Detergent-resistant Membrane Rafts Research Articles

Chemical Proteomics Reveals Nε-Fatty-Acylation of Septins by Rho Inactivation Domain (RID) of the Vibrio MARTX Toxin to Alter Septin Localization and Organization

Vibrio species, the Gram-negative bacterial pathogens causing cholera and sepsis, produce multiple secreted virulence factors for infection and pathogenesis. Among these is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin that releases several critical effector domains with distinct functions inside eukaryotic host cells. One such effector domain, the Rho inactivation domain (RID), has been discovered to catalyze long-chain Nε-fatty-acylation on lysine residues of Rho GTPases, causing inactivation of Rho GTPases and disruption of the host actin cytoskeleton. However, whether RID modifies other host proteins to exert additional functions remains to be determined. Herein, we describe the integration of bioorthogonal chemical labeling and quantitative proteomics to globally profile the target proteins modified by RID in living cells. More than 246 proteins are identified as new RID substrates, including many involved in GTPase regulation, cytoskeletal organization, and cell division. We demonstrate that RID extensively Nε-fatty-acylates septin proteins, the fourth cytoskeletal component of mammalian cells with important roles in diverse cellular processes. While affinity purification and mass spectrometry analysis show that RID-mediated Nε-fatty-acylation does not affect septin-septin interactions, this modification increases the membrane association of septins and confers localization to detergent-resistant membrane rafts. As a result, the filamentous assembly and organization of septins are disrupted by RID-mediated Nε-fatty-acylation, further contributing to cytoskeletal and mitotic defects that phenocopy the effects of septin depletion. Overall, our work greatly expands the substrate scope and function of RID and demonstrates the role of RID-mediated Nε-fatty-acylation in manipulating septin localization and organization.

Integrin-Dependent Transient Density Increase in Detergent-Resistant Membrane Rafts in Platelets Activated by Thrombin.

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbβ3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin.

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5-10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization.

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca2+-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1-101), or its short scaffolding domain (81-101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca2+-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca2+-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca2+-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca2+-signals thereby maintaining efficient phototransduction recovery and light adaptation.

Proteome and Structural Organization of the Knob Complex on the Surface of the Plasmodium Infected Red Blood Cell.

The cell membrane of the erythrocytes infected with the malaria parasite Plasmodium falciparum undergoes several changes during the course of parasite life cycle and forms protrusions known as 'knobs' on its surface during the mature trophozoite and schizont stages. The structural organization of knob components especially PfEMP1 on the iRBC surface is the main determinant for the cytoadhesive and rosetting capacity of the iRBC by binding to various host receptors as well as for the variable antigenicity, which is crucial for immunoevasion. Although several studies report individual interactions among knob constituents, a comprehensive identification of the knob proteome is lacking. The detergent-resistant membrane (DRM) rafts are isolated from the infected erythrocyte membrane and knob (KAHRP) positive fractions are subjected to proteomics analysis. In addition, structures of various knob components are modeled and assembled ab initio based on experimentally established protein interactions. Proteins of various functional classes are found to be present in the knobs including the newly identified knob constituents which include host Hsp70, elongation factor 1A, acyl CoA synthetase, and some hypothetical proteins. Ab initio structural prediction of PfEMP1, KHARP, PfEMP2, PfEMP3, and PHIST shows that these proteins are intrinsically disordered and can have varying number of protein-protein interactions depending on their lowest energy structure. Further in silico mathematical modeling of a single repeat unit of PfEMP1-PHIST is present 63-112 times along the periphery of a single knob. This study provides structural insight into the organization of the core knob components and uncovers novel proteins as knob components. This structural information can be used for the development of better vaccine design strategies or drug design to destabilize the knob structure, which is a major virulence determinant in P. falciparum malaria.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.

Characterization of Two Distinct Populations of Epididymosomes Collected in the Intraluminal Compartment of the Bovine Cauda Epididymis1

During their transit along the epididymis, mammalian spermatozoa acquire new proteins that are necessary for their acquisition of forward motility and fertility. By using the bovine model, we previously showed that small membranous vesicles named epididymosomes are secreted in the epididymal intraluminal compartment. Epididymosomes from caput and cauda are different, and interact sequentially with the transiting spermatozoa. In fact, selected proteins of epididymosomes are transferred to different subcompartments of the maturing spermatozoa. In this study, we investigate the possibility that different subpopulations of epididymosomes are present in the caudal portion of the epididymis. Through the use of discontinuous sucrose gradient ultracentrifugation, we isolated two distinct populations that differ in their protein and lipid compositions. Although they have similar diameters, the ultrastructural appearance of these two populations was very different. The low-density (Ld) vesicles are enriched in cholesterol, sphingomyelin, and ganglioside M1, suggesting the existence of detergent-resistant membrane domains or rafts. The high-density (Hd) vesicles show a high protein concentration, including ACTB and VAMP8. When each subpopulation of biotinylated cauda epididymosomes was coincubated with caput spermatozoa, a subset of biotinylated proteins was transferred to the sperm; the Ld and Hd vesicles transferring the same pattern of proteins. In vitro competition assays of protein transferred from Ld or Hd epididymosomes to sperm confirm the similarity in the selected transferred proteins. Electrospray tandem mass spectrometry (ES-MS/MS) analysis of proteins associated with the two populations of vesicles confirm the epididymal origin of some of them, the possible involvement of others in transmembrane signaling systems, and the identification of proteins for which functions in sperm physiology remain to be determined. Mass spectrometry analysis also revealed that ELSPBP1 and GBB2 were transferred from epididymosomes to spermatozoa. Results are discussed with regard to the functions of these two cauda epididymosome populations in sperm physiology.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: Role in barrier enhancement

Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-beta-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1microM, 5min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting >3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85alpha phosphatidylinositol 3-kinase (p85alphaPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens α-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Monitoring membrane rafts in colorectal cancer cells by means of correlative fluorescence electron microscopy (CFEM)

Detergent-resistant membrane (DRM) rafts have been shown to play a pivotal role in regulating key cell biological processes, such as signal transduction, cellular transport and cell survival. The fine structure of membrane rafts are studied using various different imaging approaches and the outcomes are largely dependent on the detection methodology applied. All these microscopy techniques which employ light-, laser- and photon-optics, electrons as well as atomic force probing are characterized on their turn by their strengths and limitations for membrane raft identification. This explains in part the diversity of definitions available to describe these peculiar membrane structures. We present herewith an alternative and uncomplicated microscopy tool to study fluorescently labelled DRMs with information at the transmission electron microscopical level of the same cell, enabling us to obtain a snapshot of the morpho-functional relationships between the cell's interior and DRMs. The proposed approach of correlative fluorescence electron microscopy (CFEM) can therefore be considered as an additional alternative imaging approach to unravel DRM structure-function relationships from micro- to nanometre length scales, from the cell to the molecule.

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.

Translocation of Activated Heterotrimeric G Protein Gαo to Ganglioside-enriched Detergent-resistant Membrane Rafts in Developing Cerebellum

The association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the alpha-subunit of a heterotrimeric G protein, G(o) (Galpha(o)). Using sucrose density gradient analysis of cerebellar membranes, Galpha(o), but not Gbetagamma, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5'-O-(thiotriphosphate) (GTPgammaS), which stabilizes G(o) in its active form. On the other hand, both Galpha(o) and Gbetagamma were excluded from the DRM raft fractions in the presence of guanyl-5'-yl thiophosphate, which stabilizes G(o) in its inactive form. Only Galpha(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Galpha(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Galpha(o) into the DRM rafts. Furthermore, Galpha(o) was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1alpha, a physiological ligand for the G protein-coupled receptor, stimulated [(35)S]GTPgammaS binding to Galpha(o) and caused Galpha(o) translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raft-disrupting agent methyl-beta-cyclodextrin. These results demonstrate the involvement of signal-dependent Galpha(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.

Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors

Here, we reveal a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. We show that the multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking.

Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells

Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.

Fas-, Caspase 8-, and Caspase 3-dependent Signaling Regulates the Activity of the Aminophospholipid Translocase and Phosphatidylserine Externalization in Human Erythrocytes

Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.

Transcriptional signaling from membrane raft-associated glucocorticoid receptor

The contribution of plasma membrane-associated glucocorticoid receptor (GR) to transcriptional signaling is unclear. We observed GR in low-density detergent-resistant membrane (DRM) rafts derived from human hepatoma Hep3B cells in complexes with caveolin-1, HSP90, and STAT3. In transient transfection assays, GR-stimulated transcriptional signaling was reversibly inhibited by membrane-raft disrupters filipin III and progesterone. These data provide clear evidence for a functional contribution of DRM-associated GR to transcriptional signaling.

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.