators | Demosthene, Bryan |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations, 2020- |
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