Thesis
Development of a fluorescent assay of septin filament assembly
Master of Science (M.S.), Drexel University
Aug 2020
DOI:
https://doi.org/10.17918/00000296
Abstract
Septins are cytoskeletal proteins that are involved in cytokinesis, regulation of the actin and microtubule cytoskeletons, and implicated in various diseases. In mammals, thirteen Septin proteins form complexes of defined stoichiometry, for example, a hexameric complex of SEPT2, SEPT6, and SEPT7 (SEPT2/6/7). These complexes assemble end-to-end to produce filaments. While the existence of the filaments is clear, the cellular cues controlling when and where these filaments form are poorly understood. Here, I develop a quantitative biochemical assay for Septin filament formation that will provide a key tool for the mechanistic biophysical study of this filament forming process. By improving an existing heterologous expression system for the SEPT2/6/7 complex and removing surface-exposed cysteines from all three subunits, I have enabled the introduction of novel site-specific cysteines. Using cysteine targeted fluorophores, I show that the targeted subunits can be labeled. To decide where to place fluorophores in a polymerization sensor, I demonstrated that the SEPT2/6/7 organization is not consistent with the long-standing dogma, but instead supports a recently proposed revised model. By placing cysteines near these end surfaces, I hope to generate a fluorescence sensor compatible with kinetic assays of Septin filament assembly needed to understand the dynamics of this filament forming process.
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Details
- Title
- Development of a fluorescent assay of septin filament assembly
- Creators
- Stephano Iglesias
- Contributors
- Simon Cocklin (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- viii, 100 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991014695537704721