Lipid Diffusion within Black Lipid Membranes Measured with Dual-Focus Fluorescence Correlation Spectroscopy

ChemPhysChem ◽  
2012 ◽  
Vol 13 (4) ◽  
pp. 990-1000 ◽  
Author(s):  
Kerstin Weiß ◽  
Jörg Enderlein
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Lipid Membranes ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Black Lipid Membranes ◽  
Lipid Diffusion

Quantifying Lipid Diffusion by Fluorescence Correlation Spectroscopy: A Critical Treatise

Langmuir ◽  
2012 ◽  
Vol 28 (37) ◽  
pp. 13395-13404 ◽  
Author(s):  
Fabian Heinemann ◽  
Viktoria Betaneli ◽  
Franziska A. Thomas ◽  
Petra Schwille
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Lipid Diffusion

scholarly journals Single-lipid dynamics in phase-separated supported lipid bilayers

2020 ◽  
Author(s):  
Xinxin Woodward ◽  
Christopher V. Kelly
Keyword(s):  
Lipid Bilayers ◽  
Fluorescence Correlation Spectroscopy ◽  
Cholesterol Content ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Supported Lipid Bilayers ◽  
Membrane Composition ◽  
Fluorescence Correlation ◽  
Diffusion Rates ◽  
Lipid Diffusion

ABSTRACTPhase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases.HIGHLIGHTSLipid diffusion varies with the lipid phases, temperature, and aggregationAggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopyMembrane with higher cholesterol content or at low temperature have more aggregatesA more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content

scholarly journals Bacterial pore-forming proteins induce non-monotonic dynamics due to lipid ejection and crowding

2020 ◽  
Author(s):  
Ilanila Ilangumaran Ponmalar ◽  
K. G. Ayappa ◽  
J. K. Basu
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Bacterial Virulence ◽  
Correlation Spectroscopy ◽  
Listeriolysin O ◽  
Oligomeric State ◽  
Fluorescence Correlation ◽  
Confocal Fluorescence ◽  
Membrane Bound ◽  
Lipid Diffusion ◽  
Free Area

ABSTRACTDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors require an understanding of the target cellular response to combat rising antimicrobial resistance. Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane disruption and related lipid mediated active repair processes during attack by PFTs remain largely unexplored. We report counter intuitive and non-monotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane bound oligomers of a prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO). The observed protein concentration dependent dynamical cross-over is correlated with transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events due to a preponderance of ring-like pore states. At higher protein concentrations where membrane inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodelling and mechanical property variations, providing unique insights into developing strategies to combat virulent bacterial pathogens responsible for several infectious diseases.SIGNIFICANCEDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors requires understanding target cellular responses and cellular defence strategies to combat rising antimicrobial resistant strains. While it is well understood that PFTs exist in a wide variety of oligomeric states, the underlying membrane response to these states is unexplored. Using confocal fluorescence correlation spectroscopy and a membrane free area based model we relate non-monotonic variations in the lipid diffusivity arising from an interplay of lipid ejection events and membrane crowding due to variations in concentration of membrane bound listeriolysin O. Our observations have a direct bearing on understanding cellular defense and repair mechanisms effective during initial stages of bacterial infection and intrinsically connected to the underlying membrane fluidity.

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