Fluorescent Methods to Study Biological Membranes by Yves Mély & Guy Duportail

Author:Yves Mély & Guy Duportail
Language: eng
Format: epub
Publisher: Springer Berlin Heidelberg, Berlin, Heidelberg

In particular, membrane microdomains enriched in sphingolipids and cholesterol known as lipid rafts [41] have been postulated to favor segregation of specific membrane receptors and glycosylphosphatidylinositol-anchored proteins (GPI-APs). Lipid rafts have been shown to play an important role in various biological phenomena, ranging from cell adhesion [42] to pathogen binding [43], endocytosis [44], and immune cell signaling [45]. However, there is no consensus yet on the spatiotemporal regimes regulating lipid rafts in the resting state. This is mainly because their evaluation requires tools compatible with live cell imaging and capable of resolving heterogeneities at the nanoscale, a spatial regime not accessible to standard fluorescence microscopy. Nevertheless, recent advances in biophysical techniques have led to the identification of cholesterol-dependent nanoassemblies, supporting their existence in living cells [14, 46, 47].

Besides lipid-lipid interactions that serve to target proteins to lipid rafts [3, 48], protein-lipid [49] as well as protein-protein [50] interactions can have a significant impact on lipid raft localization. It should be noted that studies have also demonstrated the formation of microdomains in activated T cells exclusively created by protein-protein networks and not maintained by interactions with lipid rafts [40]. The diffusional trapping through protein-protein interactions generated microdomains that could recruit or reject specific cell membrane proteins during signal transduction.

Protein-protein interactions can be also modulated by the presence of tetraspanins, a large family of proteins that traverse the membrane four times [51]. Tetraspanins have been found to interact strongly among each other [52] as well as with other integral proteins [53], which has led to the hypothesis that tetraspanins are involved in the lateral sorting of proteins on the cell membrane. Indeed, not only can tetraspanins organize themselves in a structure also known as the tetraspanin web [54, 55], but on endothelial cells the incorporation of a multitude of cell adhesion proteins in tetraspanin-enriched areas have been identified as preorganized adhesion platforms [56].

Another family of proteins that can form networks is represented by galectins. Galectins can generate scaffolds or lattices through interactions of their carbohydrate recognition domains (CRD). These CRDs can bind glycosylated proteins at the membrane, and therefore galectins that have multiple CRDs can recruit and compartmentalize receptors. Galectin-1, for example, has been found essential for EGFR signaling through the formation and sustaining of active H-Ras nanoclusters [48, 57]. In addition, other galectins such as galectin-3 can even form links between carbohydrate and noncarbohydrate ligands [58]. As such, galectins can act as versatile molecular organizers of the cell membrane.

Aside from lipid and protein interactions, the actin cytoskeleton that lies just below the membrane is also able to compartmentalize the membrane. Transmembrane proteins anchored to the actin cytoskeleton meshwork can act as “rows of pickets” that temporarily confine diffusing lipids and proteins [59, 60]. More recently, an exact relationship between protein dynamics and actin-defined compartments has been directly visualized [61]. In this elegant paper, the authors not only showed that the diffusion of the IgE receptor (FcεRI) is confined within actin-poor areas but also demonstrated that the size and location of these

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