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Estimating Z-ring radius and contraction in dividing Escherichia coli
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
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2010 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, no 1, 151-158 p.Article in journal (Refereed) Published
Abstract [en]

P>We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms.

Place, publisher, year, edition, pages
2010. Vol. 76, no 1, 151-158 p.
Keyword [en]
bacterial-cell-division, green fluorescent protein, assembly dynamics, crystal-structure, ftsz rings, tubulin, antibiotics, filaments, mobility, sheets
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-19345DOI: 10.1111/j.1365-2958.2010.07087.xISI: 000276036000011Scopus ID: 2-s2.0-77950208158OAI: oai:DiVA.org:kth-19345DiVA: diva2:337392
Note
QC 20110114Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
Open this publication in new window or tab >>Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, fluorescence spectroscopy was utilized to probe protein and lipid dynamics and interactions in their native, or close to native, environments. Thereby insight could be gained into the fundamentals of bacterial cell division and the innateimmune system.

A particular focus has been devoted to fluorescence fluctuations. They arise when a lower number of fluorescent molecules undergo Brownian motion through a confocal detection volume. With sensitive detectors, appropriate optics and efficient data acquisition these fluctuations can be observed and correlated. This is the heart in fluorescence correlation spectroscopy (FCS), conceived in the 1970’s. FCS has the power to quantify absolute concentrations, diffusion coefficients and to some extent binding events, and is suitable for measurements on a range of samples, including living cells.

However, FCS is not limited to solely diffusional processes. The sensitivity and time resolution allows also electron transitions within the fluorescent molecules to be probed. Of particular interest are spin transitions to and from the dark triplet state. This state is long-lived and sensitive, making it an effective sensor of the surrounding environment. We found that the triplet state could also be used to probe low frequency interactions in membranes down to a single molecule level and a theoretical model was developed that supported the observed interactions. FCS can be extended to fluorescence cross-correlation (FCCS), to handle also a second type of fluorescent molecule, fluorescing with a different colour, and whose signal is cross-correlated with the signal from the first type of fluorescent molecules. The aim was to improve and apply FCCS as a screening tool to probe proteinprotein interactions. By utilizing a methodology based on fluorescently labelled antibodies, we were able to calibrate our FCCS system and provide quantitative data on a particular receptor interaction occurring in natural killer cells. The methodology was found to increase the possibility to quantitatively analyse protein-protein interactions in the membrane of living cells.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 55 p.
Series
Trita-FYS, ISSN 0280-316X ; 22
Keyword
Fluorescence correlation spectroscopy, cytokinesis, collision theory, protein-protein interactions, lipid dynamics, single-molecule experiment
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-33842 (URN)978-91-7501-027-4 (ISBN)
Public defence
2011-06-01, FA32, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110523Available from: 2011-05-23 Created: 2011-05-19 Last updated: 2011-05-23Bibliographically approved

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