Measurement of Cell Membrane Fluidity by Laurdan GP: Fluorescence Spectroscopy and Microscopy

Scheinpflug, Kathi; Krylova, Oxana; Strahl, Henrik

doi:10.1007/978-1-4939-6634-9_10

Cited by 51 publications

(57 citation statements)

References 11 publications

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“…The average of three measurements for each preparation was calculated to determine corresponding laurdan GP values. See Scheinpflug et al 69. for detailed protocols and discussion of the laurdan-based fluidity measurements.…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

Scheinpflug

Wenzel

Krylova

et al. 2017

Sci Rep

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109

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The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides.

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Section: Methodsmentioning

confidence: 99%

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

Scheinpflug

Wenzel

Krylova

et al. 2017

Sci Rep

Self Cite

109

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“…To test this hypothesis, we analyzed the local membrane fluidity of both B. subtilis and E. coli with the fluiditysensitive membrane dye Laurdan. Laurdan exhibits a fluidity-dependent shift in its fluorescence emission spectrum, which in turn allows local membrane fluidity to be measured as Laurdan generalized polarization (GP) (Parasassi et al, 1990;Scheinpflug et al, 2017a;Wenzel et al, 2018). Indeed, when B. subtilis and E. coli cells were depleted for fluidity-promoting fatty acids, domain formation associated with local differences in membrane fluidity was observed.…”

Section: Consequences Of Low Membrane Fluidity On Membrane Homogeneitymentioning

confidence: 99%

“…(v/v) DMF) as described in detail elsewhere (Scheinpflug et al, 2017a;Wenzel et al, 2018). As in case of DPH, the staining of E. coli was carried out with Polymyxin B nonapeptide outer membranepermeabilized cells.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Gohrbandt

Lipski

Grimshaw

et al. 2019

Preprint

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Important physicochemical properties of cell membranes such as fluidity sensitively depend on fluctuating environmental factors including temperature, pH or diet. To counteract these disturbances, living cells universally adapt their lipid composition in return. In contrast to eukaryotic cells, bacteria tolerate surprisingly drastic changes in their lipid composition while retaining viability, thus making them a more tractable model to study this process. Using the model organisms Escherichia coli and Bacillus subtilis, which regulate their membrane fluidity with different fatty acid types, we show here that inadequate membrane fluidity interferes with essential cellular processes such as morphogenesis and maintenance of membrane potential, and triggers large-scale lipid phase separation that drives protein segregation into the fluid phase. These findings illustrate why lipid homeostasis is such a critical cellular process. Finally, our results provide direct in vivo support for current in vitro and in silico models regarding lipid phase separation and associated protein segregation.Keywords: Lipid phase separation, lipid domains, protein partitioning, membrane fluidity, homeoviscous adaptation, Escherichia coli, Bacillus subtilis, WALP, FOF1 ATP synthase liquid-disordered phase characterized by low packing density and high diffusion rates that forms the regular state of biological membranes, (ii) the more ordered, cholesterol/hopanoid-dependent liquidordered phase found in biological membranes in form of lipid rafts, and (iii) the gel phase characterized by dense lipid packing with little lateral or rotational diffusion, which is generally assumed to be absent in biologically active membranes (Schmid, 2017;Veatch, 2007). In fact, the temperature associated with gel phase formation has been postulated to define the lower end of the temperature range able to support vital cell functions (Burns et al., 2017;Drobnis et al., 1993;Ghetler et al., 2005). At last, the lipid phases can co-exist, resulting in separated membrane areas exhibiting distinctly different composition and characteristics (Baumgart et al., 2007;Elson et al., 2010;Heberle and Feigenson, 2011). This principal mechanism of lipid domain formation is best studied in the context of lipid rafts (Lingwood and Simons, 2010).While in vitro and in silico approaches with simplified lipid models have provided detailed insights into the complex physicochemical behavior of lipid bilayers, testing the formed hypotheses and models in the context of protein-rich biological membranes is now crucial. Bacteria tolerate surprisingly drastic changes in their lipid composition and only possess one or two membrane layers as part of their cell envelope. Consequently, bacteria are both a suitable and a more tractable model to study the fundamental biological process linked to membrane fluidity and phase separation in vivo.We analyzed the biological importance of membrane homeoviscous adaptation in Escherichia coli (phylum Proteobacteria) and Bacillus subtilis (phylum Firmic...

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“…Endocytosis can be further divided into macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and clathrin-/caveolin-independent endocytosis [19]. It is widely accepted that most nanoscaled objects are transported across the lipid bilayer through clathrin-mediated endocytosis [20][21][22], which is highly dependent upon the fluidity of the cell membrane [23]. Although the influence of nanomaterials has been extensively studied, the influence of stem cells, especially with respect to passaging, has rarely been reported.…”

Section: Introductionmentioning

confidence: 99%

Uptake of magnetic nanoparticles for adipose-derived stem cells with multiple passage numbers

et al. 2017

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With the increasingly promising role of nanomaterials in tissue engineering and regenerative medicine, the interaction between stem cells and nanoparticles has become a critical focus. The entry of nanoparticles into cells has become a primary issue for effectively regulating the subsequent safety and performance of nanomaterials in vivo. Although the influence of nanomaterials on endocytosis has been extensively studied, reports on the influence of stem cells are rare. Moreover, the effect of nanomaterials on stem cells is also dependent upon the action mode. Unfortunately, the interaction between stem cells and assembled nanoparticles is often neglected. In this paper, we explore for the first time the uptake of γ-Fe 2 O 3 nanoparticles by adipose-derived stem cells with different passage numbers. The results demonstrate that cellular viability decreases and cell senescence level increases with the extension of the passage number. We found the surface appearance of cellular membranes to become increasingly rough and uneven with increasing passage numbers. The iron content in the dissociative nanoparticles was also significantly reduced with increases in the passage number. However, we observed multiple-passaged stem cells cultured on assembled nanoparticles to have similarly low iron content levels. The mechanism may lie in the magnetic effect of γ-Fe 2 O 3 nanoparticles resulting from the field-directed assembly. The results of this work will facilitate the understanding and translation of nanomaterials in the clinical application of stem cells.

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Measurement of Cell Membrane Fluidity by Laurdan GP: Fluorescence Spectroscopy and Microscopy

Cited by 51 publications

References 11 publications

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Uptake of magnetic nanoparticles for adipose-derived stem cells with multiple passage numbers

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