Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS), a component of Gram-negative bacteria to induce production of pro-inflammatory mediators aiming at eradication of the bacteria. ...Dysregulation of the host responses to LPS can lead to a systemic inflammatory condition named sepsis. In a typical scenario, activation of TLR4 is preceded by binding of LPS to CD14 protein anchored in cholesterol- and sphingolipid-rich microdomains of the plasma membrane called rafts. CD14 then transfers the LPS to the TLR4/MD-2 complex which dimerizes and triggers MyD88- and TRIF-dependent production of pro-inflammatory cytokines and type I interferons. The TRIF-dependent signaling is linked with endocytosis of the activated TLR4, which is controlled by CD14. In addition to CD14, other raft proteins like Lyn tyrosine kinase of the Src family, acid sphingomyelinase, CD44, Hsp70, and CD36 participate in the TLR4 signaling triggered by LPS and non-microbial endogenous ligands. In this review, we summarize the current state of the knowledge on the involvement of rafts in TLR4 signaling, with an emphasis on how the raft proteins regulate the TLR4 signaling pathways. CD14-bearing rafts, and possibly CD36-rich rafts, are believed to be preferred sites of the assembly of a multimolecular complex which mediates the endocytosis of activated TLR4.
Growth and immunity are opposing processes that compete for cellular resources, and proper resource allocation is crucial for plant survival. BSK1 plays a key role in the regulation of both growth ...and immunity by associating with BRI1 and FLS2, respectively. However, it remains unclear how two antagonistic signals co-opt BSK1 to induce signal-specific activation. Here we show that the dynamic spatial reorganization of BSK1 within the plasma membrane underlies the mechanism of signal-specific activation for growth or immunity. Resting BSK1 localizes to membrane rafts as complexes. Unlike BSK1-associated FLS2 and BRI1, flg22 or exogenous brassinosteroid (BR) treatment did not decrease BSK1 levels at the plasma membrane (PM) but rather induced BSK1 multimerization and dissociation from FLS2/BSK1 or BRI1/BSK1, respectively. Moreover, flg22-activated BSK1 translocated from membrane rafts to non-membrane-raft regions, whereas BR-activated BSK1 remained in membrane rafts. When applied together with flg22, BR suppressed various flg22-induced BSK1 activities such as BSK1 dissociation from FLS2/BSK1, BSK1 interaction with MAPKKK5, and BSK translocation together with MAPKKK5. Taken together, this study provides a unique insight into how the precise control of BSK1 spatiotemporal organization regulates the signaling specificity to balance plant growth and immunity.
By using single-molecule techniques and biochemical approaches, this study demonstrates that the precise control of BSK1 spatiotemporal organization plays a pivotal role in fine-tuning the growth–defense trade-off between BRI1- and FLS2-mediated signaling.
The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane ...cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.
It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor ...tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor β (PDGFR-β) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-β-cyclodextrin (MβCD) did not affect PDGFR-β activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MβCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.
•PDGFR-β activation is affected by cholesterol depletion in a cell specific manner.•Cholesterol depletion impairs AKT and SRC signaling downstream of PDGFR-β in U2OS.•Cholesterol depletion abolish ERK signaling downstream of PDGFR-β in BJ-hTERT.•Cholesterol depletion affect cell morphology differently depending on cell type.
A wide variety of transmembrane signals are transduced by cell-surface receptors that activate intracellular signaling molecules. In particular, receptor clustering in the plasma membrane plays a ...critical role in these processes. Single-spanning or single-pass transmembrane proteins are among the most significant types of membrane receptors, which include adhesion receptors, such as integrins, CD44, cadherins, and receptor tyrosine kinases. Elucidating the molecular mechanisms underlying the regulation of the activity of these receptors is of great significance. Liquid–liquid phase separation (LLPS) is a recently emerging paradigm in cellular physiology for the ubiquitous regulation of the spatiotemporal dynamics of various signaling pathways. This study describes the emerging features of transmembrane signaling through single-spanning receptors from the perspective of phase separation. Possible physicochemical modulations of LLPS-based transmembrane signaling are also discussed.
•Liquid–liquid phase separation (LLPS) is an emerging paradigm in cell signaling.•LLPS is involved in signaling by single-spanning transmembrane receptors.•Phase separation at plasma membrane may modulate the biophysical signaling events.
•Membrane rafts have been proposed to be nm-sized, short-lived lipid-protein domains.•Methods that would allow visualizing such structures in vivo are only now beginning to emerge.•The raft concept ...maybe an oversimplification for a variety of nanostructures of heterogeneous sizes and functions.•It is wrong to equate rafts with detergent-resistant membrane fractions.•The term “raft” could be replaced with advantage by “nanodomain”.
The membrane raft hypothesis, proposed in 1997 by Simons and Ikonen, has played a paradoxical role in the history of biomembrane research. While it has generated a large amount of investigations, thus helping to increase our understanding of membranes, the object that gives name to the hypothesis, i.e. the raft itself, has been and still is an object of controversy, in which its very reality is often questioned. In this contribution I review the history of the hypothesis and its reception by membrane biologists, and summarize some of the valuable physico-chemical results that have been obtained while testing the raft hypothesis. To save a useful concept from its many misuses I propose that the expression “(transient) nanodomains” be employed instead of “rafts”.
Previous studies have shown that various glycolipids including neutral and acidic glycosphingolipids (GSLs) involve multiple biological events such as survival, neuronal development and ...differentiation processes. Moreover, recent reports have indicated that some neutral GSLs are involved in neuroinflammatory signaling and therefore involved in the occurrence of certain neurological diseases. Sphingolipids, cholesterol, and glycerophospholipids are essential constituents of neuronal membranes which function as a protective barrier of neuronal cells against the external environment and providing essential biochemical platform for proper neuronal actions and functions. At present, however, detailed molecular basis of how GSLs transmit the signals in neuroinflammatory reactions in vivo remains to be elucidated. Furthermore, we recently identified new type of autoantibodies against neutral GSLs, especially lactosylceramide in a neuroinflammatory disorder, encephalomyeloradiculoneuropathy (EMRN) and found these autoantibodies can be used as a surrogate marker for this disorder. These anti-neutral glycolipids antibodies strongly indicate that they can be a strong inducer for neuroinflammation of the brain and peripheral nervous system as well by involving innate immunity. In this review, I focused the biological and immunological activities and implications of GSLs and their autoantibodies, especially in terms of neuroinflammatory reactions in animals and human.
The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful ...which assumes that sterol‐dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes. But is the evidence clear enough to continue supporting a theoretical concept which has resisted direct proof by observation for nearly twenty years? In this essay, we revisit findings that gave rise to and substantiated the raft hypothesis, discuss its impact on recent studies, and present alternative mechanisms to account for plasma membrane heterogeneity.
An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into ...nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different – the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
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•SMA disintegrates T cell membrane into SMALPs.•Most membrane proteins are present in small SMALPs.•Some membrane raft-associated proteins are present in large SMALPs.•The large SMALPs have specific lipid composition corresponding to membrane rafts.•T cell membrane proteins present in SMALPs can be readily immunoisolated.
Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current ...superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM) require specialized equipment whereas single-molecule-based approaches e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM) involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment genetically encodable labels, and simple and rapid data acquisition, is capable of providing two-to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.
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