On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli.
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.
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology &Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands.
Jonas
J
van den Berg
School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD United Kingdom.
Heloisa
H
Galbiati
School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD United Kingdom.
Akiko
A
Rasmussen
School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD United Kingdom.
Samantha
S
Miller
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology &Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands.
Bert
B
Poolman
D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008164:Luminescent Proteins