Z-Scan Fluorescence Correlation Spectroscopy as a Tool for Diffusion Measurements in Planar Lipid Membranes

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid–lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21218149 ◽

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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Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models

10.1101/2020.03.09.983874 ◽

2020 ◽

Author(s):

R. Tzoneva ◽

T. Stoyanova ◽

A. Petrich ◽

D. Popova ◽

A. Momchilova ◽

...

Keyword(s):

Lipid Bilayers ◽

Cancer Cell ◽

Fluorescence Correlation Spectroscopy ◽

Lipid Membranes ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Membrane Lipid ◽

Correlation Spectroscopy ◽

Generalized Polarization ◽

Fluorescence Correlation

ABSTRACTAlkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, Erufosine (EPC3) is a promising drug for the treatment of several types of tumors which has not been sufficiently characterized. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy techniques (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy and generalized polarization imaging) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 might be linked to its effects on fundamental biophysical properties of lipid membranes in cancer cells.

Download Full-text