scholarly journals Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy

2022 ◽  
Author(s):  
Nirmalya Bag ◽  
Erwin London ◽  
David A Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Fluorescence Correlation Spectroscopy ◽  
Immunoglobulin E ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Membrane Diffusion ◽  
Fluorescence Correlation ◽  
The Impact

Plasma membrane hosts numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation of the plasma membrane is emerging as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the co-existence liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It is recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy we choose two well-established model systems for transbilayer interactions: crosslinking by multivalent antigen of immunoglobulin E bound to receptor FcεRI, and crosslinking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

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.

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

scholarly journals Physicochemical Properties of Chromosomes in Live Cells Characterized by Label-Free Imaging and Fluorescence Correlation Spectroscopy

2019 ◽  
Author(s):  
Tae-Keun Kim ◽  
Byong-Wook Lee ◽  
Fumihiko Fujii ◽  
Kee-Hang Lee ◽  
YongKeun Park ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Fluorescence Correlation Spectroscopy ◽  
Three Dimensional ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Optical Diffraction ◽  
Live Cells ◽  
Label Free ◽  
Mitotic Chromosomes ◽  
Fluorescence Correlation

AbstractThe cell nucleus is a three-dimensional, dynamic organelle that is organized into many subnuclear bodies, such as chromatin and nucleoli. The structure and function of these bodies is maintained by diffusion and interactions between related factors as well as dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome itself during cell division are not yet fully understood. Physical parameters, such as the refractive index (RI), volume of the mitotic chromosome, and diffusion coefficients of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser scanning microscopy and fluorescence correlation spectroscopy. Variance in these parameters correlated among various osmotic conditions, suggesting that changes in RI are consistent with those in the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobility of fluorescent proteins.

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