On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli

Jonas van den Berg, Heloisa Galbiati, Akiko Rasmussen, Samantha Miller, Bert Poolman

Research output: Contribution to journalArticle

8 Citations (Scopus)
4 Downloads (Pure)

Abstract

Bacterial mechanosensitive channels protect cells from structural damage during hypoosmotic shock. MscS, MscL and MscK are the most abundant channels in E. coli and arguably the most important ones in osmoprotection. By combining physiological assays with quantitative photo-activated localization microscopy (qPALM), we find an almost linear relationship between channel abundance and cell survival. A minimum of 100 MscL (or MscS) channels is needed for protection when a single type of channel is expressed. Under native-like conditions MscL, MscS as well as MscK distribute homogeneously over the cytoplasmic membrane and the lateral diffusion of the channels is in accordance with their relative protein mass. However, we observe cluster formation and a reduced mobility of MscL when the majority of the subunits of the pentameric channel contain the fluorescent mEos3.2 protein. These data provide new insights into the quantitative biology of mechanosensitive channels and emphasizes the need for care in analysing protein complexes even when the fluorescent tag has been optimized for monomeric behaviour.
Original languageEnglish
Article number32709
JournalScientific Reports
Volume6
DOIs
Publication statusPublished - 6 Sep 2016

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Escherichia coli
Membranes
Proteins
Microscopy
Shock
Cell Survival
Cell Membrane

Keywords

  • mechanosensitive channels
  • osmoregulation
  • super-resolution microscopy
  • lateral diffusion
  • quantitative biology
  • single-molecule analysis

Cite this

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli. / van den Berg, Jonas ; Galbiati, Heloisa; Rasmussen, Akiko; Miller, Samantha; Poolman, Bert.

In: Scientific Reports, Vol. 6, 32709, 06.09.2016.

Research output: Contribution to journalArticle

van den Berg, Jonas ; Galbiati, Heloisa ; Rasmussen, Akiko ; Miller, Samantha ; Poolman, Bert. / On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli. In: Scientific Reports. 2016 ; Vol. 6.
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abstract = "Bacterial mechanosensitive channels protect cells from structural damage during hypoosmotic shock. MscS, MscL and MscK are the most abundant channels in E. coli and arguably the most important ones in osmoprotection. By combining physiological assays with quantitative photo-activated localization microscopy (qPALM), we find an almost linear relationship between channel abundance and cell survival. A minimum of 100 MscL (or MscS) channels is needed for protection when a single type of channel is expressed. Under native-like conditions MscL, MscS as well as MscK distribute homogeneously over the cytoplasmic membrane and the lateral diffusion of the channels is in accordance with their relative protein mass. However, we observe cluster formation and a reduced mobility of MscL when the majority of the subunits of the pentameric channel contain the fluorescent mEos3.2 protein. These data provide new insights into the quantitative biology of mechanosensitive channels and emphasizes the need for care in analysing protein complexes even when the fluorescent tag has been optimized for monomeric behaviour.",
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note = "We thank Frans Bianchi and Franz Ho for assistance with molecular cloning, Tim Rasmussen for providing the pTRC-MscK plasmid, Andrew Robinson for providing the pBAD-mEos3.2 plasmid, Matthias Heinemann for assistance with the flow cytometry measurements, Paul Schavemaker for performing Smoldyn simulations and Michiel Punter for programming ImageJ plugins for PALM reconstructions and single-particle tracking. We thank Ian Booth for critical reading of the manuscript, and Christoffer {\AA}berg and Matteo Gabba for valuable discussions. The authors would like to thank David Dryden and Marcel Reuter for performing preliminary experiments from which this work has been built. The work was funded by the EU FP7 ITN-network program NICHE.",
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