scholarly journals Polarity sensitive probes for super resolution STED microscopy

2017 ◽  
Author(s):  
E Sezgin ◽  
F Schneider ◽  
V Zilles ◽  
E Garcia ◽  
D Waithe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Membrane ◽  
Membrane Lipids ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Molecular Organization ◽  
Live Cells ◽  
Sted Microscopy ◽  
Two Photon Microscopy ◽  
Polarity Sensitive

AbstractThe lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

scholarly journals STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

2021 ◽  
Author(s):  
Pablo Carravilla ◽  
Anindita Dasgupta ◽  
Gaukhar Zhurgenbayeva ◽  
Dmytro I. Danylchuk ◽  
Andrey S. Klymchenko ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Spatial Resolution ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Live Cell ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Temporal And Spatial ◽  
Polarity Sensitive

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolution, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

Phosphoinositide lipids and cell polarity: linking the plasma membrane to the cytocortex

2012 ◽  
Vol 53 ◽  
pp. 15-27 ◽  
Author(s):  
Michael P. Krahn ◽  
Andreas Wodarz
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Membrane Lipids ◽  
Leading Edge ◽  
Cell Types ◽  
Specific Protein ◽  
Molecular Organization ◽  
Apical Side ◽  
Cell Substrate ◽  
Basolateral Side

Many cell types in animals and plants are polarized, which means that the cell is subdivided into functionally and structurally distinct compartments. Epithelial cells, for example, possess an apical side facing a lumen or the outside environment and a basolateral side facing adjacent epithelial cells and the basement membrane. Neurons possess distinct axonal and dendritic compartments with specific functions in sending and receiving signals. Migrating cells form a leading edge that actively engages in pathfinding and cell-substrate attachment, and a trailing edge where such attachments are abandoned. In all of these cases, both the plasma membrane and the cytocortex directly underneath the plasma membrane show differences in their molecular composition and structural organization. In this chapter we will focus on a specific type of membrane lipids, the phosphoinositides, because in polarized cells they show a polarized distribution in the plasma membrane. They furthermore influence the molecular organization of the cytocortex by recruiting specific protein binding partners which are involved in the regulation of the cytoskeleton and in signal transduction cascades that control polarity, growth and cell migration.

scholarly journals Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles

2009 ◽  
Vol 424 (2) ◽  
pp. 163-167 ◽  
Author(s):  
Ilya Levental ◽  
Fitzroy J. Byfield ◽  
Pramit Chowdhury ◽  
Feng Gai ◽  
Tobias Baumgart ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Membrane Vesicles ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Lipid Phase ◽  
Plasma Membrane Vesicles ◽  
Liquid Ordered ◽  
Lipid Phase Separation

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

Triple-Color Super-Resolution Imaging of Live Cells: Resolving Submicroscopic Receptor Organization in the Plasma Membrane

2012 ◽  
Vol 51 (20) ◽  
pp. 4868-4871 ◽  
Author(s):  
Stephan Wilmes ◽  
Markus Staufenbiel ◽  
Domenik Liße ◽  
Christian P. Richter ◽  
Oliver Beutel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Super Resolution ◽  
Live Cells ◽  
Resolution Imaging

Triple-Color Super-Resolution Imaging of Live Cells: Resolving Submicroscopic Receptor Organization in the Plasma Membrane

2012 ◽  
Vol 124 (20) ◽  
pp. 4952-4955 ◽  
Author(s):  
Stephan Wilmes ◽  
Markus Staufenbiel ◽  
Domenik Liße ◽  
Christian P. Richter ◽  
Oliver Beutel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Super Resolution ◽  
Live Cells ◽  
Resolution Imaging

scholarly journals Phase Partitioning of Peptide Anchors in Plasma Membrane Vesicles Predicts Their Recruitment to B Cell Receptor Clusters in Live Cells

2019 ◽  
Vol 116 (3) ◽  
pp. 164a-165a
Author(s):  
Sarah A. Shelby ◽  
Ivan C. Serrano ◽  
Kandice R. Levental ◽  
Ilya Levental ◽  
Sarah L. Veatch
Keyword(s):  
Plasma Membrane ◽  
B Cell ◽  
Membrane Vesicles ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Live Cells ◽  
Plasma Membrane Vesicles ◽  
Phase Partitioning ◽  
Receptor Clusters

scholarly journals Lipid Composition but not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 415 ◽  
Author(s):  
Iztok Urbančič ◽  
Juliane Brun ◽  
Dilip Shrestha ◽  
Dominic Waithe ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Molecular Mobility ◽  
Lipid Membrane ◽  
Dynamic Properties ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Membrane Composition ◽  
Hiv 1

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from which it buds out. Previous studies have shown that the HIV-1 envelope is an environment of very low mobility, with the diffusion of incorporated proteins two orders of magnitude slower than in the plasma membrane. One of the reasons for this difference is thought to be the HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing, which has, until now, been difficult to assess experimentally. To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1–1 µm), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1–1 µm sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles’ surface. This observation suggests that the composition of the envelope rather than the particle geometry contributes to the previously described low mobility of proteins on the HIV-1 surface. Our vesicle-based study thus provides further insight into the dynamic properties of the surface of individual HIV-1 particles, as well as paves the methodological way towards better characterisation of the properties and function of viral lipid envelopes in general.

Changes in plasma-membrane lipid composition: a strategy for acclimation to copper stress

2000 ◽  
Vol 28 (6) ◽  
pp. 905-907 ◽  
Author(s):  
A. H. Berglund ◽  
M. F. Quartacci ◽  
C. Liljenberg
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Membrane Lipids ◽  
Membrane Vesicles ◽  
Membrane Lipid ◽  
Fatty Acyl ◽  
Copper Stress ◽  
Protein Ratio ◽  
Root Cells ◽  
Wheat Seedlings

Wheat seedlings were grown hydroponically in the presence of 50 μM Cu2+. The copper stress resulted in plasma-membrane (PM) changes of the root cells as altered lipid composition, a decreased phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio from 0.7 to 0.3, a decreased fatty acyl unsaturation and a decrease in the lipid/protein ratio. Membrane vesicles made from total lipid extracts of isolated PMs of wheat grown under copper excess showed a remarkably low permeability to polar molecules like glucose, as compared with the control, and no difference in proton permeability. Permeability studies of vesicles of plasma-membrane lipids, which were selectively modified by addition of specific lipids such as PC and PE, were also performed. The results are discussed with emphasis on the role of the increased PE proportion.

scholarly journals A synergistic strategy to develop photostable and bright dyes with long Stokes shift for super-resolution microscopy

2021 ◽  
Author(s):  
gangwei jiang ◽  
Tian-Bing Ren ◽  
Elisa D’Este ◽  
mengyi xiong ◽  
Bin Xiong ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Stokes Shift ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Live Cells ◽  
Sted Microscopy ◽  
New Class ◽  
Two Photon ◽  
Cellular Environment ◽  
Super Resolution Microscopy

Abstract The quality and application of super-resolution fluorescence imaging greatly lie in the properties of fluorescent probes. However, conventional fluorophores in a cellular environment often suffer from low brightness, poor photostability, and short Stokes shift (< 30 nm). Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibronic structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability as well as Stokes shift. The new fluorophore BDQF-6 exhibits around twofold greater brightness (ε × Φ = 6.6 × 104 L·mol− 1·cm− 1) and Stokes shift (56 nm) than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, BDQF-6 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine- and JF608-based probes, known as photostable fluorophores for STED imaging. More BDQF-6 derivatives were developed next, allowing us to perform wash-free organelles (mitochondria and lysosome) staining and protein labeling with ultrahigh signal-to-noise ratios (up to 106 folds) in confocal and STED microscopy of live cells, or two-photon and 3D STED microscopy of fixed cells. Furthermore, the strategy was well generalized to different types of dyes (pyronin, rhodol, coumarin, and Boranil), offering a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

scholarly journals Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

2016 ◽  
Author(s):  
Falk Schneider ◽  
Mathias P Clausen ◽  
Dominic Waithe ◽  
Thomas Koller ◽  
Gunes Ozhan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Mobility ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Correlation Spectroscopy ◽  
Diffusion Regime ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Actin Cortex ◽  
Hindered Diffusion

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling. These have been suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access the sub-diffraction diffusion regime of different fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the cellular plasma membrane, and compare it to the diffusion regime of these molecules in cell-derived actin-free giant plasma membrane vesicles (GPMVs). We show that phospholipids and sphingomyelin, which undergo hindered diffusion in the live cell membrane, diffuse freely in the GPMVs. In contrast to sphingomyelin, which is transiently trapped on molecular-scale complexes in intact cells, diffusion of the ganglioside lipid GM1 suggests transient incorporation into nanodomains, which is less influenced by the actin cortex. Finally, our data on GPI-APs indicate two molecular pools in living cells, one pool showing high mobility with trapped and compartmentalized diffusion, and the other forming immobile clusters both of which disappear in GPMVs. Our data underlines the crucial role of the actin cortex in maintaining hindered diffusion modes of most but not all membrane molecules.

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