To investigate how cytoplasmic dynein orients the mitotic spindle, we are using the budding yeast S. cerevisiae as a model cell system. This system offers facile genetics, ease of protein manipulation, and a simple astral microtubule organization enabling the dissection of dynein regulation during asymmetric cell divisions, which provides physiological relevance to human stem cell biology, development, and cancer. In previous studies, we found that dynein is targeted to the dynamic astral microtubule plus ends and is delivered via tip-tracking to its cortical anchoring protein, Num1. At the cortex, dynein uses its minus end-directed motor activity to pull an astral microtubule and the connected spindle toward the site of dynein anchorage. We discovered the critical domain in Num1 responsible for dynein interaction and function, and found She1 as a potent inhibitor of dynein motility along the astral microtubules. We will investigate how these cortical and microtubule-associated components mediate spatial and temporal regulation of dynein activity, thereby ensuring directional spindle movement into the yeast bud cell.
Aim 1. Identify the mechanism responsible for dynein inhibition along astral microtubules. To dissect the molecular basis of She1-mediated dynein regulation, we will determine how She1 interacts with dynein and analyze the subcellular localization of She1, testing the hypothesis that it is asymmetrically targeted to different areas of the cell to restrict dynein activity. We will search for specific Sh1 binding partners, investigate the functional roles of these proteins, and test the significance of their interactions with She1.
Aim 2. Determine the effect of dynein-cortex interaction on dynein motility. We hypothesize that cortical Num1 enhances dynein motility enabling dynein-dynactin to pull the mitotic spindle into the narrow bud neck. Using multiple approaches encompassing biochemistry, genetics, and in vitro single-molecule motility assays, we will dissect the molecular basis underlying the interaction between dynein, dynactin, and Num1. We will investigate the potential roles of Num1 in triggering dynein offloading or stimulating dynein minus end-directed motility.

Public Health Relevance

This research will help us understand the cellular pathways that regulate the basic functions of an ancient motor molecule called cytoplasmic dynein. In humans, this motor has many critical physiological roles ranging from maintaining healthy tissue integrity to ensuring proper divisions of many cell types including stem cells. The information obtained from this research may serve as the basis for the development of new therapies, since defects in the latter role have been linked to the genesis of abnormal divisions in cancer tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM076094-06
Application #
8438040
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2007-05-01
Project End
2017-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
6
Fiscal Year
2013
Total Cost
$378,080
Indirect Cost
$126,390
Name
University of Massachusetts Amherst
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Markus, Steven M; Omer, Safia; Baranowski, Kaitlyn et al. (2015) Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae. Traffic 16:773-86
Bezanilla, Magdalena; Gladfelter, Amy S; Kovar, David R et al. (2015) Cytoskeletal dynamics: a view from the membrane. J Cell Biol 209:329-37
Zhu, Yili; Lee, Wei-Lih (2014) The role of +TIPs in directional tip expansion. Mol Microbiol 94:486-9
Wadsworth, Patricia; Lee, Wei-Lih (2013) Microtubule motors: doin' it without dynactin. Curr Biol 23:R563-5
Markus, Steven M; Kalutkiewicz, Katelyn A; Lee, Wei-Lih (2012) Astral microtubule asymmetry provides directional cues for spindle positioning in budding yeast. Exp Cell Res 318:1400-6
Collins, Elizabeth S; Balchand, Sai Keshavan; Faraci, Jessica L et al. (2012) Cell cycle-regulated cortical dynein/dynactin promotes symmetric cell division by differential pole motion in anaphase. Mol Biol Cell 23:3380-90
Markus, Steven M; Kalutkiewicz, Katelyn A; Lee, Wei-Lih (2012) She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements. Curr Biol 22:2221-30
Tang, Xianying; Germain, Bryan St; Lee, Wei-Lih (2012) A novel patch assembly domain in Num1 mediates dynein anchoring at the cortex during spindle positioning. J Cell Biol 196:743-56
Markus, Steven M; Plevock, Karen M; St Germain, Bryan J et al. (2011) Quantitative analysis of Pac1/LIS1-mediated dynein targeting: Implications for regulation of dynein activity in budding yeast. Cytoskeleton (Hoboken) 68:157-74
Markus, Steven M; Lee, Wei-Lih (2011) Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation. Bioarchitecture 1:209-215

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