Quantifying Localizations and Dynamics in Single Bacterial Cells

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Quantifying Localizations and Dynamics in Single Bacterial Cells

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dc.contributor.advisor Paulsson, Johan Martin
dc.contributor.author Landgraf, Dirk
dc.date.accessioned 2012-11-19T15:33:18Z
dash.embargo.terms 2014-10-05 en_US
dash.embargo.terms 2014-10-05
dc.date.issued 2012-11-19
dc.date.submitted 2012
dc.identifier.other http://dissertations.umi.com/gsas.harvard:10612 en
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:9920184
dc.description.abstract Levels 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.iso en_US en_US
dash.license LAA
dc.subject Clp proteases en_US
dc.subject RpoS en_US
dc.subject systematic biology en_US
dc.subject biophysics en_US
dc.subject microbiology en_US
dc.subject fluorescent proteins en_US
dc.subject microscopy en_US
dc.subject stress response en_US
dc.subject protein degradation en_US
dc.title Quantifying Localizations and Dynamics in Single Bacterial Cells en_US
dc.type Thesis or Dissertation en_US
dash.depositing.author Landgraf, Dirk
dash.embargo.until 2014-10-05
thesis.degree.date 2012 en_US
thesis.degree.discipline Systems Biology en_US
thesis.degree.grantor Harvard University en_US
thesis.degree.level doctoral en_US
thesis.degree.name Ph.D. en_US
dc.contributor.committeeMember Mitchison, Timothy en_US
dc.contributor.committeeMember Rudner, David en_US
dc.contributor.committeeMember Lahav, Galit en_US
dc.contributor.committeeMember Cluzel, Philippe en_US

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