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

doi:10.1038/srep32709

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

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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.

Original language	English
Article number	32709
Journal	Scientific Reports
Volume	6
DOIs	https://doi.org/10.1038/srep32709
Publication status	Published - 6 Sep 2016

Keywords

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

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10.1038/srep32709Licence: CC BY

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli
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Final published version, 1.1 MBLicence: CC BY

Samantha Miller
- School of Medicine, Medical Sciences & Nutrition, Medical Sciences - Senior Lecturer
- Institute of Medical Sciences
Person: Academic

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

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. ",

keywords = "mechanosensitive channels, osmoregulation, super-resolution microscopy, lateral diffusion, quantitative biology, single-molecule analysis",

author = "{van den Berg}, Jonas and Heloisa Galbiati and Akiko Rasmussen and Samantha Miller and Bert Poolman",

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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doi = "10.1038/srep32709",

language = "English",

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AU - van den Berg, Jonas

AU - Galbiati, Heloisa

AU - Rasmussen, Akiko

AU - Miller, Samantha

AU - Poolman, Bert

N1 - 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 Åberg 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.

PY - 2016/9/6

Y1 - 2016/9/6

N2 - 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.

AB - 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.

KW - mechanosensitive channels

KW - osmoregulation

KW - super-resolution microscopy

KW - lateral diffusion

KW - quantitative biology

KW - single-molecule analysis

U2 - 10.1038/srep32709

DO - 10.1038/srep32709

M3 - Article

VL - 6

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 32709

ER -

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

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Samantha Miller

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