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STUDIES OF DYNEIN ANCHORING PROTEIN IN BUDDING YEAST SACCHAROMYCES CEREVISIAE

Abstract
Inheritance of equal genomic content is vital for cellular growth and survival. During cell division, the mitotic spindle is built to ensure equal chromosome segregation between dividing cells. Fidelity of segregation is achieved by the precise positioning of the mitotic spindle by force generators located at the cell cortex where they exert pulling on microtubule (MT). Cortical dynein, a minus end directed motor, generates pulling forces via microtubule end-on capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with plasma membrane (PM), endoplasmic reticulum (ER), and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. I employed yeast genetics, live cell fluorescence microscopy and image analysis to identify key regulators of Num1 localization and to dissect the mechanism of dynein-MT interaction in various contexts. I show that Num1 form distinct populations, and loss of Scs2 and Scs22, two major ER-to-PM tethering proteins, results in defective Num1 distribution along the cell cortex. Loss of Scs2 and Scs22 also resulted in loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER-independent population of Num1 at the polar ends of the cell, dynein motor activity, and the CAP-Gly domain of dynactin Nip100/p150Glued subunit, but not the MT plus end depolymerase Kip3 or Kar3. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.
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