Spectrin assembly in avian erythroid development is determined by competing reactions of subunit homo- and hetero-oligomerization

Erythroid differentiation entails the biogenesis of a membrane skeleton, a network of proteins underlying and interacting with the plasma membrane, whose major constituent is the heterodimeric protein spectrin, composed of two structurally similar but distinct subunits, alpha (relative molecular mass (Mr) 240,000) and beta (Mr 220,000), which interact side-on with each other to form a long rod-like molecule. Interaction of this network with the membrane is mediated by the binding of the beta subunit to ankyrin, which in turn binds to the cytoplasmic domain of the transmembrane anion transporter (also referred to as band 3). Purified alpha and beta subunits of spectrin from the membrane of mature red blood cells will spontaneously heterodimerize, suggesting that assembly of the spectrin-actin skeleton is a simple self-assembly process, but in vivo studies with developing chicken embryo erythroid cells have indicated that assembly in vivo is more complex. We now present evidence that newly synthesized spectrin subunits in vivo or in vitro rapidly adopt one of two competing conformations, a heterodimer or a homo-oligomer. These competing reactions seem to determine the overall extent of spectrin assembled during erythroid development by determining which conformation will assemble onto the membrane-skeleton (the heterodimer) and which conformations are targeted for degradation (the homo-oligomers).     

Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate

The actin-binding protein gelsolin requires micromolar concentrations of calcium ions to sever actin filaments, to potentiate its binding to the end of the filament and to promote the polymerization of monomeric actin into filaments. Because transient increases in both intracellular [Ca2+] and actin polymerization accompany the cellular response to certain stimuli, it has been suggested that gelsolin regulates the reversible assembly of actin filaments that accompanies such cellular activations. But other evidence suggests that these activities do not need increased cytoplasmic [Ca2+] and that once actin-gelsolin complexes form in the presence of Ca2+ in vitro, removal of free Ca2+ causes dissociation of only one of two bound actin monomers from gelsolin and the resultant binary complexes cannot sever actin filaments. The finding that cellular gelsolin-actin complexes can be dissociated suggests that a Ca2+-independent regulation of gelsolin also occurs. Here we show that, like the dissociation of profilin-actin complexes, phosphatidylinositol 4,5-bisphosphate, which undergoes rapid turnover during cell stimulation, strongly inhibits the actin filament-severing properties of gelsolin, inhibits less strongly the nucleating ability of this protein and restores the potential for filament-severing activity to gelsolin-actin complexes.     

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.     

Partial deficiency of erythrocyte spectrin in hereditary spherocytosis

Hereditary spherocytosis (HS) is a common, clinically heterogeneous haemolytic anaemia in which the primary erythrocyte defect is believed to be some abnormality in the spectrin-actin membrane skeleton, leading to loss of surface membrane. Recessively inherited spectrin deficiency with extreme erythrocyte fragility and spherocytosis has been identified in certain mutant mice and two severely anaemic humans. Although suspected, deficiency of spectrin has not been demonstrated in less severe forms of human HS. We not report the quantitation of erythrocytes spectrin by radioimmunoassay. We found that normal erythrocytes contained 240,000 copies of spectrin heterodimer, whereas erythrocytes from 14 patients with a variety of types of HS were all partially deficient in spectrin (range 74,000-200,000 copies), the magnitude of the deficiency correlating with the severity of the disease. Spectrin deficiency of varying degrees is common in HS and probably represents the principal structural defect leading to loss of surface membrane.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide

Many of the physical properties of the erythrocyte membrane appear to depend on the membrane skeleton, which is attached to the membrane through associations with transmembrane proteins. A membrane skeletal protein, protein 4.1, is pivotal in the assembly of the membrane skeleton because of its ability to promote associations between spectrin and actin. Protein 4.1 also binds to the membrane through at least two sites: a high-affinity site on the glycophorins and a site of lower affinity associated with band 3 (ref. 11). The glycophorin-protein 4.1 association has been proposed to be involved in maintenance of cell shape. Here we show that the association between glycophorin and protein 4.1 is regulated by a polyphosphoinositide cofactor. This observation suggests a mechanism which may explain the recently reported dependence of red cell shape on the level of polyphosphoinositides in the membrane.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.     

In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte.

The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.     

Regulation of glutamate receptor binding by the cytoskeletal protein fodrin

The erythrocyte cytoskeleton, which consists primarily of a meshwork of spectrin and actin, controls cell shape and the disposition of proteins within the membrane. Proteins similar to spectrin have recently been found in diverse cells and tissues, and it is possible that they mediate the capping of cell-surface receptors, although this has not been demonstrated directly. In neurones, the spectrin-like protein fodrin lines the cortical cytoplasm and may link actin filaments to the membrane. Fodrin has been hypothesized to regulate the number of receptor binding sites on neuronal membranes for the putative neurotransmitter L-glutamate. Micromolar calcium concentrations activate the thiol protease calpain I, induce fodrin degradation and more than double the density of glutamate binding sites; these effects are all blocked by thiol protease inhibitors. We have now used specific antibodies to examine further the role of fodrin proteolysis in regulating glutamate receptors. We report that fodrin antibodies block the fodrin degradation and increase in glutamate binding normally induced by calcium, and so provide direct evidence for control of membrane receptors by a non-erythroid spectrin.     

Ca2+-sensitive gelation of actin filaments by a new protein factor

Two protein factors which bind to, and induce gelation of, actin filaments were purified from Ehrlich tumour cells. Filamin induced Ca2+-insensitive gelation, whereas a new protein factor ('actinogelin') was found to induce Ca2+-sensitive gelation.     

An interaction between vinculin and talin

In cultured fibroblasts, microfilament bundles terminate at adhesion plaques (focal contacts), the specialized regions where the cells adhere most tightly to the underlying substrate. Vinculin is a protein concentrated in adhesion plaques and has been suggested as a possible link between the ends of the bundles of actin filaments and the plasma membrane. If vinculin is one protein in a chain of attachment between the bundles of microfilaments and the plasma membrane, it is important to identify other components which interact with vinculin. We have recently discovered a new protein in adhesion plaques which we refer to as talin. Here we show that talin binds to vinculin, which suggests that talin may be involved with vinculin in the attachment of microfilament bundles to the plasma membrane at the adhesion plaques.     

Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane, and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. Here, we show that the sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. Our data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, our results show that there is negative feedback between ERM activation and activity of the Rho pathway.     

Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells

Chromaffin cells release catecholamine- and peptide-containing granules by exocytosis, by a mechanism involving movement of secretory granules towards the cell membrane, their apposition to it and the fusion of the granule membrane with the plasma membrane. One of the two subunits of membrane-associated brain spectrin, alpha-fodrin is an actin-binding protein which is found at the periphery of chromaffin cells and may be involved in secretion. Because cultured chromaffin cells can be permeabilized with detergents, giving pores large enough to permit the entry of immunoglobulin molecules, we used permeabilized cells to test the effect of specific antibodies on secretory mechanisms. Incubation of permeabilized cells with polyclonal immunoaffinity-purified monospecific anti-alpha-fodrin antibody or its Fab fragments did not modify basal release but did specifically inhibit Ca2+-induced catecholamine release by exocytosis. Our observations indicate that fodrin and the cytoskeleton participate in the release mechanism.     

Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.     

Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.     

Formation of reverse rigor chevrons by myosin heads

The uniform angle and conformation of myosin subfragment 1 (S1) bound to actin filaments (F-actin) attest to the precise alignment and stereospecificity of the binding of these two contractile proteins. Because actin filaments are polar, myosin heads must swing or rotate about the head-tail junction in order to bind. Electron microscopy of isolated thick filaments and of myosin molecules suggests that the molecules are flexible, but myosin fragments and crossbridges have been reported not to interact with inappropriately oriented actin filaments. Here we describe myofibrillar defects engendered by a site-directed mutation within the flight-muscle-specific actin gene of the fruitfly Drosophila. The mutation apparently retards sarcomere assembly: peripheral thick and thin filaments are misregistered and not incorporated into the Z-line. Therefore, a myosin filament encounters thin filaments with the 'wrong' polarity. We show that myosin heads tethered in a single thick filament can bind with opposite rigor crossbridge angles to flanking thin filaments, which are apparently of opposite polarities. Preservation of identical actomyosin interfaces requires that sets of heads originating from opposite sides of the thick filament swivel 180 degrees relative to each other, implying that myosin crossbridges are as flexible as isolated molecules.     

Effect of ATP on actin filament stiffness

Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function.     

The RickA protein of Rickettsia conorii activates the Arp2/3 complex

Actin polymerization, the main driving force for cell locomotion, is also used by the bacteria Listeria and Shigella and vaccinia virus for intracellular and intercellular movements. Seminal studies have shown the key function of the Arp2/3 complex in nucleating actin and generating a branched array of actin filaments during membrane extension and pathogen movement. Arp2/3 requires activation by proteins such as the WASP-family proteins or ActA of Listeria. We previously reported that actin tails of Rickettsia conorii, another intracellular bacterium, unlike those of Listeria, Shigella or vaccinia, are made of long unbranched actin filaments apparently devoid of Arp2/3 (ref. 4). Here we identify a R. conorii surface protein, RickA, that activates Arp2/3 in vitro, although less efficiently than ActA. In infected cells, Arp2/3 is detected on the rickettsial surface but not in actin tails. When expressed in mammalian cells and targeted to the membrane, RickA induces filopodia. Thus RickA-induced actin polymerization, by generating long actin filaments reminiscent of those present in filopodia, has potential as a tool for studying filopodia formation.     

Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage

Many observations suggest the presence of transmembrane linkages between the cytoskeleton and the extracellular matrix. In fibroblasts both light and electron microscopic observations reveal a co-alignment between actin filaments at the cell surface and extracellular fibronectin. These associations are seen at sites of cell matrix interaction, frequently along stress fibres and sometimes where these bundles of microfilaments terminate at adhesion plaques (focal contacts). Non-morphological evidence also indicates a functional linkage between the cytoskeleton and extracellular matrix. Addition of fibronectin to transformed cells induces flattening of the cells and a reorganization of the actin cytoskeleton, with the concomitant appearance of arrays of stress fibres. Conversely, disruption of the actin cytoskeleton by treatment with cytochalasin B leads to release of fibronectin from the cell surface. As yet, there is no detailed knowledge of the molecules involved in this transmembrane linkage, although several proteins have been suggested as candidates in the chain of attachment between bundles of actin filaments and the cytoplasmic face of the plasma membrane: these include vinculin, alpha-actinin and talin, each one having been identified at regions where bundles of actin filaments interact with the plasma membrane and underlying cell-surface fibronectin. Recently, the cell-substrate attachment (CSAT) antigen has been identified as a plasma membrane receptor for fibronectin, raising the possibility that this glycoprotein complex may serve as a bridge between fibronectin and one or more of the underlying cytoskeletal components mentioned. Here we have investigated the interaction of the purified CSAT antigen with these cytoskeletal components, and we demonstrate an interaction specifically between the CSAT antigen and talin.