High-speed single-molecule imaging reveals signal transduction by induced transbilayer raft phases

Abstract

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 <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.

Membrane phase separation drives organization at B cell receptor clusters

“…Our observations, combined with other recent studies (Kinoshita et al, 2017;Koyama-Honda et al, 2020;Li et al, 2020;Stone et al, 2017), put to rest a long history of debate regarding the existence of ordered membrane domains often referred to as 'lipid rafts' in cells. Our findings clearly demonstrate that ordered domains can and do exist in intact cells, that they can emerge in response to stimuli (Fig 1), and that their presence impacts the concentration and dynamics of membrane proteins (Figs 4,6).…”

“…Despite decades of research, direct evidence for phase separation within the intact plasma membrane has proven elusive, and the simplistic models used to rationalize rafts either lack utility or are easily disproved, leading to valid skepticism (Kenworthy, 2008;Munro, 2003). Recent years have brought several promising measurements using methods sensitive to single molecule dynamics, molecular-scale organization, or membrane material properties (Eggeling et al, 2009;Koyama-Honda et al, 2020;Sanchez et al, 2012;Sengupta et al, 2007), though directly relating these observations to principles of membrane phase separation has proven difficult. In contrast, plasma membranes isolated from cells can clearly separate into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases (Baumgart et al, 2007), and there is clear evidence that membrane composition is carefully tuned to support a wide variety of biological functions (Ernst et al, 2016;Lingwood and Simons, 2010).…”

Membrane Rafts: Portals for Viral Entry

“…Rafts may induce conformational changes in resident proteins, affecting their activity ( Sezgin et al, 2017 ). Because of this, lipid rafts are essential for maintenance of cellular functions such as signal transduction ( Koyama-Honda et al, 2020 ), receptor activation ( Shi and Ruan, 2020 ), intracellular lipid and protein trafficking ( Ouweneel et al, 2020 ), spatial organization of the plasma membrane ( van IJzendoorn et al, 2020 ), endocytosis ( Nichols, 2003a ) and extracellular vesicle formation ( Skryabin et al, 2020 ). As a consequence of their broad involvement in cell physiology, lipid rafts play an important role in complex processes including immune response ( Varshney et al, 2016 ), host–pathogen interaction ( Bukrinsky et al, 2020 ), cancer development ( Greenlee et al, 2020 ), and cardiovascular disorders ( Das and Das, 2009 ).…”

Transporters Beam Signals from Cell Membranes

“…Live-cell single-molecule fluorescence imaging has become a powerful analytical tool to investigate such cellular processes [134]. Recent advances in single-molecule imaging were developed, which greatly enhanced the imaging time resolutions down to 5.0 nm, an improvement by a factor of 6.7 [135]. The resolution substantially refined the detection of signal transduction mediated by CD59 cluster rafts and raftophilic lipid rafts in the outer leaflet of the PM.…”

“…Dependence on the lateral raft-lipid interactions with cholesterol and molecules with saturated alkyl chains can now be measured by following the stabilized raft-lipid clusters upon the recruitment and activation of cytoplasmic signaling molecules, such as Lyn, H-Ras, and ERK, at the inner leaflet [135]. The result suggests a direct involvement of cholesterol in signal transduction.…”