Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectinss, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. pupurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixture of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to move together with those for concanavalin A. A method for thentitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.     

Lateral Distribution of the Cytochrome b(6)/f and Coupling Factor ATP Synthetase Complexes of Chloroplast Thylakoid Membranes

We have visualized directly the distribution of the cytochrome b₆/f and coupling factor ATP synthetase complexes in thylakoid membranes of embedded, thin-sectioned, intact chloroplasts by using rabbit antibodies directed against each complex, followed by ferritin-conjugated goat anti- (rabbit immunoglobulin G) antibodies. The labeling patterns indicate that in spinach (Spinacia oleracea) chloroplasts the cytochrome b₆/f complex is distributed laterally throughout both stacked grana and unstacked stroma membrane regions, whereas the coupling factor ATP synthetase complex is found exclusively in stroma thylakoids and in the marginal and end membranes of grana.     

Spectrin involvement in a 40 degrees C structural transition of the red blood cell membrane

Proteins involved in a structural transition detected in red blood cell membranes at 40 degrees C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40 degrees C transition detected in ghosts by both a stearic acid spin label (16-doxyl stearic) and a sulfhydryl-specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25-30 degrees C and a massive transition with an onset temperature of 48 and 40 degrees C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40 degrees C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti-4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1:1 molar ratio. The involvement in this transition of a localized spectrin domain(s) containing few exposed sulfhydryl groups is proposed.     

Member-associated changes during erythropoiesis. On the mechanism of maturation of reticulocytes to erythrocytes

The mature mammalian erythrocyte has a unique membranoskeleton, the spectrin-actin complex, which is responsible for many of the unusual membrane properties of the erythrocyte. Previous studies have shown that in successive stages of differentiation of the erythropoietic series leading to the mature erythrocyte there is a progressive increase in the density of spectrin associated with the membranes of these cells. An important stage of this progression occurs during the enucleation of the late erythroblast to produce the incipient reticulocyte, when all of the spectrin of the former cell is sequestered to the membrane of the reticulocyte. The reticulocyte itself, however, does not exhibit a fully formed membranoskeleton. In particular, the in vitro binding of multivalent ligands to specific membrane receptors on the reticulocyte was shown to cause a clustering of some fractions of these ligand-receptor complexes into special mobile domains on the cell surface. These domains of clustered ligand-receptor complexes became invaginated and endocytosed as small vesicles. By immunoelectron microscopic experiments, these invaginations and endocytosed vesicles were found to be specifically free of spectrin on their cytoplasmic surfaces. These earlier findings then raised the possibility that the maturation of reticulocytes to mature erythrocytes in vivo might involve a progressive loss of reticulocyte membrane free of spectrin, thereby producing a still more concentrated spectrin-actin membranoskeleton in the erythrocyte than in the reticulocyte. This proposal is tested experimentally in this paper. In vivo reticulocytes were observed in ultrathin frozen sections of spleens from rabbits rendered anemic by phenylhydrazine treatment. These sections were indirectly immunolabeled with ferritin-antibody reagents directed to rabbit spectrin. Most reticulocytes in a section had one or more surface invaginations and one or more intra-cellular vesicles that were devoid of spectrin labeling. The erythrocytes in the same sections did not exhibit these features, and their membranes were everywhere uniformly labeled for spectrin. Spectrin-free surface invaginations and intracellular vesicle were also observed with reticulocytes within normal rabbit spleens. Based on these results, a scheme for membrane remodeling during reticulocyte maturation in vivo is proposed.     

Occurrence of lipid receptors inferred from brain and erythrocyte spectrins binding NaOH-extracted and protease-treated neuronal and erythrocyte membranes

It was previously shown in model systems that brain spectrin binds membrane phospholipids. In the present study, we analysed binding of isolated brain spectrin and red blood cell spectrin to red blood or neuronal membranes which had been treated as follows: (1). extracted with low ionic-strength solution, (2). the above membranes extracted with 0.1 M NaOH, and (3). membranes treated as above, followed by protease treatment and re-extraction with 0.1 M NaOH. It was found that isolated, NaOH-extracted, protease-treated neuronal and red blood cell membranes bind brain and red blood cell spectrin with moderate affinities similar to those obtained in model phospholipid membrane-spectrin interaction experiments. Moreover, this binding was competitively inhibited by liposomes prepared from membrane lipids. The presented results indicate the occurrence of receptor sites for spectrins that are extraction- and protease-resistant, therefore most probably of lipidic nature, in native membranes.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : II. Antispectrin-Induced Transmembrane Aggregation of the Binding Sites for Positively Charged Colloidal Particles

The effects of affinity-purified antispectrin γ-globulins on the topographic distribution of anionic residues on human erythrocytes membranes was investigated using collo ida iron hydroxide labeling of mounted, fixed, ghost membranes. Antispectrin γ-globulins were sequestered inside ghosts by hemolysis and the ghosts were incubated for 30 min at 37°C and then fixed with glutaraldehyde. The topographic distribution of colloidal iron hydroxide clusters on ghosts incubated with low (<0.05 mg/ml) or high (>5–10 mg/ml concentrations of sequestered antispectrin was dispersed, but the distribution at intermediate concentrations (0.1–5 mg/ml) was highly aggregated. The aggregation of colloidal iron hydroxide binding sites was time and temperature dependent and required the sequestering of cross-linking antibodies (antispectrin Fab could not substitute for γ-globulin antibodies) inside the ghosts. Prior glutaraldehyde fixation or fixation at the time of hemolysis in antispectrin solutions prevented the antispectrin-induced colloidal iron site aggregation. The antispectrin reacted exclusively at the inner ghost membrane surface and the colloidal iron hydroxide bound to N-acetylneuraminic acid residues on the outer membrane surface which are overwhelming on the sialoglycoprotein glycophorin. These results were interpreted as evidence for a structural transmembrane linkage between the inner surface peripheral protein spectrin and the integral membrane component glycophorin.     

αI-spectrin represents evolutionary optimization of spectrin for red blood cell deformability

Spectrin tetramers of the membranes of enucleated mammalian erythrocytes play a critical role in red blood cell survival in circulation. One of the spectrins, αI, emerged in mammals with enucleated red cells after duplication of the ancestral α-spectrin gene common to all animals. The neofunctionalized αI-spectrin has moderate affinity for βI-spectrin, whereas αII-spectrin, expressed in nonerythroid cells, retains ancestral characteristics and has a 10-fold higher affinity for βI-spectrin. It has been hypothesized that this adaptation allows for rapid make and break of tetramers to accommodate membrane deformation. We have tested this hypothesis by generating mice with high-affinity spectrin tetramers formed by exchanging the site of tetramer formation in αI-spectrin (segments R0 and R1) for that of αII-spectrin. Erythrocytes with αIIβI presented normal hematologic parameters yet showed increased thermostability, and their membranes were significantly less deformable; under low shear forces, they displayed tumbling behavior rather than tank treading. The membrane skeleton is more stable with αIIβI and shows significantly less remodeling under deformation than red cell membranes of wild-type mice. These data demonstrate that spectrin tetramers undergo remodeling in intact erythrocytes and that this is required for the normal deformability of the erythrocyte membrane. We conclude that αI-spectrin represents evolutionary optimization of tetramer formation: neither higher-affinity tetramers (as shown here) nor lower affinity (as seen in hemolytic disease) can support the membrane properties required for effective tissue oxygenation in circulation.     

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.     

Integral protein linkage and the bilayer-skeletal separation energy in red blood cells

Stabilization of the lipid bilayer membrane in red blood cells by its association with an underlying membrane-associated cytoskeleton has long been recognized as critical for proper red blood cell function. One of the principal connections between skeleton and bilayer is via linkages between band 3, the integral membrane protein that transports anions across the cell surface, and membrane skeletal elements including ankyrin, adducin, spectrin, and the junctional complex of the skeleton. Here, we use membrane tether formation coupled with fluorescent labeling of membrane components to examine the importance of band 3 in stabilizing the bilayer-skeletal association. In membranes from a patient deficient in band 3, the energy associated with the bilayer skeleton is approximately zero, whereas when band 3 is immobilized by ligation with the monoclonal antibody R10, the energy of association approximately doubles. Fluorescence images of tethers reveal that ∼40% of the band 3 on the normal cell surface can be pulled into the tether, confirming a lateral segregation of membrane components during tether formation. These results validate a critical role for band 3 in stabilizing the bilayer-skeletal association in red cells.     

Cross-linkings between spectrin and band 3 in human erythrocyte membranes

A specific structural association between spectrin component 1 and band 3 in human erythrocyte membrane has been demonstrated by covalent cross-linkings, specific labeling, and the technique of two-dimensional gel electrophoresis. A complex of 330,000 daltons, representing 1 + 3, was produced in mildly oxidized membranes at physiologic pH and isotonic conditions but not at hypotonic conditions (< 10 mM KCl or NaCl). The yield of this complex decreased dramatically as the monovalent cation concentration decreased from 90 mM to 30 mM. The presence of Mg⁺⁺ or Ca⁺⁺ (2 mM) at low ionic strength promoted 1 + 3 cross-linking in an amount similar to that produced at isotonic conditions. The specific segment of band 3 involved in the cross-linking was also investigated by means of chymotrypsin digestion of band 3 in the intact red cells. The results showed the cross-links between spectrin component 1 and the 55,000-dalton fragment of band 3 at physiologic pH and isotonic conditions. This is consistent with the idea that band 3 is anchored on or contacted with the submembrane meshwork at the cytoplasmic membrane surface.     

ELECTRON MICROSCOPY OF THE OXYNTIC CELL IN THE GASTRIC GLANDS OF THE BULLFROG (RANA CATESBIANA) : I. The Non-Acid-Secreting Gastric Mucosa

The fine structure of the oxyntic cell from the gastric glands of the bullfrog was studied in lead hydroxide—stained sections of gastric mucosa fixed in buffered osmium tetroxide and embedded in n-butyl methacrylate. The oxyntic cell in non-acid-secreting stomachs (gastric juice pH, 7.4–7.8) is characterized by: (a) numerous closely packed smooth surfaced vesicular and tubular profiles disposed randomly in the cell; some of these elements show interconnections making it possible to identify this component with smooth surfaced endoplasmic reticula of certain other cell types, (b) a small percentage of rough surfaced profiles characteristic of endoplasmic reticula possessing RNP particles on the outer membrane surfaces, (c) a Golgi complex consisting of multiple isolated non-polarized arrays of smooth surfaced parallel elongated profiles and associated vesicular elements, (d) a sparse granular component (140 A) scattered freely in the cytoplasmic matrix, (e) numerous mitochondria with a dense matrix and containing an unusually large number of closely approximated cristae, (f) a number of zymogen granules consisting of either a dense body limited by a membrane or surrounded by a halo of less dense material which is in turn limited by a membrane, and (g) a number of granules (~260 A) containing several smaller granules (~80 A) identified presumably as glycogen. Intracellular canaliculi were not observed. Instead the free surface of the oxyntic cell facing the lumen of the gastric gland shows a complicated plication of the plasma membrane. Intercellular canaliculi are seen frequently between adjacent oxyntic cells. The walls of these canaliculi are made up of folded and ruffled cell membranes. The basal surface of the cell also exhibited this type of configuration. Occasional smooth surfaced profiles are seen communicating with the free surface, the wall of an intercellular canaliculus, or the basal surface of the cell. Although nerve endings were not found in association with oxyntic cells, unmyelinated nerves were observed in the vicinity of the gastric glands.     

Protein 4.1 is involved in a structural thermotropic transition of the red blood cell membrane detected by a spin-labeled stearic acid

Proteins involved in a structural transition in red blood cell membranes detected at 8 +/- 1.5 degrees C by a stearic acid spin-label have been investigated. Calcium loading of red blood cells with ionophore A23187 caused the disappearance of the 8 degrees C transition. Protein 4.1 appears to be the most susceptible protein to Ca2+ treatment. Antibodies specific for spectrin, band 3 (43K cytoplasmic domain), and protein 4.1 have been utilized as specific probes to modify membrane thermotropic properties. The 8 degrees C transition was eliminated by anti-4.1 protein antibodies but was not modified by the other antibodies. To further characterize the protein(s) involved in the transition, ghosts were subjected to sequential extraction of skeletal proteins. The extraction of band 6, spectrin, and actin did not modify the 8 degrees C transition. In contrast, high-salt extraction (1 M KCl) of spectrin-actin-depleted vesicles, a procedure that extracts proteins 2.1 and 4.1, was able to eliminate the 8 degrees C transition. Rebinding of purified protein 4.1 to the high salt extracted vesicles restored the 8 degrees C transition. These results indicate the involvement of protein 4.1 in the transition and suggest a functional membrane association of this protein. The binding of protein 4.1 to the membrane seems to contribute significantly to the thermotropic properties of red blood cells.     

THE FINE STRUCTURE AND IMMUNOLOGICAL LABELING OF THE ACHROMATIC MITOTIC APPARATUS AFTER DISRUPTION OF CELL MEMBRANES

After treatment of HeLa and L cells with vinblastine sulfate the material of microtubules (tubulin) was reorganized into (a) large paracrystals (PC) of tightly packed tubules; (b) smaller aggregates of tubules with greater diameter whose walls are constituted from well defined, helically arranged morphological subunits; and (c) microtubules associated with helices of polyribosomes of uniform size. All of these structures survived disruption of cellular membranes by means of a nonionic detergent. Following a thorough stripping of membranes there remained a subcellular fraction sedimenting at 1,500 g for 15 min, in which were contained nuclei, centrioles, and the above mentioned microtubular elements, maintained as a complex of organelles by an interconnecting network of 80 Å microfibrils. As a result of membrane disruption it was possible to localize precisely in the electron microscope the binding of ferritin antibody conjugates. Specific labeling at the surface of PC and microtubule aggregates could be demonstrated. This result was substantiated by means of the immunoperoxidase method of labeling the PC. A concentrated deposit of ferritin was also found in the vicinity of centrioles and related structures, the annuli of the nuclear pore complex and the annulate lamellae. However, the specificity of the label on these organelles remains questionable because ferritin, albeit in lower concentration, was also present on them in control preparations reacted with preimmune sera.     

Spectrin rearrangement early in erythrocyte ghost endocytosis

The endocytic vacuoles induced in white ghosts were found to be depleted of spectrin and therefore it was proposed that they arose from spectrin-free areas in the erythrocyte membrane. To follow changes in spectrin distribution during endocytosis, affinity-purified rabbit antispectrin antibodies were produced. Quantitative techniques were developed for the use of a highly specific 125I-F(ab')2 antispectrin, and these showed that before the appearance of vacuoles, as assessed by phase microscopy, there was a reproducible decrease in immunoreactive spectrin. To determine whether this spectrin decrease represented a local or diffuse spectrin loss or a spectrin rearrangement, morphologic studies were undertaken using transmission electron microscopy on samples treated with rabbit antispectrin and ferritin-conjugated goat anti-rabbit immunoglobulin. These studies showed that endocytosis was preceded by the creation of extensive spectrin-free areas separated by discrete spectrin-containing zones. Pretreatment of ghosts with alkaline phosphatase blocked all forms of endocytosis and prevented the creation of spectrin-free areas. Therefore, it is proposed that under the impetus of endocytosis inducers, phosphorylated spectrin is redistributed so that spectrin-free zones are created, and that endocytic vacuoles form and fuse in spectrin-free areas.     

αI-spectrin represents evolutionary optimization of spectrin for red blood cell deformability

Spectrin tetramers of the membranes of enucleated mammalian erythrocytes play a critical role in red blood cell survival in circulation. One of the spectrins, αI, emerged in mammals with enucleated red cells after duplication of the ancestral α-spectrin gene common to all animals. The neofunctionalized αI-spectrin has moderate affinity for βI-spectrin, whereas αII-spectrin, expressed in nonerythroid cells, retains ancestral characteristics and has a 10-fold higher affinity for βI-spectrin. It has been hypothesized that this adaptation allows for rapid make and break of tetramers to accommodate membrane deformation. We have tested this hypothesis by generating mice with high-affinity spectrin tetramers formed by exchanging the site of tetramer formation in αI-spectrin (segments R0 and R1) for that of αII-spectrin. Erythrocytes with αIIβI presented normal hematologic parameters yet showed increased thermostability, and their membranes were significantly less deformable; under low shear forces, they displayed tumbling behavior rather than tank treading. The membrane skeleton is more stable with αIIβI and shows significantly less remodeling under deformation than red cell membranes of wild-type mice. These data demonstrate that spectrin tetramers undergo remodeling in intact erythrocytes and that this is required for the normal deformability of the erythrocyte membrane. We conclude that αI-spectrin represents evolutionary optimization of tetramer formation: neither higher-affinity tetramers (as shown here) nor lower affinity (as seen in hemolytic disease) can support the membrane properties required for effective tissue oxygenation in circulation.     

Insulin degradation XXIII. Distribution of glutathione-insulin transhydrogenase in isolated rat hepatocytes as studied by immuno-ferritin and electron microscopy

The distribution of glutathione-insulin transhydrogenase (glutathione: protein-disulphide oxidoreductase, EC 1.8.4.2) in isolated rat hepatocytes that had been first treated with rabbit antiserum against purified rat liver transhydrogenase and then with ferritin-conjugated goat anti-rabbit γ-globulin was examined by electron microscopy. In cells with antact plasma membrane, the immunoferritin labeling of glutathione-insulin transhydrogenase was observed on a few external microvillous projections at the outside of the cell. In cells with breaks in the plasma membrane, the immunoferritin labeling appeared extensively on smooth vesicles just inside the plasma membrane and on smooth endoplasmic reticulum extending to and including the outer nuclear membrane, in addition to the external microvillous projections. There was some immunoferritin labeling on rough endoplasmic reticulum and on the inner surface of the plasma membrane. The mitochondria and the outer surface of the plasma membrane of the cell did not show the ferritin labeling. Control parallel samples in which the antiserum was substituted with normal (i.e. non-immune) serum or with neutralized antiserum (prepared by absorption with the transhydrogenase) showed little or no immunoferritin labeling. These results are consistent with the idea that gluthalione-insulin transhydrogenase probably synthesized in the endoplasmic reticulum and that the transhydrogenase accessible to cell surface (or found in the isolated plasma membrane preparations) probably represents a functional continuity between the endoplasmic reticulum and the plasma membrane.     

THE PROPERTIES OF SPECIFIC STAINS FOR ELECTRON MICROSCOPY PREPARED BY THE CONJUGATION OF ANTIBODY MOLECULES WITH FERRITIN

In order to take full advantage of recent developments in the electron microscopic examination of cellular ultrastructure and composition, it is necessary to develop specific electron stains capable of identifying and localizing a wide variety of macromolecular components of cells. To this end, antibody conjugates have been prepared by chemically coupling the highly electron-scattering ferritin molecule to antibody. Antigen-antibody precipitations with these ferritin-antibody conjugates have demonstrated that under the appropriate conditions they retain the specific binding properties of the antibody from which they are prepared. An electron microscopic study has been made of aggregates of tobacco mosaic virus and its ferritin-conjugated antibody. The aggregates were prepared in solution and then sprayed onto specimen screens. The electron micrographs reveal that the conjugate specifically attached to, and delineated, the virus rods. The chemistry, structure, and resolving power of the ferritin-antibody conjugates, the specificity of their reactions with homologous antigen, and the nature of the problems to be faced in application of these conjugates to the study of the internal antigens of cells are discussed.     

Phosphorylation and dephosphorylation of spectrin

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of ³²Pi from [³²P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [³²P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.     

An assay of malaria parasite invasion into human erythrocytes: The effects of chemical and enzymatic modification of erythrocyte membrane components

Invasion of erythrocytes by malaria parasites is known to be blocked by proteolytic digestion of merozoite receptors allegedly present in red cell membranes. This information was used in the present work to develop a simple and convenient assay for parasite invasion into red blood cells and for evaluating the role played by red cell membrane components in this process. Synchronized in vitro cultures of Plasmodium falciparum containing only ring stages were subjected to either trypsin or pronase digestion, a treatment that neither affected ring development into schizonts nor mature merozoite release. Cells from this culture were not invaded by the released merozoites. However, upon addition of untreated human red blood cells, marked invasion was observed, either microscopically or as [³H]isoleucine incorporation. The new assay circumvents the need for separating schizonts from uninfected cells and provides a convenient means for assessing how chemical and biochemical manipulation of red blood cells affects their invasiveness by parasites. Using this assay, we verified that sheep and rabbit erythrocytes were resistant to invasion, as were human erythrocytes which had been treated with trypsin, pronase or neuraminidase. Chymotrypsin digestion of human erythrocytes was without effect on invasion. Human erythrocytes which were chemically modified with the impermeant amino reactive reagent H₂DIDS, or with the crosslinker of spectrin, TCEA, were found to resist invasion. The results underscore the involvement of surface membrane components as well as of elements of the cytoskeleton in the process of parasite invasion into erythrocytes.     

Expression of red cell membrane proteins in erythroid precursor cells

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50–60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70–80% of all cells in the spleen of anemic animals, while only 1–2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.