Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements

Pokorná, Šárka; Ventura, Ana E.; Santos, T. C.; Hof, Martin; Prieto, Manuel; Futerman, Anthony H.; Silva, Liana C.

doi:10.1016/j.jphotobiol.2022.112404

Cited by 15 publications

(9 citation statements)

References 70 publications

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“…FRET measurements to quantify the membrane potential were carried-out via spectral imaging, instead of the standard, filter-based method [ 49 ]. The spectral imaging approach and detection in the photon-counting mode were previously shown to provide more information and to improve the accuracy and sensitivity of the measurements [ 70 , 71 ]. For the FRET analysis, ROIs at the equatorial plane of isolated membrane regions were chosen within the average intensity map and the RG ratio was quantified via the spectral information from each pixel.…”

Section: Resultsmentioning

confidence: 99%

Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane

Petrich,

Chiantia

2023

Viruses

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The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid–lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.

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Section: Resultsmentioning

confidence: 99%

Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane

Petrich,

Chiantia

2023

Viruses

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“…Comparable effects were already observed in the literature. [43][44][45][46] Our long-term goal is to nd more general rules that could be a guide in the development of new antibacterial agents acting on the bacterial cytoplasmic membrane. The current aim was to explore structure-activity relationships of LEGO-LPPOs molecules differing specically in the lengths of their hydrophobic modules when other parts of the molecules remained unchanged.…”

Section: Discussionmentioning

confidence: 99%

LEGO-lipophosphonoxins: length of hydrophobic module affects permeabilizing activity in target membranes of different phospholipid composition

Dugić,

Brzobohatá,

Mojr

et al. 2024

RSC Adv.

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“…For Laurdan, the protocol was defined as follows: 410 nm excitation, 5 nm excitation bandpass, emission one at 440 nm, 10 nm emission bandpass, emission two at 490 nm, 10 nm emission bandpass, and a target measurement error of 1%. Although Laurdan is customarily excited at 350 nm, excitation at longer wavelengths improved the signal/noise ratio, which is theorized to be a consequence of photo-selection [ 25 , 31 , 34 ]. All fluorescence intensity measurements were carried out in triplicate and at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Multiple fluorescent probes for evaluating membrane status and lipid organization are commonly used, and many alternatives have been proposed for adoption. Among these, 6-dodecanoyl-2-dimethylaminonaphtalene (Laurdan) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been extensively used as reporters in numerous natural and artificial membrane systems [ 26 , 27 , 33 , 34 , 35 , 36 , 37 , 38 ]. While neither probe directly responds to lipid ordering, their optical responses adjust to changes in the physical environment.…”

Section: Introductionmentioning

confidence: 99%

“…While neither probe directly responds to lipid ordering, their optical responses adjust to changes in the physical environment. Laurdan’s dipolar relaxation leads to spectral shifts [ 5 , 30 , 31 , 32 , 34 , 38 , 39 ], and DPH’s anisotropy is affected by the rotational diffusion of the dye [ 29 , 36 , 37 ]. Both mechanisms are influenced by the ordering of the lipids in the membranes in which the probes reside.…”

Section: Introductionmentioning

confidence: 99%

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Hypo-Osmotic Stress and Pore-Forming Toxins Adjust the Lipid Order in Sheep Red Blood Cell Membranes

Whiting,

Stanton,

Kucheriava

et al. 2023

Membranes

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Lipid ordering in cell membranes has been increasingly recognized as an important factor in establishing and regulating a large variety of biological functions. Multiple investigations into lipid organization focused on assessing ordering from temperature-induced phase transitions, which are often well outside the physiological range. However, particular stresses elicited by environmental factors, such as hypo-osmotic stress or protein insertion into membranes, with respect to changes in lipid status and ordering at constant temperature are insufficiently described. To fill these gaps in our knowledge, we exploited the well-established ability of environmentally sensitive membrane probes to detect intramembrane changes at the molecular level. Our steady state fluorescence spectroscopy experiments focused on assessing changes in optical responses of Laurdan and diphenylhexatriene upon exposure of red blood cells to hypo-osmotic stress and pore-forming toxins at room temperature. We verified our utilized experimental systems by a direct comparison of the results with prior reports on artificial membranes and cholesterol-depleted membranes undergoing temperature changes. The significant changes observed in the lipid order after exposure to hypo-osmotic stress or pore-forming toxins resembled phase transitions of lipids in membranes, which we explained by considering the short-range interactions between membrane components and the hydrophobic mismatch between membrane thickness and inserted proteins. Our results suggest that measurements of optical responses from the membrane probes constitute an appropriate method for assessing the status of lipids and phase transitions in target membranes exposed to mechanical stresses or upon the insertion of transmembrane proteins.

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Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements

Cited by 15 publications

References 70 publications

Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane

Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane

LEGO-lipophosphonoxins: length of hydrophobic module affects permeabilizing activity in target membranes of different phospholipid composition

Hypo-Osmotic Stress and Pore-Forming Toxins Adjust the Lipid Order in Sheep Red Blood Cell Membranes

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