Independent mobility of proteins and lipids in the plasma membrane of <scp><i>E</i></scp><i>scherichia coli</i>

Nenninger, Anja; Mastroianni, Giulia; Robson, Alexander; Lenn, Tchern; Xue, Qing; Leake, Mark C.; Mullineaux, Conrad W.

doi:10.1111/mmi.12619

Cited by 56 publications

(70 citation statements)

References 54 publications

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“…Therefore, as for GHUV, an intermediate diffusion coefficient value is found, and shows that despite the asymmetric character of the membrane, the lipid lateral diffusion coefficient is lowered by the copolymer chains, suggesting some interdigitation between lipids and copolymer chains in the membrane. The lipid lateral diffusion has been also evaluated in aGHUV at 37 °C and is found to be 2.3 ± 0.7 µm 2 s −1 confirming the impact of temperature on membrane's fluidity,30, 71, 72 and the relevance of our systems, with a lateral diffusion coefficient close to those reported in cells' membrane.…”

Section: Resultssupporting

confidence: 72%

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

et al. 2017

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Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion‐centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)‐b‐poly(ethylene oxide) (PBut‐b‐PEO) and outer monolayer of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half‐life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(lissamine rhodamine B sulfonyl) (DOPE‐rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm2 s−1 at 25 °C and D = 2.3 ± 0.7 μm2 s−1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell‐like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

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Section: Resultssupporting

confidence: 72%

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

et al. 2017

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“…However, a value of ∼ 9 μm 2 s −1 is even faster than measured for membrane protein diffusion in giant‐unilamellar vesicles at very low protein to lipid ratios (Ramadurai et al ., ). We consider it unlikely that 9 μm 2 s −1 represents a true value of diffusion coefficient of a membrane protein in a living bacterium, as to the best of our knowledge no other measurements of membrane protein diffusion report values higher than 0.5 μm 2 s −1 (Deich et al ., ; Nenninger et al ., ), even when measured by SMT.…”

Section: Discussionmentioning

confidence: 90%

Impact of osmotic stress on protein diffusion in Lactococcus lactis

Mika

Schavemaker

Krasnikov

et al. 2014

Molecular Microbiology

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SummaryWe measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Grampositive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria.

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“…We assigned the faster diffusion state of CusA mE to be dominated by free CusA trimers that are not assembled into CusCBA complexes and that might even have a few CusB protein molecules attached ( Fig. 2A (34)(35)(36). Second, the interaction affinity of CusA and CusB in their apo forms is in the micromolar range (24); with cellular CusA protein concentration in the submicromolar range (Fig.…”

Section: Results and Analysismentioning

confidence: 99%

Adaptor protein mediates dynamic pump assembly for bacterial metal efflux

Santiago

Chen

Genova

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.multicomponent efflux complex | substrate-responsive dynamic assembly | periplasmic adaptor protein | metal sensing | single-molecule tracking B acteria are often exposed to harsh environments, including high metal ion concentrations and toxic organic molecules. Efflux of metal ions helps bacteria maintain appropriate intracellular concentrations of essential metals while removing toxic ones (1-6). Efflux of organic molecules, including antibiotics, is a key mechanism for bacterial multidrug resistance (7-14). The tripartite resistance-nodulation-division (RND) family efflux pumps confer major clinically relevant drug resistance in Gram-negative bacteria such as Escherichia coli and the infectious Pseudomonas aeruginosa (7-14). They are composed of a proton-motive-force-driven inner-membrane pump, a periplasmic adaptor protein, and an outer-membrane channel. Once assembled, these pumps traverse the cell periplasm, providing a direct extrusion pathway from the periplasm (and cytoplasm) to the outside of the cell. However, these direct pathways also tightly link the inner and outer membranes, which, if overly stable, would impede the periplasm's plasticity and ability to respond dynamically to external and internal stimuli to buffer the cell from changes in its surroundings (15).How can tripartite efflux pumps operate without compromising the dynamic nature of the periplasm? One possibility is that these efflux complexes are dynamic structures and assemble only in the presence of their substrates. This mechanism has been hypothesized for the E. coli HlyBD-TolC complex (16), in which HlyB is a ATP-binding cassette superfamily efflux pump. However, experimental validation of this mechanism, as well as its relevance to the RND family efflux pumps, remains elusive, partly due to the difficulty in studying two membrane proteins together with a periplasmic protein under physiologically relevant conditi...

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Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli

Cited by 56 publications

References 54 publications

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

Asymmetric Hybrid Polymer–Lipid Giant Vesicles as Cell Membrane Mimics

Impact of osmotic stress on protein diffusion in Lactococcus lactis

Adaptor protein mediates dynamic pump assembly for bacterial metal efflux

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