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Biological Insights from Single-Particle Tracking in Living Cells
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time.

Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s−1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers.

Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1159
Keyword [en]
single-particle tracking, intracellular diffusion, nucleoid exclusion, transcription-translation coupling, antibiotics
National Category
Biophysics
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-229342ISBN: 978-91-554-8991-5 (print)OAI: oai:DiVA.org:uu-229342DiVA: diva2:736592
Public defence
2014-09-05, lecture hall B42, Uppsala Biomedical Centre, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-08-15 Created: 2014-08-06 Last updated: 2014-09-08Bibliographically approved
List of papers
1. Single-molecule investigations of the stringent response machinery in living bacterial cells
Open this publication in new window or tab >>Single-molecule investigations of the stringent response machinery in living bacterial cells
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2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 31, E365-E373 p.Article in journal (Refereed) Published
Abstract [en]

The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism.

Keyword
cytosolic diffusion, single particle tracking, photoactivated localization microscopy, stroboscopic illumination
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-157244 (URN)10.1073/pnas.1102255108 (DOI)000293385700009 ()
Available from: 2011-08-31 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved
2. Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
Open this publication in new window or tab >>Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
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2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, 11413-11418 p.Article in journal (Refereed) Published
Abstract [en]

Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

Keyword
nucleoid exclusion, transcription-translation coupling, antibiotics, single-molecule tracking, single-molecule imaging
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-229101 (URN)10.1073/pnas.1411558111 (DOI)000339807200043 ()25056965 (PubMedID)
Available from: 2014-07-30 Created: 2014-07-30 Last updated: 2017-12-05Bibliographically approved

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