No 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.

words ~40 wordsHigh-speed single-molecule imaging indicated that CD59-cluster rafts and GM1-cluster rafts stably induced in the plasma membrane outer leaflet generated nano-scale transbilayer raft phases, which continually and transiently recruited Lyn and H-Ras in the inner leaflet by cooperative raft-lipid interactions. 5 Abstract 160 words ≤ 160 words Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59-cluster rafts and GM1-cluster rafts stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK, 10 continually recruited Lyn and H-Ras right beneath them in the inner leaflet, with dwell lifetimes of <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the non-raftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Since 15 GM1-cluster rafts recruited Lyn and H-Ras as efficiently as CD59-cluster rafts, and the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipidanchored signaling molecules by lateral raft-lipid interactions, and thus serve as a key signal transduction platform. 15 al., 2011; Suzuki et al., 2012;Kusumi et al., 2014;Raghupathy et al., 2015). Nevertheless, exactly how raft domains or raft-based lipid interactions participate in the transbilayer signal transduction of GPI-ARs remains unknown. Indeed, raft-based interactions might even be involved in the signal transduction by transmembrane receptors (Coskun et al., 2011;Chung et al., 2016;Shelby et al., 2016). 20In giant unilamellar vesicles undergoing liquid-ordered (Lo)/liquid-disordered (Ld)phase separation, the Lo/Ld-phase domains in the outer leaflet spatially match the same domains in the inner leaflet, indicating strong interbilayer coupling due to phase separation across the bilayer (Collins and Keller, 2008;Blosser et al., 2015). In living cells, the long-chain phosphatidylserine present in the PM inner leaflet was proposed to play key 25 roles in the transbilayer coupling (Raghupathy et al., 2015). However, the mechanisms of 4 / 57 transbilayer coupling in the PM for the induction of signal transduction are not well understood.Using CD59 as a prototypical GPI-AR, our previous single fluorescent-molecule imaging showed that nanoparticle-induced CD59 clusters form stabilized raft domains with diameters on the order of 10 nm in the PM outer leaflet, which in turn continually recruit 5 Giα, Lyn, and PLCγ2 molecules one after another in a manner dependent on raft-lipid interactions, triggering the IP3-Ca 2+ signaling pathway. Therefore, the CD59 clusters were termed "CD59-cluster signaling rafts" or simply "CD59-cluster rafts" (Stefanova et al., 1991;Suzuki et al., 2007a; Suzuki et al., 2007b;Simons and Gerl, 2010; Su...

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes &lt;0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft–lipid interactions and thus serve as a key signal transduction platform.

The DAX-1 (also known as AHC) gene encodes an unusual member of the nuclear hormone receptor superfamily. DAX-1 plays a critical role during gonadal and adrenal differentiation since mutations of the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. In recent studies, DAX-1 was reported to function as a transcriptional suppressor of Ad4BP/SF-1, a critical transcription factor in gonadal and adrenal differentiation. With respect to implication of Ad4BP/SF-1 in the transcriptional regulation of the DAX-1 gene, inconsistent findings have been previously reported. We investigated the upstream region of the mouse Dax-1 (also known as Ahch) gene and identified a novel Ad4/SF-1 site by transient transfection and electrophoretic mobility shift assays. In addition, immunohistochemical analyses with a specific antibody to Dax-1 indicated the presence of immunoreactive cells in steroidogenic tissues, pituitary gland, and hypothalamus. Although the distributions of Dax-1 and Ad4BP/SF-1 were very similar, they were not completely identical. The expression of Dax-1 was significantly impaired in knock-out mice of the Ftz-f1 gene, which encodes Ad4BP/ SF-1. Taken together, our findings indicate that Ad4BP/SF-1 controls the transcription of the Dax-1 gene.