Membrane-altering effects of velnacrine and N-methylacridinium: relevance to tacrine and Alzheimer's disease.

The interaction of pharmacological agents potentially useful in Alzheimer's disease, 9-amino-1,2,3,4-tetrahydroacridine (THA or tacrine) and its major metabolite velnacrine (or HP-029), along with related compounds with cytoskeletal proteins in human erythrocyte membrane was investigated using electron paramagnetic resonance spin labeling techniques. The results suggest that: (1) the position of the positive charge of tacrine may be important in the mechanism of its interaction with the membrane cytoskeleton; (2) like tacrine, velnacrine also strengthens cytoskeletal protein-protein interactions in erythrocyte membranes, but appears to be only about half as potent as tacrine. These results are discussed with relevance to therapeutic use of these agents in Alzheimer's disease.     

Membrane-cytoskeleton associations during myogenesis deviate from traditional definitions

Plasma membrane-cytoskeleton associations involving four membrane proteins (A5, H58, H36, and I20) were studied in developing L8E63 rat skeletal muscle cells using immunofluorescence microscopy and photometry on the basis of three criteria: Triton-insolubility, colocalization with cytoskeletal components, and sensitivity to cytoskeleton-directed drugs. The results presented demonstrate that there are developmental stage-specific associations between membrane proteins and the cytoskeleton during skeletal myogenesis. Several inconsistencies were found with traditional expectations of membrane-cytoskeleton associations. For example, although A5 is Triton-insoluble and sensitive to cytochalasin, its distribution generally does not correspond with any known cytoskeletal structure. Furthermore, the topography of A5 is dependent on the integrity of the plasma membrane. H36 and I20 are completely soluble in Triton and therefore by accepted definitions would not be expected to be associated with any cytoskeletal component. Yet H36 and actin codisrupt in the presence of cytochalasin, while I20, whose distribution does not correspond with microtubules, is uniquely sensitive to their disruption. These results demonstrate that (i) neither Triton-solubility nor colocalization alone predicts all membrane-cytoskeleton associations; some associations between the membrane and cytoskeleton are unstable in nonionic detergent; (ii) the native distribution of proteins in the membrane may not reflect their cytoskeletal associations; and (iii) the topography of some membrane proteins with no apparent association with the cytoskeleton may be greatly influenced by the cell cytoskeleton.     

The brush border cytoskeleton is not static: in vivo turnover of proteins

The shape and stability of intestinal epithelial cell microvilli are maintained by a cytoskeletal core composed of a bundle of actin filaments with several associated proteins. The core filaments are intimately associated with the overlying plasma membrane, in which there occur rapid turnover of proteins and constant incorporation of new membrane. Previous work has shown that starvation or inhibition of protein synthesis results in modulation of microvillar length, which indicates that there may be cytoskeletal protein turnover. We demonstrate herein, by means of in vivo pulse labeling with radioactive amino acids, that turnover of brush border cytoskeletal proteins occurs in mature absorptive cells. Turnover of cytoskeletal proteins appears to be quite slow relative to membrane protein turnover, which suggests that the turnover of these two microvillar compartments is not coupled. We thus conclude that cytoskeletal protein turnover may be a factor used to maintain normal length and stability of microvilli and that the cytoskeleton cannot be considered a static structure.     

Differential effects of arylating and oxidizing analogs of N-acetyl-p-benzoquinoneimine on red blood cell membrane proteins

Incubation of human red blood cell membranes (white ghosts) with N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolite of acetaminophen, or with either an arylating or an oxidizing analog of NAPQI, resulted in the inhibition of membrane ion transporting systems and the modification of cytoskeletal proteins. NAPQI and 2,6-dimethyl-NAPQI, which primarily arylates protein thiols, inhibited the calmodulin-activated Ca pump ATPase activity, the basal (calmodulin-independent) Ca pump ATPase activity and the Na,K pump ATPase activity. In contrast, 3,5-dimethyl-NAPQI, which primarily oxidizes protein thiols, caused selective inhibition of the calmodulin-activated Ca pump ATPase activity. Sodium dodecyl sulfate gel electrophoresis of red blood cell (RBC) membrane proteins revealed that NAPQI and 2,6-dimethyl-NAPQI, but not 3,5-dimethyl-NAPQI, decreased the intensity of band 3 corresponding to the anion transporter, whereas NAPQI as well as 2,6-dimethyl-NAPQI, and to a lesser extent 3,5-dimethyl-NAPQI, caused a decrease of cytoskeletal protein bands, including spectrin, actin, and bands 4.1 and 4.2. These modifications were associated with increased formation of high molecular weight protein aggregates that did not enter the gel. Treatment of 3,5-dimethyl-NAPQI-exposed ghosts with the reducing agent dithiothreitol (DTT), resulted in the recovery of the affected cytoskeletal protein bands. Conversely, the modifications caused by NAPQI and 2,6-dimethyl-NAPQI were only partially reversed by DTT treatment. Taken together our results suggest that NAPQI and its two analogs modified ion transporting systems and cytoskeletal proteins by reacting with protein thiols. Both oxidation and arylation of protein thiols can alter the functional properties of important RBC membrane proteins. Of the two reactions, arylation appeared to be the less specific and more damaging event.     

A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

细胞膜蛋白与细胞骨架蛋白相互作用研究进展

细胞膜蛋白与胞浆骨架蛋白的相互作用对于维持细胞正常形态 ,细胞粘附与信号传导有重要作用。含有 4 .1 JEF结构域的蛋白 4 .1超家族与含有PDZ结构域的MAGUK蛋白家族能结合多种膜蛋白胞内区与胞浆蛋白 ,在膜蛋白与胞浆蛋白之间建立联系 ,对于细胞、细胞 -细胞间连接的正常结构与功能的维持有着重要作用。     

Submembraneous microtubule cytoskeleton: interaction of TRPP2 with the cell cytoskeleton

TRPP2, also called polycystin-2, the gene product of PKD2, is a membrane protein defective in 10-15% of cases of autosomal dominant polycystic kidney disease. Mutations in PKD2 are also associated with extrarenal disorders, such as hepatic cystogenesis and cardiovascular abnormalities. TRPP2 is a Ca-permeable nonselective cation channel present in the endoplasmic reticulum and plasma membrane, as well as in cilia of renal epithelial and embryonic nodal cells, in which it likely forms part of a flow sensor. Recent studies have identified a number of TRPP2-interacting proteins, of which many are cytoskeletal components. Work from our and other laboratories indicates that cytoskeletal partner proteins seem to play important, albeit highly complex, roles in the regulation of TRPP2 expression, localization and channel function. This minireview covers current knowledge about cytoskeletal interactions with TRPP2, and suggests that mutations in proteins of the TRPP2-cytoskeleton complex may be implicated in the pathogenesis of autosomal dominant polycystic kidney disease.     

Platelet cytoskeleton — Membrane interactions

The order of attachment of the purified platelet cytoskeletal proteins, F-actin, α-actinin and Actin Binding Protein (ABP) to the isolated platelet membrane has been investigated. Of the three proteins, only F-actin would directly interact with a membrane preparation that had been extensively washed to remove associated cytoskeletal proteins. α-Actinin would only add to the membrane to which F-actin had been re-attached and ABP only if both F-actin and α-actinin were present on the membrane. These studies provide some insight into the nature of the attachment of the platelet cytoskeleton to the cytoplasmic side of the membrane.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Neutrophil chemoattractant receptors and the membrane skeleton

Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process which involves participation of cytoskeletal elements. Evidence exists suggesting that the cytoskeleton and/or the membrane skeleton controls the distribution of FPR in the plane of the plasma membrane, thus controlling the accessibility of FPR to different proteins in functionally distinct domains. In desensitized cells, FPR are restricted to domains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inaccessible to the agonist-occupied receptor, preventing cell activation. The mechanism of interaction of FPR with the membrane skeleton is poorly understood but evidence is accumulating that suggests a direct binding of FPR (and other receptors) to cytoskeletal proteins such as actin.     

Cytoskeletal mechanisms of neuronal morphogenesis

The cytoskeleton is the major intracellular structure that determines the morphology of a neuron. Thus, mechanisms that ensure a precisely regulated assembly of cytoskeletal elements in time and space have an important role in the development from a morphologically simple neuronal precursor cell to a complex polarized neuron that can establish contacts to several hundreds of other cells. Here, cytoskeletal mechanisms that underlie the formation of neurites, directed elongation and stabilization of neuronal processes are summarized. It has become evident that different cytoskeletal elements are highly crosslinked with each other by several classes of specific linker proteins. Of these, microtubule-associated proteins (MAPs) appear to have an important role in connecting the microtubule skeleton to other cytoskeletal filaments and plasma membrane components during neuronal morphogenesis. Future experiments will have to elucidate the function and the regulation of the neuronal cytoskeleton in an authentic nervous system environment during development. Recent approaches are discussed at the end of this article.     

Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.     

Hemin-promoted peroxidation of red cell cytoskeletal proteins

Hemin-induced crosslinking of the erythrocyte membrane proteins was analyzed at three levels: (i) whole membranes, (ii) integrated or dissociated cytoskeletons, and (iii) isolated forms of the three main cytoskeletal proteins, spectrin, actin, and protein 4.1. Addition of H2O2 and hemoglobin to resealed membranes from without did not affect any of the membrane proteins. Hemin that can transport across the membrane induced, in the presence of H2O2, crosslinking of protein 4.1 and spectrin. Both free hemin and hemoglobin added with H2O2 induced crosslinking of integer cytoskeletons and mixtures of isolated cytoskeletal proteins, but hemin was always more active. Of the three major cytoskeletal proteins, spectrin and protein 4.1 were most active while the participation of actin was only minor. The yield of crosslinked products was increased in all reaction mixtures with pH, with an apparent pK above 9.0. Replacement of H2O2 by phenylhydrazine and tert-butyl hydroperoxide resulted in crosslinking of the same proteins, but with lower activity than H2O2. Bityrosines, which were identified by their specific fluorescence emission characteristics, were formed in reaction mixtures containing hemin and hydrogen peroxide and either spectrin or protein 4.1, but not actin. On the basis of fact that bityrosines were revealed only in reaction mixtures that produced protein adducts, formation of intermolecular bityrosines was analyzed to be involved in crosslinking of the cytoskeletal proteins. Since the levels of membrane-intercalated hemin are correlated with aggregation of membrane proteins, it is suggested that the peroxidative properties of hemin are responsible for its toxicity.     

Force profiles of protein pulling with or without cytoskeletal links studied by AFM

To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.     

Cell Shape Can Mediate the Spatial Organization of the Bacterial Cytoskeleton

The bacterial cytoskeleton guides the synthesis of cell wall and thus regulates cell shape. Because spatial patterning of the bacterial cytoskeleton is critical to the proper control of cell shape, it is important to ask how the cytoskeleton spatially self-organizes in the first place. In this work, we develop a quantitative model to account for the various spatial patterns adopted by bacterial cytoskeletal proteins, especially the orientation and length of cytoskeletal filaments such as FtsZ and MreB in rod-shaped cells. We show that the combined mechanical energy of membrane bending, membrane pinning, and filament bending of a membrane-attached cytoskeletal filament can be sufficient to prescribe orientation, e.g., circumferential for FtsZ or helical for MreB, with the accuracy of orientation increasing with the length of the cytoskeletal filament. Moreover, the mechanical energy can compete with the chemical energy of cytoskeletal polymerization to regulate filament length. Notably, we predict a conformational transition with increasing polymer length from smoothly curved to end-bent polymers. Finally, the mechanical energy also results in a mutual attraction among polymers on the same membrane, which could facilitate tight polymer spacing or bundling. The predictions of the model can be verified through genetic, microscopic, and microfluidic approaches.     

Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis

Endocytosis is an important way for cells to take up liquids and particles from their environment. It requires membrane bending to be coupled with membrane fission, and the actin cytoskeleton has an active role in membrane remodelling. Here, we review recent research into the function of Bin-Amphiphysin-Rvs (BAR) domain proteins, which can sense membrane curvature and recruit actin to membranes. BAR proteins interact with the endocytic and cytoskeletal machinery, including the GTPase dynamin (which mediates vesicle fission), N-WASP (an Arp2/3 complex regulator) and synaptojanin (a phosphoinositide phosphatase). We describe three classes of BAR domains, BAR, N-BAR and F-BAR, providing examples of each discussing and how they function in linking membranes to the actin cytoskeleton in endocytosis.     

Proteomic analysis of a membrane skeleton fraction from human liver

The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called "membrane skeletons". Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons.     

Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and β-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and β/γ-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.     

Identification of tubulin-binding proteins of the chicken erythrocyte plasma membrane skeleton which colocalize with the microtubular marginal band

The plasma membrane of nucleated erythrocytes contains a microtubular marginal band which appears to be associated with the plasma membrane skeleton. In this report, we identify two families of cytoskeletal proteins which may be involved in such an association. These proteins, of molecular mass 78 kDa and 48 kDa on SDS-PAGE, are shown to bind tubulin based on a 125I-labeled tubulin binding assay. Solubilization of isolated chicken erythrocyte plasma membranes in Triton X-100 shows that these proteins centrifuge with the pellet, indicating that they are bound to the membrane skeleton. Finally, immunofluorescence studies using antisera raised against the 78 kDa and 48 kDa proteins show that they colocalize with the marginal band in intact cells. Colocalization of cytoskeletal tubulin-binding proteins with the marginal band favors a hypothesis suggesting that the 78 kDa and 48 kDa proteins are involved in the association of the two molecular superstructures.     

Effects of membrane lipids and -proteins and cytoskeletal proteins on the kinetics of cholesterol exchange between high density lipoprotein and human red blood cells, ghosts and microvesicles.

To better understand the effects of plasma membrane lipids and proteins and the cytoskeleton on the kinetics of cellular cholesterol efflux, the effects of (1), selectively depleting either sphingomyelin (SM) or phosphatidylcholine (PC); (2), cross-linking the cytoskeleton, and (3), removing certain cytoskeletal and integral membrane proteins on radiolabelled cholesterol efflux from red blood cells (RBC) have been studied. When RBC were treated with either phospholipase A2 or sphingomyelinase C to hydrolyze either 30-40% of the PC or 40-50% of the SM, respectively, the halftimes (t1/2) for cholesterol efflux to excess HDL3 were not significantly altered, with the values being 4.4 +/- 0.8 h or 3.7 +/- 0.4 h, respectively, compared to 4.6 +/- 0.6 h for control RBC. To investigate the effects of the cytoskeleton on the rate of free cholesterol (FC) desorption from the plasma membrane, the cytoskeletal proteins were cross-linked by either heat-treatment or exposure to diamide and cholesterol efflux from ghosts of these cells was measured. Cross-linking the cytoskeletal proteins by diamide treatment resulted in no significant change in t1/2 for treated (3.6 +/- 0.6 h) compared to control (4.2 +/- 0.4 h) ghosts: this suggests that the cytoskeleton does not play a large role in modulating cholesterol efflux. To investigate the effects of membrane proteins on cholesterol efflux, RBC microvesicles, containing mainly band 3 and 4 proteins and little of the cytoskeletal proteins, such as spectrin (bands 1,2) or actin (band 5), were obtained by incubation with the ionophore A23187. With excess HDL3 present, microvesicles exhibited a t1/2 of 4.2 +/- 1.9 h (compared to the t1/2 of 4.2 +/- 0.4 h for control ghosts). The results described in this paper suggest that neither changing the SM/PC ratio in the membrane nor cross-linking the cytoskeletal proteins nor removing the cytoskeleton changes the t1/2 for cholesterol efflux to excess HDL3. Presumably, the cholesterol-phospholipid interactions are insensitive to these perturbations in membrane structure.