Date of Graduation

Summer 2008

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Kyoungtae Kim

Keywords

endocytosis, dynamin, Vps1p, vesicles, vacuole, internalization

Subject Categories

Biology

Abstract

Endocytosis is a process by which cells take up extracellular materials, recycle the plasma membrane, and down-regulate the expression of membrane receptor molecules. In mammals, a GTPase protein (dynamin) is responsible for scission of endocytic vesicles from the plasma membrane. Among three dynamin-like proteins (Dnm1p, Mgm1p and Vps1p) in budding yeast, S. cerevisiae, a previous study showed that Vps1p interacts with Sla1p and is involved in actin organization, providing the first evidence for its role in endocytosis. The goal of this study was to identify or characterize potential endocytic defects caused by the loss of Vps1p during the entire process of endocytosis. FM4-64 pulse chase labeling showed that loss of Vps1p results in a delay in the formation of the vacuole at the end of a 90 min incubation period at 30 °;C in Vps1Δ cells compared to WT cells. Particle tracking analysis of green fluorescent protein (GFP)-fused stage-specific endocytic markers Ede1p, Sla1p, and Abp1p showed that the loss of Vps1p results in significant prolongation of their lifetimes on the membrane. Additionally, Abp1-GFP patch motility in Vps1Δ cells was slower and non-directional compared to WT cells. Particle tracking analysis of Abp1-GFP patches in Dnm1Δ and Mgm1Δ cells further showed that the other two dynamin-like proteins Dnm1p and Mgm1p do not play a role in the internalization step of endocytosis in yeast. By staining with Rhodamine-Phalloidin (Rh-Ph) dye, it was observed that the loss of Vps1p results in fragmentation of actin cables at a non-permissive temperature of 37 °;C. Consistent with the findings of Rh-Ph staining, particle tracking of FM4-64 vesicles revealed that, the loss of Vps1p results in slower motility of endocytic vesicles in the cytoplasm in Vps1Δ cells compared to WT cells at 37 °;C. In spite of the observed delay in internalization of endocytic vesicles and delay observed in post-internalization motility of endocytic vesicles in Vps1Δ cells, the possibility of a defect in fusion of endocytic vesicle to the vacuole is yet to be elucidated.

Copyright

© Srikant Nannapaneni

Campus Only

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