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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01kh04ds865
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dc.contributor.advisorPetry, Sabine
dc.contributor.authorRale, Michael Joseph
dc.contributor.otherMolecular Biology Department
dc.date.accessioned2022-06-16T20:34:54Z-
dc.date.available2022-06-16T20:34:54Z-
dc.date.created2022-01-01
dc.date.issued2022
dc.identifier.urihttp://arks.princeton.edu/ark:/88435/dsp01kh04ds865-
dc.description.abstractTo establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules generated by γTuRCs. However, the field to date has no real-time, direct observation of this activation effect.In this dissertation, I first present a new high-yield, high-stringency method for purifying endogenous γTuRC from Xenopus egg extract. This method builds on prior work utilizing the known interaction between the γTuNA domain and γTuRC, improving the final yield into the hundreds of nanomolar final concentration. Next, I utilize Xenopus egg extract and in vitro single molecule imaging assays to show that γTuNA activates microtubule nucleation in extract and directly activates γTuRC in vitro. This is the first real-time, direct observation of this activation effect. Furthermore, I explore the nature of this direct interaction via mutation analysis and find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA’s L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, I find that γTuNA’s activating effect opposes inhibitory regulation by stathmin. In sum, my work helps illuminates how γTuRC is controlled in space and time in order to build specific cytoskeletal structures.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherPrinceton, NJ : Princeton University
dc.relation.isformatofThe Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: <a href=http://catalog.princeton.edu>catalog.princeton.edu</a>
dc.subjectCDK5RAP2
dc.subjectcentrosome
dc.subjectgamma-tubulin ring complex
dc.subjectGolgi
dc.subjectmicrotubules
dc.subjectsingle molecule imaging
dc.subject.classificationBiochemistry
dc.subject.classificationCellular biology
dc.subject.classificationMolecular biology
dc.titleDissecting the activation of microtubule nucleation by the γ-tubulin ring complex (γTuRC).
dc.typeAcademic dissertations (Ph.D.)
pu.date.classyear2022
pu.departmentMolecular Biology
Appears in Collections:Molecular Biology

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