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dc.contributor.advisorPaulsson, Johan Martin
dc.contributor.authorLandgraf, Dirk
dc.date.accessioned2012-11-19T15:33:18Z
dash.embargo.terms2014-10-05en_US
dash.embargo.terms2014-10-05
dc.date.issued2012-11-19
dc.date.submitted2012
dc.identifier.citationLandgraf, Dirk. 2012. Quantifying Localizations and Dynamics in Single Bacterial Cells. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard:10612en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:9920184
dc.description.abstractLevels of macromolecules fluctuate both spatially and temporally in individual cells. Such heterogeneity could be exploited for bet hedging in uncertain environments, or be suppressed by negative feedback if perturbations are deleterious. For the master stress-response regulator in Escherichia coli, RpoS, both of these scenarios have been suggested. RpoS levels are also exceedingly low and controlled by the ClpXP protease, which reportedly displays extreme spatial heterogeneity. However, little is known quantitatively about RpoS dynamics. This is partly because no functional protein fusions exist, but also because the quantitative tools for studying fluctuations and localizations are limited, particularly ones that can be independently validated. Here I develop such methods and begin applying them to RpoS. Protein localization measurements increasingly rely on fluorescent protein fusions and are difficult to verify independently. I designed a non-intrusive method for validating localization patterns in live bacterial cells by exploiting post-division heterogeneity in downstream processes. Applying this assay to the ClpXP protease, widely reported to form biologically relevant foci, revealed in fact that the protease molecules are not specifically localized inside cells, as confirmed by four independent methods. I further evaluated 20+ commonly used fluorescent reporters and found that many cause severe mislocalization when fused to homo-oligomers, likely due to avidity effects. Further reinvestigating other foci-forming proteins strongly suggests that the previously reported foci were all caused by the fluorescent proteins used. For mRNAs – which are often present in low numbers per cell and major sources of non-genetic heterogeneity – existing single-cell assays have unknown accuracy: the experimental counting errors could completely over-shadow the natural variation. I therefore optimized and cross-evaluated two single-molecule mRNA detection methods. Several problems were identified and solutions discussed. I succeeded in building a functional RpoS protein fusion, and used bulk methods to show that the RpoS feedback loop is effectively not operating during exponential- phase growth. Mathematical analyses and initial experiments in a microfluidic device further suggest that the RpoS system has several unusual properties contributing towards extremely fast stress response. A stochastic analysis further suggests that the RpoS feedback loop cannot suppress spontaneous fluctuations, and preliminary experiments indicate that large deviations might indeed play important roles.en_US
dc.language.isoen_USen_US
dash.licenseLAA
dc.subjectClp proteasesen_US
dc.subjectRpoSen_US
dc.subjectsystematic biologyen_US
dc.subjectbiophysicsen_US
dc.subjectmicrobiologyen_US
dc.subjectfluorescent proteinsen_US
dc.subjectmicroscopyen_US
dc.subjectstress responseen_US
dc.subjectprotein degradationen_US
dc.titleQuantifying Localizations and Dynamics in Single Bacterial Cellsen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorLandgraf, Dirk
dc.date.available2014-10-06T07:30:36Z
thesis.degree.date2012en_US
thesis.degree.disciplineSystems Biologyen_US
thesis.degree.grantorHarvard Universityen_US
thesis.degree.leveldoctoralen_US
thesis.degree.namePh.D.en_US
dc.contributor.committeeMemberMitchison, Timothyen_US
dc.contributor.committeeMemberRudner, Daviden_US
dc.contributor.committeeMemberLahav, Galiten_US
dc.contributor.committeeMemberCluzel, Philippeen_US
dash.contributor.affiliatedLandgraf, Dirk


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