title: Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.
abstract: The rod-shaped Gram-negative bacterium Escherichia coli multiplies by elongation followed by binary fission. Longitudinal growth of the cell envelope and synthesis of the new poles are organized by two protein complexes called elongasome and divisome, respectively. We have analyzed the spatio-temporal localization patterns of many of these morphogenetic proteins by immunolabeling the wild type strain MC4100 grown to steady state in minimal glucose medium at 28°C. This allowed the direct comparison of morphogenetic protein localization patterns as a function of cell age as imaged by phase contrast and fluorescence wide field microscopy. Under steady state conditions the age distribution of the cells is constant and is directly correlated to cell length. To quantify cell size and protein localization parameters in 1000s of labeled cells, we developed 'Coli-Inspector,' which is a project running under ImageJ with the plugin 'ObjectJ.' ObjectJ organizes image-analysis tasks using an integrated approach with the flexibility to produce different output formats from existing markers such as intensity data and geometrical parameters. ObjectJ supports the combination of automatic and interactive methods giving the user complete control over the method of image analysis and data collection, with visual inspection tools for quick elimination of artifacts. Coli-inspector was used to sort the cells according to division cycle cell age and to analyze the spatio-temporal localization pattern of each protein. A unique dataset has been created on the concentration and position of the proteins during the cell cycle. We show for the first time that a subset of morphogenetic proteins have a constant cellular concentration during the cell division cycle whereas another set exhibits a cell division cycle dependent concentration variation. Using the number of proteins present at midcell, the stoichiometry of the divisome is discussed.
authors.affiliation: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam Amsterdam, Netherlands.
authors.forename: Norbert O E
authors.initials: NO
authors.lastname: Vischer
authors.affiliation: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam Amsterdam, Netherlands.
authors.forename: Jolanda
authors.initials: J
authors.lastname: Verheul
authors.affiliation: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam Amsterdam, Netherlands ; Molecular Cytology, Swammerdam Institute for Life Sciences, Faculty of Sciences, University of Amsterdam Amsterdam, Netherlands.
authors.forename: Marten
authors.initials: M
authors.lastname: Postma
authors.affiliation: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam Amsterdam, Netherlands ; Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University Amsterdam, Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: van den Berg van Saparoea
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.
authors.forename: Elisa
authors.initials: E
authors.lastname: Galli
authors.affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas Madrid, Spain.
authors.forename: Paolo
authors.initials: P
authors.lastname: Natale
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK ; Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Kenn
authors.initials: K
authors.lastname: Gerdes
authors.affiliation: Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University Amsterdam, Netherlands.
authors.forename: Joen
authors.initials: J
authors.lastname: Luirink
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Vollmer
authors.affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas Madrid, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Vicente
authors.affiliation: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam Amsterdam, Netherlands.
authors.forename: Tanneke
authors.initials: T
authors.lastname: den Blaauwen
keywords: FtsN; FtsZ; LpoA; PBP1B; divisome; image analysis; immunolocalization; non-destructive marking