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.

Cooperative response and clustering: Consequences of membrane-mediated interactions among mechanosensitive channels

“…Although all these studies suggest cluster formation for native channels, the debate around this question is still open. A recent study, through use of PALM (photo-activated localization microscopy) and SPT (single particle tracking), had shown strong indications that labeling with fluorescent molecules predisposes MscL channels to form clusters [34]. In either case, if there is any form of channel aggregation in bacterial cells, the collective phenomena we describe here may be directly relevant for the osmotic response of bacteria.…”

Dynamic clustering regulates activity of mechanosensitive membrane channels

“…Driven by membrane tension, the helices are thought to tilt with respect to one another, creating a space between them (up to 3 nm in diameter) for small solutes to non-selectively pass through [15]. Recent studies debate the existence and the role of spontaneous clustering of one of the MSCs found in Escherichia coli, MSC of large conductance (MscL) [16,17]. Indeed, membrane clustering appears to be a common mechanism in cellular signaling, and has been observed for many transmembrane proteins and signaling receptors [18].…”

“…Clustering of MSC in vitro has been shown to result in collective, non-linear gating behavior [16], suggesting that it could tamper with cell's passive response during a hypoosmotic shock recovery. However, assessing the extent of MscL aggregation via imaging techniques in vivo has proven to be difficult due to the potential artifacts of the MscL tags on the process [17], and hence opens a need to explore orthogonal ways to investigate the aggregation phenomenon. Here, by developing a minimal computer model of MSCs embedded in a fluid membrane, we investigate the physical mechanisms behind the MSC cluster formation and cooperative gating, and their implications on cell-volume regulation.…”

“…redAs we did not observed any MSC clustering due to pure membrane-mediated interactions, we chose a top-down strategy. We know that: (i) MSC aggregation has been reported in vitro [16], and in vivo while working with MSCs labelled with a small covalent dye [17], (ii) direct inter-protein interactions, such as polar and electrostatic interactions and packing of small apolar side chains [24][25][26][27][28][29], can lead to attractions of trans-membrane proteins [18]. In addition, local lipid phase separation around the protein can yield effective inter-protein attraction and protein aggregation.…”

“…Our prediction on the role of clustering for the cell volume regulation can be probed experimentally by tracing the volume recovery posthypoosmotic shock for different extents of the clustering. The MSC clustering can be enhanced by tagging the channels with fluorescent proteins [17] or modulated by expressing the channels to different levels.…”