Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Amarachi Grace Ochiobi - University of Georgia, Athens
Co-Author(s): Steven Markus, Colorado State University, Fort Collins, CO
Dynein is a motor protein that walks towards the minus end of microtubules. This project focuses on understanding how dynein positions the spindle during mitosis, a key event during various developmental processes. For this study, we used the model organism Saccharomyces cerevisiae (budding yeast) in which dynein’s only function is to position the spindle. To perform its function, dynein binds to the plus end of an astral microtubule, and stays there until it is offloaded to the cortex. Once at the cortex, dynein grasps onto an astral microtubule, generating tension on the astral microtubules, which pulls the spindle into the proper position prior to cytokinesis. The positioning of the spindle during mitosis is an essential process that ensures equal distribution of genetic materials between the mother and daughter cell.
The ability of dynein to carry out this function is dependent on several regulatory proteins. In this project, we studied the regulation of dynein by She1. In yeast cells, She1 affects the direction with which dynein can position the spindle by spatially restricting dynein activity to the daughter cell. The mechanism by which She1 can differentially affect dynein activity in the mother versus the daughter cell is unknown. We set out to test the hypothesis that She1 localizes more prominently in the mother cell than in the daughter cell, and thereby differentially affects dynein activity in the mother versus daughter cell. To test our hypothesis, we took advantage of a recently developed fluorescent protein marker (SUN Tag) to tag She1 in cells, which would allow us to determine the localization of She1. SUN-tag technology utilizes a GFP-single chain fragment variable (scFv) antibody fragment (GFP-scFv) in conjunction with a small epitope tag that is fused to the protein of interest (e.g., She1). Although we managed to generate a SUN-tagged She1 yeast strain, we were unable to observe any GFP-labeled She1, possibly due to defective expression of the GFP-scFv. Future studies will focus on optimizing expression of the GFP-scFv in yeast cells, which will permit us to ultimately observe She1.
References: Markus S.M., Kalutkiewicz K.A., Lee W.L (2012). She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements.Curr. Biol. 22:2221-30.
Markus S.M., Lee W.L (2011). Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation. BioArchitecture. 1:5, 209-215.
Funder Acknowledgement(s): I thank the director of the program Dr. Paul Laybourn for making it possible for me to participate in this program, and also to my Principal investigator and mentor Dr. Steven Markus for dedicating so much of his time to this project. I would also like to thank all members of Markus Lab for their support and patience, especially to Lindsay Lammers for being my backbone and guide all through this program. This study was supported by NSF grant #1460507 awarded to Paul Laybourn PhD, Director of Colorado State University Molecular Biosciences REU Site, Fort Collins, GA.
Faculty Advisor: Steven Markus,