Tween-20 Induces the Structural Remodeling of Single Lipid Vesicles

Dresser, Lara; Graham, Sarah; Miller, Lisa; Schaefer, Charley; Conteduca, Donato; Johnson, Steven; Leake, Mark C.; Quinn, Steven D.

doi:10.1021/acs.jpclett.2c00704

Cited by 23 publications

(10 citation statements)

References 64 publications

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“…Fluorescence decays were recorded peak emission for GFP of 509 nm at the magic angle and until a peak of 10 000 photon counts were collected. Fluorescence decays were then fitted after reconvolution with the instrument response function using a tri-exponential model (figure 4 b ), where it is the intensity at time, t , normalized to the intensity at t = 0, and t i and a i represent the fluorescence lifetime and fractional amplitude of the i th decay components, as previously [47].…”

Section: Methodsmentioning

confidence: 99%

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

et al. 2022

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In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae . It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

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Section: Methodsmentioning

confidence: 99%

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

et al. 2022

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“…Earlier, Oheim et al discussed the analysis of the integrated intensity and the line intensity plots of single particles by imaging 100 nm fluorescent beads with the TIRF microscopy technique . The authors and multiple other researchers − in the field of single-particle fluorescence imaging with TIRF illuminations have shown that the intensity of single particles fits the Gaussian distribution, and the typical line width of the distribution is between 0.3 and 1 μm. Together, the slightly broader intensity distribution of the labeled virus, the broader width of the line intensity plots of higher intensity particles, and our DLS data indicate an extent of aggregation/size heterogeneity of virus particles in our sample.…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 Binding to Terminal Sialic Acid of Gangliosides Embedded in Lipid Membranes

et al. 2023

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Multiple recent reports indicate that the S protein of SARS-CoV-2 specifically interacts with membrane receptors and attachment factors other than ACE2. They likely have an active role in cellular attachment and entry of the virus. In this article, we examined the binding of SARS-CoV-2 particles to gangliosides embedded in supported lipid bilayers (SLBs), mimicking the cell membrane-like environment. We show that the virus specifically binds to sialylated (sialic acid (SIA)) gangliosides, i.e., GD1a, GM3, and GM1, as determined from the acquired single-particle fluorescence images using a time-lapse total internal reflection fluorescence (TIRF) microscope. The data of virus binding events, the apparent binding rate constant, and the maximum virus coverage on the ganglioside-rich SLBs show that the virus particles have a higher binding affinity toward the GD1a and GM3 compared to the GM1 ganglioside. Enzymatic hydrolysis of the SIA−Gal bond of the gangliosides confirms that the SIA sugar unit of GD1a and GM3 is essential for virus attachment to the SLBs and even the cell surface sialic acid is critical for the cellular attachment of the virus. The structural difference between GM3/GD1a and GM1 is the presence of SIA at the main or branched chain. We conclude that the number of SIA per ganglioside can weakly influence the initial binding rate of SARS-CoV-2 particles, whereas the terminal or more exposed SIA is critical for the virus binding to the gangliosides in SLBs.

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“…Fluorescence decays were then fitted after reconvolution with the instrument response function using a tri-exponential model (Fig. 4B), where It is the intensity at time, t , normalized to the intensity at t = 0, and t i and a i represent the fluorescence lifetime and fractional amplitude of the i th decay components, as previously (48).…”

Section: Methodsmentioning

confidence: 99%

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

Lecinski

Shepherd

Bunting

et al. 2022

Preprint

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In eukaryotes, intracellular physical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, meso- and macro- length scale molecular diffusion, and protein folding. However, mapping the molecular crowding and viscoelastic landscapes in living cells remains challenging. One principal tool to measure viscoelasticity is passive rheology, in which the diffusion of exogenous fluorescent particles internalised in living cells is tracked though time and the physical properties inferred from a marker particle's mean square displacement. Recently, the crGE2.3 Förster Resonance Energy Transfer (FRET) based biosensor has been developed to quantify crowding in cells, though it is unclear how this readout depends on viscous and elastic properties and the molecular weight of the crowder. Here, we present correlative multi-technique and multidimensional data to explore the diffusion and molecular crowding characteristics of common molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as candidate in situ viscoelasticity markers in the cytoplasm of live budding yeast cells, Saccharomyces cerevisiae, and we discuss the limitations of this approach.

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Tween-20 Induces the Structural Remodeling of Single Lipid Vesicles

Cited by 23 publications

References 64 publications

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

SARS-CoV-2 Binding to Terminal Sialic Acid of Gangliosides Embedded in Lipid Membranes

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

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