Publication Date
2010
Document Type
Thesis
Committee Members
Nancy J. Bigley (Committee Member), Barbara Hull (Committee Member), Mill W. Miller (Advisor)
Degree Name
Master of Science (MS)
Abstract
Rev is a regulatory protein that plays an important role in the replication of HIV virus by post-transcriptionally promoting expression of viral proteins late in infection. Rev expression also slows cell growth, leads to an accumulation of cells in G2/M specifically before the spindle checkpoint, and can produce changes in ploidy. Because Rev is capable of depolymerizing microtubules (MTs) in vitro, possibly by a mechanism shared with Kinesin-13 proteins, themselves potent cellular MT depolymerases, I tested the hypothesis that these cellular defects were due to an interaction between Rev and tubulin.
To this end, Rev and select Rev mutants defective in RNA binding and nuclear import (M6), nuclear export (M10), and Rev multimerization (M4) were expressed in HeLa cells. Rev's ability to interact with tubulin was monitored by reciprocal co-immunoprecipitation experiments using antibodies specific for tubulin and the Rev transgene. Results from these experiments are consistent with this hypothesis as Rev and tubulin can be detected in the same immunoprecipitates.
To extend these results, deconvolution microscopy was used to colocalize Rev and spindle microtubules. Whereas Rev, M4, M6, and M10 fused to green or yellow fluorescent protein are largely dispersed throughout the cytoplasm of mitotic cells, the use of colocalization software indicates there is a shallow gradient of Rev accumulation proximal to the spindle. Some M6 appears to colocalize at or near spindle poles although this is also seen in control cells. However, while these data suggest there is a potential for substantial colocalization between Rev and tubulin, visual inspection shows there is little compelling colocalization with spindle MTs. However, because immunostaining readily detects tubulin polymerized into MTs and not soluble tubulin heterodimers, the results of the colocalization and co-immunoprecipitation assays are both consistent with the hypothesis that Rev and Rev mutants are interacting with the heterodimer and not the polymerized tubulin. Intriguingly, significant amounts of wild-type Rev, M4 and M10 accumulate perichromosomally where a large fraction of spindle MTs nucleates early in mitosis. Thus Rev is spatially positioned within the cell to affect spindle assembly during early mitosis. Indeed, the previously discovered cell cycle defects of wild-type Rev, M4, M6, and M10 are all consistent with this hypothesis. Taken together, these results suggest that cells have the ability to correct spindle defects that occur during prometaphase. In conclusion, these results suggest that Rev and Rev mutants interact with tubulin heterodimers and might interfere with cell cycle progression.
Since Rev expressing cells accumulate in G2/M phase, the mitotic defects in cells expressing Rev and Rev mutants were examined. Previous research has suggested that expression of Rev and Rev mutants alters progression through mitosis with cells accumulating before the spindle assembly checkpoint. These results suggest that Rev expression may interfere with chromosomal congression and therefore alter tension across the spindle and between kinetochores. To investigate this, the distances between spindle poles and interkinetchore distances were measured in metaphase cells. No significant differences were found between cells expressing Rev or Rev mutants and control cells.
Page Count
81
Department or Program
Microbiology and Immunology
Year Degree Awarded
2010
Copyright
Copyright 2010, some rights reserved. My ETD may be copied and distributed only for non-commercial purposes and may be modified only if the modified version is distributed with these same permissions. All use must give me credit as the original author.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.
