The Bacterial Cytoskeleton Spatially Confines Functional Membrane Microdomains

Abstract

22Cell membranes laterally segregate into microdomains enriched in certain lipids and scaffold 23 proteins. Membrane microdomains modulate protein-protein interactions and are essential for cell 24 polarity, signaling and membrane trafficking. How cells organize their membrane microdomains, and 25 the physiological importance of these microdomains, is unknown. In eukaryotes, the cortical actin 26 cytoskeleton is proposed to act like a fence, constraining the dynamics of membrane microdomains. 27Like their eukaryotic counterparts, bacterial cells have functional membrane microdomains (FMMs) 28 that act as platforms for the efficient oligomerization of protein complexes. In this work, we used the 29 model organism Bacillus subtilis to demonstrate that FMM organization and movement depend 30 primarily on the interaction of FMM scaffold proteins with the domains' protein cargo, rather than with 31 domain lipids. Additionally, the MreB actin-like cytoskeletal network that underlies the bacterial 32 membrane was found to frame areas of the membrane in which FMM mobility is concentrated. 33Variations in membrane fluidity did not affect FMM mobility whereas alterations in cell wall organization 34 affected FMM mobility substantially. Interference with MreB organization alleviates FMM spatial 35 confinement whereas, by contrast, inhibition of cell wall synthesis strengthens FMM confinement. The 36 restriction of FMM lateral mobility by the submembranous actin-like cytoskeleton or the extracellular 37 wall cytoskeleton appears to be a conserved mechanism in prokaryotic and eukaryotic cells for 38 localizing functional protein complexes in specific membrane regions, thus contributing to the 39 organization of cellular processes. 40 41 42 43 48 actually heterogeneous fluids, with different membrane lipid species laterally segregating into sub-49 micrometer domains, driven by their physico-chemical properties 2-4 . This heterogeneous organization 50 of membrane lipids leads to a heterogeneous distribution of the embedded membrane proteins, which 51 appears essential for their functionality 5 . Membrane microdomains are widely present, mobile 52 structures that vary in size from transient protein clusters measured in nm 6 to large micron-sized stable 53 domains such as desmosomes 7 . These domains harbor proteins specialized in membrane trafficking, 54 cell-cell adhesion, and signal transduction, and they provide targets for viral entry 8 . 56It is now appreciated that membrane proteins can significantly impact membrane organization, in 57 particular stabilizing membrane domains. For instance, the scaffold activity of the flotillin proteins FLO1 58 and FLO2 plays an important role in stabilizing some eukaryotic membrane rafts ("lipid rafts") 9-11 ; it 59 would seem that this raft organization occurs in part through the capture and stabilization of specific 60 lipids by flotillins 12,13 . In addition, the activity of the flotillin scaffold contributes to the recruitment of 61 other raft-associated proteins, thus facilitatin...

Membrane-partitioned cell wall synthesis in mycobacteria

“…In B. subtilis and E. coli , actin homologs like MreB direct peptidoglycan synthesis along the lateral cell surface ( Daniel and Errington, 2003 ; Iwai et al, 2002 ; Shi et al, 2018 ; Zhao et al, 2017 ). They also organize the membrane into domains of increased ( Oswald et al, 2016 ; Strahl et al, 2014 ) and decreased fluidity ( Wagner et al, 2020 ). Global reductions in membrane fluidity interfere with the assembly and motion of B. subtilis MreB ( Zielińska et al, 2020 ; Gohrbandt et al, 2019 ; Kurita et al, 2020 ), potentially indicating a feedback loop between the physical state of the membrane and MreB-directed cell wall elongation.…”

“…We wondered whether the peptidoglycan polymer itself might contribute to the membrane partitioning that organizes its synthesis. In B. subtilis , for example, enzymatic removal of the cell wall delocalizes membrane staining by a lipophilic fluorescent dye ( Muchová et al, 2011 ) and enhances the mobility of membrane domain-associated flotillin proteins ( Wagner et al, 2020 ). To test this hypothesis in M. smegmatis , we spheroplasted bacteria that expressed MurG-Dendra2 or DivIVA-eGFP-ID and mCherry-GlfT2.…”

The cell wall polymer initiates plasma membrane partitioning in mycobacteria

“…One factor that maintains the behavior of membrane components across different domains of life is physical connection to extracellular surfaces. For example, enzymatic removal of neuronal extracellular matrix (Frischknecht et al, 2009) or the plant or bacterial cell wall (Daněk et al, 2020; Feraru et al, 2011; Martinière et al, 2012; McKenna et al, 2019; Wagner et al, 2020) can alter some of which are normally enriched in domains. Genetic or pharmacological inhibition of cell wall synthesis sometimes, but not always, disrupts membrane partitioning or proxies thereof (Feraru et al, 2011)(Daněk et al, 2020; Feraru et al, 2011; García-Heredia et al, 2021; Hayashi et al, 2018; Wagner et al, 2020).…”

“…For example, enzymatic removal of neuronal extracellular matrix (Frischknecht et al, 2009) or the plant or bacterial cell wall (Daněk et al, 2020; Feraru et al, 2011; Martinière et al, 2012; McKenna et al, 2019; Wagner et al, 2020) can alter some of which are normally enriched in domains. Genetic or pharmacological inhibition of cell wall synthesis sometimes, but not always, disrupts membrane partitioning or proxies thereof (Feraru et al, 2011)(Daněk et al, 2020; Feraru et al, 2011; García-Heredia et al, 2021; Hayashi et al, 2018; Wagner et al, 2020). The molecular mechanisms by which physical, bilayer-extrinsic interactions control membrane partitioning are not known.…”

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides

“…MreB also co-localizes with RIFs both in B. subtilis and E. coli, but less strictly than MurG and PlsX (Strahl et al, 2014;Oswald et al, 2016). In contrast, flotillins are proteins that are thought to be associated with and stabilize lipid rafts, may be used as proxies for rigid membrane domains (Lopez and Kolter, 2010;Bach and Bramkamp, 2013;Wagner et al, 2020). In B. subtilis known flotillins are the integral membrane protein FloA and the peripheral FloT (Figure 4) (Dempwolff et al, 2012).…”