Off-campus UMass Amherst users: To download dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users, please click the view more button below to purchase a copy of this dissertation from Proquest.
(Some titles may also be available free of charge in our Open Access Dissertation Collection, so please check there first.)
Microtubule dynamics in interphase and mitotic cells
Abstract
Microtubules are dynamic polymers which play important roles in mitosis, cell locomotion, and the determination and maintenance of cell polarity. To understand the role of microtubule assembly and disassembly in these processes I have localized sites of microtubule growth by injecting labeled tubulin subunits into cells during spindle elongation (anaphase B) and chromosome separation (anaphase A). The behavior of microtubules in interphase cells was also examined by injection of fluorescent tubulin subunits, and observation of the resulting fluorescent microtubules using low light level fluorescence microscopy. These experiments demonstrate that rapid assembly of interzonal microtubules occurs concomitantly with the maximal rate of spindle elongation. Furthermore, the rate of interzonal microtubule elongation is greater than the rate of spindle pole separation, and the pattern of microtubule assembly is most consistent with the dynamic instability model of microtubule behavior. These observations demonstrate that interzonal microtubule assembly is not directly coupled to spindle elongation in these cells and place constraints on existing models of spindle pole separation. Electron microscopic and confocal fluorescence observations of cells injected during anaphase A reveal that injection of biotin-tubulin can induce the assembly of kinetochore microtubules at their plus-ends--normally the site of microtubule disassembly at this stage--in a concentration dependent manner. The elongation of kinetochore microtubules is accompanied by the reversal of chromosome-to-pole motion. Thus, anaphase onset does not prevent addition of tubulin subunits at the plus-end of kinetochore microtubules, and some aspects of mitosis can be regulated by the concentration of free tubulin in vivo. Finally, fluorescent microtubules in living CHO fibroblasts and PtK$\sb1$ epithelial cells have been examined using a 2 second observation interval. These observations confirm results of earlier studies which demonstrate that microtubules turn over more rapidly in fibroblasts than in epithelial cells. Surprisingly, quantitative analysis of individual microtubule behavior reveals that microtubules in epithelial cells undergo depolymerization more frequently than microtubules in fibroblasts. However, microtubules undergoing disassembly are rapidly rescued in epithelial cells but not fibroblasts. These results demonstrate that microtubule behavior is regulated in a cell type specific manner and suggest that epithelial cells contain regulatory factors not found in fibroblasts.
Subject Area
Cellular biology
Recommended Citation
Shelden, Eric Aaron, "Microtubule dynamics in interphase and mitotic cells" (1992). Doctoral Dissertations Available from Proquest. AAI9305893.
https://scholarworks.umass.edu/dissertations/AAI9305893