Thesis
Towards in-vitro reconstitution of actin comet tail formation and septin caging in Shigella
Master of Science (M.S.), Drexel University
Aug 2024
DOI:
https://doi.org/10.17918/00010587
Abstract
Intracellular pathogens, both bacterial and viral, manipulate the host cytoskeleton by creating actin comet tails, which are dense, branching structures that aid their movement within host cells. Shigella, a deadly cause of bacterial diarrhea worldwide, utilizes actin comet tails to spread cell-to-cell. Host cells counteract this by recruiting proteins like septins to cage the bacteria and induce autophagy. Previous research has recreated actin tail formation in vitro using purified proteins on inanimate surfaces and bacterial pathogens. In addition, the septin caging process has been recapitulated with purified proteins on live Shigella. Despite this, a key question remains, does the septin cage directly prevent actin comet tail formation? This project aims at establishing an in vitro reconstitution system that closely resembles the actin comet tail produced by Shigella in the native environment. To achieve this, I have combined six different proteins expressed and purified in E. coli BL21 (DE3) and two different proteins purified from endogenous sources which are then added together in a set concentration with a motility buffer and polystyrene bead coated with GST-VCA or GST-Cdc42, depending on experimental goals. This is a challenging system to reconstitute and here I describe how I was able to reconstitute the actin comet tail assembly process in anticipation of combining it with later septin assembly on Shigella.
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Details
- Title
- Towards in-vitro reconstitution of actin comet tail formation and septin caging in Shigella
- Creators
- Sarath Reghunathan
- Contributors
- Shae B. Padrick (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xii, 137 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991021901914704721