Effect of different agents onto multidrug resistant tumor cells revealed by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has been used to analyze the plasma membrane fluidity and heterogeneity of multidrug resistant cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been explored. Firstly, we reveal a modification of plasma membrane heterogeneities according to the addition of a fluidity modulator, benzyl alcohol. On the other hand, revertants such as verapamil and cyclosporin-A appear to act more specifically on the slow diffusion sites, such as lipids microdomains.

Download Full-text

Application of fluorescence correlation spectroscopy to the measurement of agonist binding to a G-protein coupled receptor at the single cell level

Faraday Discussions ◽

10.1039/b307407b ◽

2004 ◽

Vol 126 ◽

pp. 197 ◽

Cited By ~ 23

Author(s):

Stephen J. Briddon ◽

Richard J. Middleton ◽

Andrew S. Yates ◽

Michael W. George ◽

Barrie Kellam ◽

...

Keyword(s):

Single Cell ◽

G Protein ◽

Fluorescence Correlation Spectroscopy ◽

Correlation Spectroscopy ◽

Single Cell Level ◽

Agonist Binding ◽

G Protein Coupled Receptor ◽

Cell Level ◽

Fluorescence Correlation ◽

G Protein Coupled

Download Full-text

3N1330 Analysis of the interaction between protein kinase c and plasma membrane by using fluorescence correlation spectroscopy

Seibutsu Butsuri ◽

10.2142/biophys.42.s197_1 ◽

2002 ◽

Vol 42 (supplement2) ◽

pp. S197

Author(s):

K. Saito ◽

M. Tamura ◽

M. Kinjo

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Fluorescence Correlation Spectroscopy ◽

Correlation Spectroscopy ◽

Fluorescence Correlation ◽

Kinase C

Download Full-text

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

10.1101/163766 ◽

2017 ◽

Cited By ~ 1

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.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0114 ◽

2011 ◽

Vol 22 (18) ◽

pp. 3498-3507 ◽

Cited By ~ 61

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.

Download Full-text