Raft-based interactions of gangliosides with a GPI-anchored receptor

Abstract

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

“…In comparison, identically labeled phosphatidylethanolamine appeared to diffuse freely in the membrane (Eggeling et al, 2009), which implied that lipid-cytoskeleton interactions were not responsible for the anomalous cholesterol-dependent sphingolipid diffusion. Noteworthy, this finding of unhindered phosphatidylethanolamine diffusion conflicts with a previous single molecule tracking study (Fujiwara et al, 2002), and subsequent STED-FCS and single molecule tracking studies reported by these authors and others (Andrade et al, 2015; Fujiwara et al, 2016; Komura et al, 2016). Although, the authors of the STED study never concluded that the cholesterol-dependent trapping of sphingomyelin, GM1 and GPI-anchored proteins was indicative of tiny lipid rafts, their results were often cited by others as support for the lipid raft hypothesis (Lingwood and Simons, 2010; Levental and Veatch, 2016).…”

“…Instead, alternative hypotheses that do not involve cohesive sphingolipid-cholesterol interactions must be developed, investigated, and discarded if they prove inconsistent with experimental results. These alternative hypotheses should account for the following observations: The diffusion and distribution of proteins and lipids is influenced by the actin cytoskeleton (Fujiwara et al, 2002, 2016; Mueller et al, 2011; D'Auria et al, 2013; Frisz et al, 2013a,b; Honigmann et al, 2014; Andrade et al, 2015; Sevcsik et al, 2015; Komura et al, 2016). …”

The Total Synthesis of Starfish Ganglioside GP3 Bearing a Unique Sialyl Glycan Architecture

“…[8] Meanwhile, arabinosyld onor 14 was reactedw ith galactosyl acceptor 13 upon the activation by Crich's promoter system, [17] yielding Arafa (1,4)Gal sequence 18 in excellent yield without destroying the allyl ether moiety.A gain, the disaccharide underwent conversion into glycosyli midate donor 20 by following as imilar procedure to that for 17.…”

The Continuing Mystery of Lipid Rafts

“…Indeed, most SR experiments are conducted in chemically fixed cells or membrane sheets 133, 152, 153 , and these preparations can be prone to artifacts because lipids and even some membrane proteins are not immobilized by most fixation protocols 154 . Experiments in live cells are possible, but require experimental and analytical methods that anticipate the fast dynamics expected for raft structures 155, 156 .…”

“…Several approaches that do not rely in image reconstruction have had some success in this area. A recent advance definitively correlated three distinct measures of raft association – DRMs association, ordered phase portioning in GPMVs, and surface patching in live cells – across more than a dozen probes, along with probing the lifetime of the small entities in unperturbed cells, using multi-color single particle tracking 156 . This study has provided some of the strongest evidence to date to link classical raft observations with novel model membranes and modern imaging techniques.…”