scholarly journals Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

10.3791/54756 ◽  
2017 ◽  
Author(s):  
Elina Staaf ◽  
Sunitha Bagawath-Singh ◽  
Sofia Johansson
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Plasma Membranes ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

2017 ◽  
Author(s):  
Dominic Waithe ◽  
Falk Schneider ◽  
Jakub Chojnacki ◽  
Mathias Clausen ◽  
Dilip Shrestha ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Spatial Heterogeneity ◽  
Fluorescence Correlation Spectroscopy ◽  
Large Scale ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
The Other ◽  
Statistical Accuracy ◽  
Fluorescence Correlation ◽  
Diffusion Dynamics

AbstractScanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

scholarly journals Diffusion Coefficient of Fluorescent Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane of Cells

2008 ◽  
Vol 19 (4) ◽  
pp. 1663-1669 ◽  
Author(s):  
Urszula Golebiewska ◽  
Marian Nyako ◽  
William Woturski ◽  
Irina Zaitseva ◽  
Stuart McLaughlin
Keyword(s):  
Diffusion Coefficient ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Fluorescence Correlation Spectroscopy ◽  
Plasma Membranes ◽  
Diffusion Constant ◽  
Cell Types ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Outer Leaflet

Phosphatidylinositol 4,5-bisphosphate (PIP2) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP2 on the inner leaflet of the plasma membrane. If a significant fraction of PIP2 is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP2 diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP2 into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average ± SD value from all cell types was D = 0.8 ± 0.2 μm2/s (n = 218; 25°C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 ± 0.8 μm2/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP2 on inner leaflet of these plasma membranes is bound reversibly.

scholarly journals Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites

2020 ◽  
Vol 21 (21) ◽  
pp. 8149
Author(s):  
Christian Kleusch ◽  
Cornelia Monzel ◽  
Krishna Chander Sridhar ◽  
Bernd Hoffmann ◽  
Agnes Csiszár ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Lipid Membranes ◽  
Plasma Membranes ◽  
Focal Adhesions ◽  
Model System ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Adhesion Sites ◽  
Lipid Mobility ◽  
Associated Proteins

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s).

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