Actin is an essential cytoskeletal protein that plays key roles in several cellular functions such as phagocytosis and cell motility with the help of actin binding proteins (ABPs). Gelsolin is a calcium regulated ABP that severs and caps actin filaments. Gelsolin helps control actin filament assembly dynamics that are required for cell survival. Cleavage products of gelsolin lead to Familial Amyloidosis, Finnish type, and conformational changes to gelsolin are implicated in disease progression. The majority of in vitro studies of gelsolin and actin have been performed in dilute buffer conditions which do not simulate the molecular interactions occurring in the intracellular environment. The intracellular space is packed with many macromolecules such as carbohydrates and other proteins. These macromolecules induce steric hindrance and excluded volume effects and have been shown to alter protein-protein interactions and protein conformations. We hypothesize that gelsolin and actin filaments present in crowded environments will produce greater gelsolin severing activity due to steric hinderance and induced conformational changes. To test this hypothesis, we have visualized actin filament severing by gelsolin in solution with macromolecular crowders utilizing total internal reflection fluorescence (TIRF) microscopy. Steady-state average filament lengths and filament length distributions were analyzed to determine the effect crowding has on gelsolin-mediated filament severing. Real-time filament severing assays visualized by TIRF allowed us to compare gelsolin's severing efficiency in the presence of crowders to those in dilute buffer conditions. Co-sedimentation assays were performed in order to determine the effect of crowding on gelsolin binding to actin filaments. Taken together, this study demonstrates that macromolecular crowding modulates gelsolin-mediated actin filament severing activities, offering insights into the interactions between actin and gelsolin inside the cell. These insights will deepen our understanding of in vivo cytoskeletal regulation which is linked to cell physiology and may aid researchers studying actin-related diseases.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2020-1228 |
| Date | 01 January 2020 |
| Creators | Heidings, James |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations, 2020- |
Page generated in 0.0023 seconds