Author

Sandhya Jain

Date of Graduation

Spring 2009

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Kyoungtae Kim

Keywords

eisosome, endocytosis, slm1, slm2, vesicles

Subject Categories

Biology

Abstract

Endocytosis is the process by which cells recycle the plasma membrane and internalize extracellular components. Sites of endocytosis can be randomly formed or may be selected by protein factors called eisosomes. Pil1 is the main organizer of eisosomes. Slm1, Slm2, Lsp1, and Sur7 are a few components associated with eisosomes. Previous studies have shown that Sur7 colocalizes with Slm1 and Pil1, but the details of how Slm1 is involved in eisosome assembly is unknown. Hence I wanted to determine the spatial relationship between Slm1 and Pil1. I found that Slm1 and Pil1 colocalize with each other. Loss of Pil1 caused Slm1 to occur as big aggregates whereas localization of Pil1 was not affected by loss of Slm1. There was a weak genetic interaction observed between SLM1 and PIL1 genes indicating that these genes might share a functional pathway. SLM genes have a PH domain which interacts with PIP2 thus helps in organizing the actin cytoskeleton. In Slmts mutants, the actin cytoskeleton is disrupted. Since actin is crucial for endocytosis, I wanted to know if endocytosis is impaired in Slmts mutants. To determine that, liquid phase endocytosis (FM4-64 pulse chase labeling) and receptor mediated endocytosis (Abp1-RFP) was performed. Deletion of Slm1 and Slm2 alone didn't show any defect in endocytosis, whereas endocytosis was delayed in Slmts mutants even after 30 minutes of incubation. The motility of endocytic vesicle Abp1-RFP was determined using a fluorescence microscope with the help of software called Image J, generating a mean squared displacement plot vs time. The endocytic vesicles in the wild type form on the membrane, and move into the cytoplasm in a directed motion. The Abp1 patches in Slmts mutants formed albeit slowly, and exhibited a random motion. Taken together, the data obtained in this study show clear evidence that Slm proteins are required for proper endocytosis.

Copyright

© Sandhya Jain

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