Dynamics of putative raft-associated proteins at the cell surface

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.     

脂筏与精子膜信号传导

脂筏是细胞膜内由特殊脂质与蛋白质构成的微域。小窝是脂筏的一种形式,小窝标记蛋白有小窝蛋白和小窝舟蛋白。脂筏或小窝与生物信号传导、细胞蛋白转运和胆固醇平衡有关。最近实验证实哺乳动物精子膜具有脂筏结构,脂筏与膜胆固醇外逸对于启动受精的信号传导具有重要作用。     

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.     

Evidence for leptin receptor isoforms heteromerization at the cell surface

Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin’s effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

Structured summary

MINT-7714817: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by anti tag co-immunoprecipitation (MI:0007)MINT-7714785: LEPRc (uniprotkb:P48357-2) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714951, MINT-7714744: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714859: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714885, MINT-7714672: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by bioluminescence resonance energy transfer (MI:0012)MINT-7714835: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714914, MINT-7714723, MINT-7714759: LeprB (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714703, MINT-7714936, MINT-7714772: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714872: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by anti tag co-immunoprecipitation (MI:0007)     

The cellular prion protein: biochemistry,topology, and physiologic functions

Studies on the transmission from man to animals of Creutzfeld-Jacob disease (CJD) led Prusiner to identify a proteinaceous infectious particle lacking nucleic acid, which was called prion. The identification of the infectious prion (PrPsc) then led to the discovery of the normal cellular counterpart (PrPc). One of the still enigmatic aspects regarding prion diseases is actually how, where, and when the transformation PrPc/PrPsc is occurring, this being due to the result of a large extent to the fact that so far most studies have been dedicated to the formation and transmission of PrPsc, whereas the understanding of physiologic roles of PrPc are in their infancy. In this review, we hope to identify the most reliable hypotheses for future experiments on PrPc. This is relevant not only for the understanding of PrPc functions but also to unravel the enigmatic nature of PrPc/PrPsc conversion.     

Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor.

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.     

Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.     

Regulation of expression and function of Lck tyrosine kinase by high cell density

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.     

Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts

We have evaluated the sizes and lifetimes of rafts in the plasma membrane from the existing literature, with a special attention paid to their intrinsically broad distributions and the limited time and space scales that are covered by the observation methods used for these studies. Distinguishing the rafts in the steady state (reserve rafts) from those after stimulation or unintentional crosslinking of raft molecules (stabilized receptor-cluster rafts) is critically important. In resting cells, the rafts appear small and unstable, and the consensus now is that their sizes are smaller than the optical diffraction limit (250 nm). Upon stimulation, the raft-preferring receptors are clustered, inducing larger, stabilized rafts, probably by coalescing small, unstable rafts or cholesterol-glycosphingolipid complexes in the receptor clusters. This receptor-cluster-induced conversion of raft types may be caused by suppression of alkyl chain isomerization and the lipid lateral diffusion in the cluster, with the aid of exclusion of cholesterol from the bulk domain and the boundary region of the majority of transmembrane proteins. We critically inspected the possible analogy to the boundary lipid concept. Finally, we propose a hypothesis for the coupling of GPI-anchored receptor signals with lipid-anchored signaling molecules in the inner-leaflet raft.     

Rafts: scale-dependent, active lipid organization at the cell surface

Rafts have been conceptualized as lateral heterogeneities in the organization of cholesterol and sphingolipids, endowed with sorting and signaling functions. In this review we critically examine evidence for the main tenet of the 'raft hypothesis', namely lipid-dependent segregation of specific membrane components in the plasma membrane. We suggest that conventional approaches to studying raft organization wherein membranes are treated as passive, thermally equilibrated systems are unlikely to provide an adequate framework to understand the mechanisms of raft-organization in vivo. An emerging view of raft organization is that it is spatio-temporally regulated at different scales by the cell. This argues that rafts must be defined by simultaneous observation of components involved in particular functions. Recent evidence from the study of glycosylphosphatidyl inositol-anchored proteins, a common raft-marker, supports this picture in which larger scale, more stable rafts are induced from preexisting small-scale lipid-dependent structures actively maintained by cellular processes.     

Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition. © 1994 Wiley-Liss, Inc.     

Organization of GPI-anchored proteins at the cell surface and its physiopathological relevance

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrP^C, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.     

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells

MAL is an integral protein component of the machinery for apical transport in epithelial Madin-Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.     

Accumulation of a GPI-anchored protein at the cell surface requires sorting at multiple intracellular levels

Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositol-anchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady-state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high-pressure frozen and freeze-substituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo- and exocytosis in this organism, is 48-52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum --> Golgi complex --> cell surface) or three enrichment steps (endoplasmic reticulum --> Golgi complex --> endosomes --> cell surface), suggesting sorting between several membrane compartments.     

T-cell antigen receptor triggering and lipid rafts: a matter of space and time scales. Talking Point on the involvement of lipid rafts in T-cell activation

T-cell antigen receptor triggering mechanisms and lipid rafts are of broad interest, but are also controversial topics. Here, we review some recent progress in these two research fields, which has been accomplished mostly in live cells and with the use of advanced technologies. We then discuss the potential relationship between membrane-domain organization and T-cell antigen receptor-triggering mechanisms. On the basis of the relevant experimental observations, we argue that the key to achieving a better understanding of both processes is the ability to monitor the molecular dynamics and interactions taking place in the membrane of T cells at a spatial scale of tens to hundreds of nanometres, with a subsecond-to-second temporal resolution.     

Arabidopsis capping protein senses cellular phosphatidic acid levels and transduces these into changes in actin cytoskeleton dynamics

Plants respond rapidly and precisely to a broad spectrum of developmental, biotic and abiotic cues. In many instances, signaling cascades involved in transducing this information result in changes to the cellular architecture and cytoskeletal rearrangements. Based originally on paradigms for animal cell signaling, phospholipids have received increased scrutiny as key intermediates for transmitting information to the actin cytoskeleton. Significantly, a wealth of biochemical data for plant actin-binding proteins (ABPs) demonstrates that many of these interact with phosphoinositide lipids in vitro. Moreover, phosphatidic acid (PA) has been identified not only as an abundant structural lipid in plants, but also as an intermediary in developmental and stress signaling pathways that lead to altered actin organization. Several years ago, the heterodimeric capping protein (CP) from Arabidopsis was demonstrated to bind PA and is negatively regulated by this lipid in vitro. Whether this form of regulation occurs in cells, however, remained a mystery. A new study, that combines live-cell imaging of cytoskeletal dynamics with reverse-genetic analyses in Arabidopsis, provides compelling new evidence that CP is inhibited from binding filament ends in the presence of PA in vivo. This allows rapid actin polymerization and increases in filament abundance following stimulation and could be one key factor in the physiological responses of plant cells to environmental stimuli.     

Conformation of the Ras-binding domain of Raf studied by molecular dynamics and free energy simulations

Recognition of Ras by its downstream target Raf is mediated by a Ras-recognition region in the Ras-binding domain (RBD) of Raf. Residues 78–89 in this region occupy two different conformations in the ensemble of NMR solution structures of the RBD: a fully α-helical one, and one where 87–90 form a type IV β-turn. Molecular dynamics simulations of the RBD in solution were performed to explore the stability of these and other possible conformations of both the wild-type RBD and the R89K mutant, which does not bind Ras. The simulations sample a fully helical conformation for residues 78–89 similar to the NMR helical structures, a conformation where 85–89 form a 3₁₀-helical turn, and a conformation where 87–90 form a type I |iB-turn, whose free energies are all within 0.3 kcal/mol of each other. NOE patterns and H^α chemical shifts from the simulations are in reasonable agreement with experiment. The NMR turn structure is calculated to be 3 kcal/mol higher than the three above conformations. In a simulation with the same implicit solvent model used in the NMR structure generation, the turn conformation relaxes into the fully helical conformation, illustrating possible structural artifacts introduced by the implicit solvent model. With the Raf R89K mutant, simulations sample a fully helical and a turn conformation, the turn being 0.9 kcal/mol more stable. Thus, the mutation affects the population of RBD conformations, and this is expected to affect Ras binding. For example, if the fully helical conformation of residues 78–89 is required for binding, its free energy increase in R89K will increase the binding free energy by about 0.6 kcal/mol. Proteins 31:186–200, 1998. © 1998 Wiley-Liss, Inc.     

橙色荧光蛋白的研究进展及其应用

荧光蛋白(Fluorescent protein,FPs)可作为探针用以探究细胞内分子间相互作用,追踪特定代谢物的代谢途径,对活细胞内的各种代谢过程和细胞通路进行详细、准确的描述。目前已有的FPs几乎已经覆盖了从紫外光到远红外光的所有光谱波段,这些FPs借助高分辨率显微技术应用于生命科学的诸多领域,为生物学的发展作出巨大贡献。橙色FPs通常指光谱区间在540–570nm的FPs,近几年来关于橙色FPs的研究进展较快,并且其作为标记蛋白以及荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET)中的荧光受体在生物学及医学领域得到较多的应用。文中综述了近15年橙色FPs领域的相关研究,重点聚焦橙色FPs的发展和应用,为今后橙色FPs的研究提供依据。     

Studies of distribution,location and dynamic properties of EGFR on the cell surface measured by image correlation spectroscopy

In this work, we have studied the distribution and dynamic properties of Epidermal Growth Factor (EGF) receptors in the plasma membrane of fixed and live cells as well as the extent of co-localization of this transmembrane protein with proteins specific for three-membrane microdomains: membrane rafts, caveolae and clathrin-coated pits. This was achieved using a family of image-processing tools called image correlation spectroscopy (ICS), image cross-correlation spectroscopy (ICCS) and dynamic image correlation spectroscopy (DICS). Our results indicate that EGFR is diffusely distributed on the cell surface at 37°C and aggregates as the temperature is lowered to 4°C. This aggregation takes place within 15 min and is reversible. Changes in temperature also affect the diffusion of EGFR by two orders of magnitude. The dynamic properties of EGFR are similar to the dynamic properties of a GPI-anchored protein known to be present in membrane rafts, which motivated us to explore the extent of co-localization of EGFR with this membrane raft protein using ICCS. Our results indicate that more than half of the EGFR population is present in membrane rafts and smaller percentages are present in caveolae and clathrin-coated pits.     

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.