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

Christian Kleusch,Rudolf Merkel,Cornelia Monzel,Krishna Chander Sridhar,Agnes Csiszár,Bernd Hoffmann

doi:10.3390/ijms21218149

Abstract

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).

Highlights

Adhesion of mammalian cells to the extracellular matrix is decisive for physiological and pathological processes such as tissue maintenance and repair, cell locomotion, and scar formation
As was verified by immunostaining cardiac myofibroblasts could be clearly identified within the mixed co-culture using phase contrast microscopy based on their larger size and rounder shape when compared to myocytes
Analyzing FAs, reflection interference microscopy (RIM) clearly showed many dark streaks (0.5–2 μm width, 1–6 μm length) all over the cell body (Figure 1A). These structures are typical for focal adhesions [1]. This assignment was verified by imaging cardiac myofibroblasts transfected with green fluorescent protein (GFP)-vinculin (Figure 1A), which is a well-established and reliable marker protein of focal adhesions [44]

Summary

Introduction

Adhesion of mammalian cells to the extracellular matrix is decisive for physiological and pathological processes such as tissue maintenance and repair, cell locomotion, and scar formation In all these processes, mechanical forces have to be transmitted across the cell membrane. As the membrane is fluid and cannot sustain static forces, transmembrane protein linkages are indispensable for this task They are furnished by focal adhesions (FAs) that are seen in cultured cells as micron-sized, protein-rich plaques [1]. The central elements of FAs are heterodimeric cell adhesion molecules of the integrin family They are composed of one α and one β chain that span the plasma membrane and together form binding domains for molecular motifs on the extracellular matrix (ECM). Integrins are connected to a plaque of many different adaptor molecules such as vinculin and talin that together form connections to the intracellular actin cytoskeleton [9,10,11]

Methods

Results

Discussion

Conclusion