Thesis
Septin filament assembly conditions and fluorescent assay development
Master of Science (M.S.), Drexel University
Dec 2023
DOI:
https://doi.org/10.17918/00001908
Abstract
Septins are a family of GTP-binding proteins that have important roles in cytokinesis and the regulation of other cytoskeletal proteins such as microtubules and actin. Changes in septin organization have been associated with pathogenic states including neurodegenerative disorders, cancers, and viral infections. Despite our growing understanding of the roles septins play in cytoskeletal activities and diseases, we understand relatively little about how septin polymerization is controlled and guided to specific intracellular locations. Using biochemical assays, I have characterized how changing salt and pH conditions influence mammalian septin assembly in bulk solution. I have found that pH sensitivity of the system is substantial, which was previously unrecognized. These experiments provide insight into how septin polymerization may be regulated in cells. Moreover, I use this new knowledge to develop a novel fluorescent sensor for septin assembly. Using the known hexamer structure and polymerization interface, the Padrick lab has designed septin complexes with unique surface cysteine mutations. I use these to introduce environmentally sensitive fluorophores in targeted locations. Our current design reports spectral changes that mirror the assembly as detected using other metrics and functions even at low labeling fraction. We hope to use this improved sensor to quantify septin filament assembly in solution, on membranes, and on other cytoskeletal surfaces, revealing how septin filament assembly is controlled in the cell.
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Details
- Title
- Septin filament assembly conditions and fluorescent assay development
- Creators
- Eric David Brinckman
- Contributors
- Shae B. Padrick (Advisor)Patrick J. Loll (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- vii, 76 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991021819215504721