Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitro-benzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of [14C]AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. [14C]AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.     

Neurospecific cytoskeletal proteins

Data available in literature on neurospecific proteins of cytoskeletal structures--microtubules, microfilaments and intermediate filaments are generalized. Properties of tissue-specific cytoskeletal proteins which are typical of nerve cells are summarized. The structure, physicochemical properties, cell localization, metabolism and function of cytoskeletal proteins are characterized. The coexpression and interaction of different cytoskeletal structures are considered. An analysis of neurospecific cytoskeletal proteins is of great practical importance for neurobiology, neurooncology, neurosurgery. The proteins can be used as markers of different pathologies in the nervous system.     

Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca-induced membrane fusion events in chicken erythrocytes

The profound morphological changes which follow the treatment of chicken erythrocytes with the ionophore A23187 and Ca²⁺ are associated with a concomitant breakdown of certain membrane-associated proteins including α-spectrin, goblin and microtubule-associated proteins (MAPS) which undergo a limited proteolysis to give large, well-defined fragments. The Ca²⁺-sensitive protease responsible for these changes appears to be present in the soluble fraction of the cells. Treatment with TLCK or iodoacetamide inhibits both the major morphological changes and the proteolytic events but these agents do not prevent the dissociation of microtubules or the activation of endogenous sphingomyelinase which occur in cells with raised levels of intracellular Ca²⁺. It is suggested that the sphingomyelinase is activated as a consequence of a Ca²⁺-induced loss of phospholipid asymmetry in the plasma membrane.     

The cytoskeletal system of mammalian primitive erythrocytes: studies in developing marsupials

Seeking to resolve conflicting literature on cytoskeletal structure in mammalian "primitive" generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short-tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population at birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface-associated network (membrane skeleton), closely resembled that of non-mammalian vertebrate erythrocytes. By day 2 or 3, much smaller anucleate erythrocytes of "definitive" morphology, lacking marginal bands, appeared in abundance. These accounted for greater than 90% of the circulating population of both species by day 6-8. Non-nucleated erythrocytes of a different type, constituting 1-6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non-mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long-suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non-mammalian vertebrates.     

Formin family proteins in cytoskeletal control

Functions of the cell cortex, including motility, adhesion, and cytokinesis, are mediated by the reorganization of the actin cytoskeleton. The assembly of the cytoskeletal components at cortical sites is regulated dynamically in a temporal and spatial manner. Recent evidence indicates that the formin family proteins play a crucial role in the reorganization of the cytoskeleton. In this review, recent advance in the understanding of the functions of formin family proteins is discussed.     

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.     

Regulation of cytoskeletal dynamics by actin-monomer-binding proteins

The actin cytoskeleton is a vital component of several key cellular and developmental processes in eukaryotes. Many proteins that interact with filamentous and/or monomeric actin regulate the structure and dynamics of the actin cytoskeleton. Actin-filament-binding proteins control the nucleation, assembly, disassembly and crosslinking of actin filaments, whereas actin-monomer-binding proteins regulate the size, localization and dynamics of the large pool of unpolymerized actin in cells. In this article, we focus on recent advances in understanding how the six evolutionarily conserved actin-monomer-binding proteins - profilin, ADF/cofilin, twinfilin, Srv2/CAP, WASP/WAVE and verprolin/WIP - interact with actin monomers and regulate their incorporation into filament ends. We also present a model of how, together, these ubiquitous actin-monomer-binding proteins contribute to cytoskeletal dynamics and actin-dependent cellular processes.     

Selective interaction of cytoskeletal proteins with liposomes

A protein kinase activity analogous to that found in interferon-treated HeLa cells is detectable in human plasma rich in platelets. This kinase activity is manifested by the phosphorylation of an endogenous Mr 72000 protein which could be conveniently assayed after partial purification on poly(G)-Sepharose. Here, we show that the protein kinase system in the plasma consists of at least 2 components. The protein kinase is found to be localised in the platelets whereas most of the substrate (the Mr 72000 protein) is found free in the plasma and a fraction of it associated with the surface of platelets.     

The interaction of lymphocyte membrane proteins with the lymphocyte cytoskeletal matrix

Lymphocyte membrane proteins are important in the transduction of signals across the plasma membrane. Visual and biophysical studies have shown that after ligand binding, membrane proteins may become immobile in the plane of the membrane and may cap. In intact cells, binding of cross-linking ligands to surface immunoglobulin converts it to a detergent-insoluble state (77% insoluble). This conversion is positively correlated with the transmission of a mitogenic signal. Class II histocompatibility proteins (Ia) and thy-1 remain predominantly detergent soluble (60 to 97% soluble). Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons with 0.34 M sucrose, 0.5 mM ATP, 0.5 mM dithiothreitol, 1 mM EDTA, or 3 X 10(-5) M DNAase I, 1 mM EDTA. To determine if the membrane-associated cytoskeleton contains the sufficient components for ligand-induced receptor insolubilization, experiments were done with a crude plasma membrane fraction. The results with whole cells or crude plasma membranes were comparable. These studies support the view that ligand-induced insolubilization of membrane proteins is due to their interaction with cytoskeletal structures.     

Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca2+-induced membrane fusion events in chicken erythrocytes

The profound morphological changes which follow the treatment of chicken erythrocytes with the ionophore A23187 and Ca2+ are associated with a concomitant breakdown of certain membrane-associated proteins including alpha-spectrin, goblin and microtubule-associated proteins (MAPS) which undergo a limited proteolysis to give large, well-defined fragments. The Ca2+-sensitive protease responsible for these changes appears to be present in the soluble fraction of the cells. Treatment with TLCK or iodoacetamide inhibits both the major morphological changes and the proteolytic events but these agents do not prevent the dissociation of microtubules or the activation of endogenous sphingomyelinase which occur in cells with raised levels of intracellular Ca2+. It is suggested that the sphingomyelinase is activated as a consequence of a Ca2+-induced loss of phospholipid asymmetry in the plasma membrane.     

The cytoskeletal system of nucleated erythrocytes. I. Composition and function of major elements

We have studied the dogfish erythrocyte cytoskeletal system, which consists of a marginal band of microtubules (MB) and trans-marginal band material (TBM). The TBM appeared in whole mounts as a rough irregular network and in thin sections as a surface-delimiting layer completely enclosing nucleus and MB. In cells incubated at 0 degrees C for 30 min or more, the MB disappeared but the TBM remained. MB reassembly occurred with rewarming, and was inhibited by colchicine. Flattened elliptical erythrocyte morphology was retained even when MBs were absent. Total solubilization of MB and TBM at low pH, or dissolution of whole anucleate cytoskeletons, yielded components comigrating with actin, spectrin, and tubulin standards during gel electrophoresis. Mass-isolated MBs, exhibiting ribbonlike construction apparently maintained by cross-bridges, contained four polypeptides in the tubulin region of the gel. Only these four bands were noticeably increased in the soluble phase obtained from cells with 0 degrees C- disassembled MBs. The best isolated MB preparations contained tubulin but no components comigrating with high molecular weight microtubule- associated proteins, spectrin, or actin. Actin and spectrin therefore appear to be major TBM constituents, with tubulin localized in the MB. The results are interpreted in terms of an actin- and spectrin- containing subsurface cytoskeletal layer (TBM), related to that of mammalian erythrocytes, which maintains cell shape in the absence of MBs. Observations on abnormal pointed erythrocytes containing similarly pointed MBs indicate further that the MB can deform the TBM from within so as to alter cell shape. MBs may function in this manner during normal cellular morphogenesis and during blood flow in vivo.     

Peripheral proteins of human erythrocytes

Water soluble, nonglycosylated proteins have been extracted from human erythrocyte membranes by two different methods and characterized immunochemically and by PAGE. The spectrin peripheral protein complex (PAGE bands 1 + 2) has been equated with two antigens of intermediate mobility in immunoelectrophoretic analysis of crude spectrin developed with antiserum to bands 1 + 2 purified by elution from gels. Nonspectrin proteins, including catalase, remain in close association with isolated membranes, and display solubility properties similar to those of spectrin. Along with spectrin, they may also function in the intact cell as peripheral proteins.     

Monoclonal antibodies to red cell cytoskeletal proteins

Monoclonal antibodies to human red cell cytoskeletal proteins were produced following immunization of mice with Triton shells produced from intact red cells. Two lines producing antibodies binding to spectrin and actin, respectively, were subcloned and further characterized. Clones producing the anti-spectrin antibody were stable. The antibody was monoclonal and specific for spectrin band 2. The anti-actin clones were unstable.     

Force generation by cytoskeletal filament end-tracking proteins

Force generation in several types of cell motility is driven by rapidly elongating cytoskeletal filaments that are persistently tethered at their polymerizing ends to propelled objects. These properties are not easily explained by force-generation models that require free (i.e., untethered) filament ends to fluctuate away from the surface for addition of new monomers. In contrast, filament end-tracking proteins that processively advance on filament ends can facilitate rapid elongation and substantial force generation by persistently tethered filaments. Such processive end-tracking proteins, termed here filament end-tracking motors, maintain possession of filament ends and, like other biomolecular motors, advance by means of 5'-nucleoside triphosphate (NTP) hydrolysis-driven affinity-modulated interactions. On-filament NTP hydrolysis/phosphate release yields substantially more energy than that required for driving steady-state assembly/disassembly of free filament ends (i.e., filament treadmilling), as revealed by an energy inventory on the treadmilling cycle. The kinetic and thermodynamic properties of two simple end-tracking mechanisms (an end-tracking stepping motor and a direct-transfer end-tracking motor) are analyzed to illustrate the advantages of an end-tracking motor over free filament-end elongation, and over passive end-trackers that operate without the benefit of NTP hydrolysis, in terms of generating force, facilitating rapid monomer addition, and maintaining tight possession of the filament ends. We describe an additional cofactor-assisted end-tracking motor to account for suggested roles of cofactors in the affinity-modulated interactions, such as profilin in actin-filament end-tracking motors and EB1 in microtubule end-tracking motors.     

The cytoskeletal system of nucleated erythrocytes. III. Marginal band function in mature cells

Marginal bands (MBs) of microtubules are believed to function during morphogenesis of nonmammalian vertebrate erythrocytes, but there has been little evidence favoring a continuing role in mature cells. To test MB function, we prepared dogfish erythrocytes with and without MBs at the same temperature by (a) stabilization of the normally cold- labile MB at 0 degree C by taxol, and (b) inhibition of MB reassembly at room temperature by nocodazole or colchicine. We then compared the responses of these cells to mechanical stress by fluxing them through capillary tubes. Before fluxing , cells with or without MBs had normal flattened elliptical shape. After fluxing , deformation was consistently observed in a much greater percentage of cells lacking MBs. The difference in percent deformation between the two cell types was highly significant. That the MB is an effector of cell shape was further documented in studies of the formation of singly or doubly pointed dogfish erythrocytes that appear during long-term incubation of normal cells at room temperature. On-slide perfusion experiments revealed that the pointed cells contain MBs of corresponding pointed morphology. Incubation of cells with and without MBs showed that they become pointed only when they contain MBs, indicating that the MB acts as a flexible frame which can deform and support the cell surface from within. To test this idea further, cells with and without MBs were exposed to hyperosmotic conditions. Many of the cells without MBs collapsed and shriveled , whereas those with MBs did not. The results support the view that the MB has a continuing function in mature erythrocytes, resisting deformation and/or rapidly returning deformed cells to an efficient equilibrium shape in the circulation.