title: Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins.
abstract: Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro-and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Dempwolff
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Physics, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Felix K
authors.initials: FK
authors.lastname: Schmidt
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Ana B
authors.initials: AB
authors.lastname: Hervás
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Alex
authors.initials: A
authors.lastname: Stroh
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rösch
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Biology, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Cornelius N
authors.initials: CN
authors.lastname: Riese
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Dersch
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Biology, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Heimerl
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Daniella
authors.initials: D
authors.lastname: Lucena
authors.affiliation: Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Nikola
authors.initials: N
authors.lastname: Hülsbusch
authors.affiliation: Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Claudia A O
authors.initials: CA
authors.lastname: Stuermer
authors.affiliation: University of Tsukuba, Faculty of Life and Environmental Sciences, Tsukuba, Ibaraki, Japan.|Department of Microbiology, Institute for Applied Bioscience, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Norio
authors.initials: N
authors.lastname: Takeshita
authors.affiliation: Department of Microbiology, Institute for Applied Bioscience, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Fischer
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Physics, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Bruno
authors.identifier: 0000-0003-4050-3254
authors.initials: B
authors.lastname: Eckhardt
authors.affiliation: SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.|Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Graumann
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003902:Detergents / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D021381:Protein Transport / Q000502:physiology
chemical_list: D003902:Detergents; D008565:Membrane Proteins; C106354:flotillins