Crosslinking of isolated cytoskeletal proteins with hemoglobin: a possible damage inflicted to the red cell membrane

Crosslinking of isolated red cell membrane cytoskeletal proteins and hemoglobin mediated by H2O2 was studied. The products of spectrin and hemoglobin interaction were demonstrated electrophoretically to be high-molecular-weight polypeptides crosslinked by nondisulfide covalent bonds. The molecular weight of the protein bands correlated with various combinations of spectrin and hemoglobin chains and the relative amount of the different products was dependent on the molar ratio of the interacting proteins. Free hemin caused spectrin crosslinking as well, but globin in the absence of hemin was inactive. Since the H2O2-mediated reaction resulted in reduction of the spectrin tryptophan fluorescence, the latter was used to monitor the reaction progress under various conditions. Both oxyhemoglobin and methemoglobin were found to be most efficient, whereas cyanmethemoglobin and hemichrome were relatively inactive. Analysis of the data implied that tryptophan oxidation as well as spectrin conformational changes follow an iron-induced crosslinking of the interacting proteins. Actin, the second major protein in the red cell cytoskeleton, behaved similarly to spectrin. The intrinsic fluorescence intensity of both G- and F-actin was decreased upon addition of H2O2 to the mixture of hemoglobin and each of the actin forms. SDS-polyacrylamide gel electrophoresis revealed that G-actin crosslinked one or two hemoglobin chains. F-actin-hemoglobin interaction induced by H2O2 produced very high aggregates that could not penetrate the gel. It is suggested that crosslinking of cytoskeletal proteins in red cells containing membrane-associated hemoglobin provides a rationale for the loss of membrane flexibility.     

Long-term intercalation of residual hemin in erythrocyte membranes distorts the cell

The effect of long-term incubation of residual globin-free hemin on whole red blood cell and isolated cytoskeletal proteins was studied. Hemin at concentrations found in pathological red cells was inserted to fresh erythrocytes. Increased hemolysis developed in the hemin-containing cells after a few days at 37 degrees C and after about four weeks at 4 degrees C. Since lipid and hemoglobin peroxidation did not depend on the presence of hemin, time-dependent effects on the cytoskeleton proteins were studied. Observations were: (1) spectrin and protein 4.1 exhibited a time-dependent increasing tendency to undergo hemin-induced peroxidative crosslinking. (2) The ability of the serum proteins, albumin and hemopexin, to draw hemin from spectrin, actin and protein 4.1 decreased with time of incubation with hemin. These results were attributed to time-dependent hemin-induced denaturation of the cytoskeletal proteins. Albumin taken as a control for physiological hemin trap was unaffected by hemin. Small amounts of hemo-spectrin (2-5%) were analyzed in circulating normal cells, and this in vivo hemo-spectrin also failed to release hemin. It was concluded that slow accumulation of hemin, a phenomenon increased in pathological cells, is a toxic event causing erythrocyte destruction.     

Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.     

Disintegration of red cell membrane cytoskeleton by hemin

Spectrin and actin were isolated and their oligomeric state after association with hemin at various conditions was studied. Intact cytoskeletons were prepared by Triton X-100 extraction of red blood cells and incubated with hemin and their stability analyzed by the appearance of dissociated proteins in the supernatant. The cytoskeletons dissociated in a time, temperature and hemin concentration-dependent manner. Following 18 hours incubation in the presence of 0.3 mM hemin there was no dissociation at 4 degrees C, while at the same hemin concentration after 2 hours complete dissociation of the cytoskeletons occurred at 37 degrees C. Microscopy indicated that the cytoskeletons incubated with hemin lost their "cell like" shapes in a time dependent manner. Hemin applied to intact cells also caused dissociation of their cytoskeletons as judged by the failure to separate integer cytoskeletons from red cells treated with hemin. From hemin-induced dissociation profiles of separated actin, spectrin and whole cytoskeletons under various conditions, a mechanism of cytoskeleton breakdown was analyzed, as a release of band 4.1 in the first step which is followed by spectrin dimerization and eventually dissociation of the entire cytoskeletons.     

Hemin-mediated dissociation of erythrocyte membrane skeletal proteins

Spectrin tetramers and oligomers in normal erythrocytes are cross-linked by actin and protein 4.1 to form a two-dimensional membrane skeletal network. In the present study, we find that hemin, a breakdown product of hemoglobin, progressively (a) alters the conformation of spectrin as revealed by electron microscope studies and by the decreased resistance of spectrin to proteolytic degradation, (b) alters the conformation of protein 4.1 as revealed by the increased mobility of protein 4.1 on nondenaturing gel electrophoresis, (c) weakens spectrin dimer alpha beta-dimer alpha beta, spectrin alpha-spectrin beta, as well as spectrin-protein 4.1 associations as analyzed by nondenaturing gel electrophoresis, and (d) diminishes the structural stability of erythrocyte membrane skeletons (i.e. Triton-insoluble ghost residues) subjected to mechanical shearing. Since hemin may be liberated from oxidized or unstable mutant hemoglobin under pathological conditions, these hemin-induced effects on spectrin, protein 4.1, and membrane skeletal stability may play a role in the membrane lesion of these erythrocytes.     

Composition of the ternary protein complex of the red cell membrane cytoskeleton

The red cell membrane skeletal network is constructed from actin, spectrin and protein 4.1 in a molar ratio of actin subunits/spectrin heterodimer/protein 4.1 of 2:1:1. This represents saturation of the actin filaments, since incubation with extraneous spectrin and protein 4.1 leads to no binding of additional spectrin, either to the inner surface of ghost membranes or to lipid-free membrane cytoskeletons. Partial extraction of spectrin from the membrane is accompanied by release of actin under all conditions. Regardless of the proportion of spectrin extracted, the molar ratio of spectrin dimers/actin subunits is constant at 1:2. This is not the result of release or cooperative breakdown of whole lattice junctions from the network, for the number of actin filaments, judged by capacity to nucleate polymerisation of added G-actin, remains unchanged even when as much as 60% of the total spectrin has been lost. A similar 1:2:1 stoichiometry characterises the complex formed when G-actin is allowed to polymerise in the presence of varying amounts of spectrin and protein 4.1. When this complex is treated with the depolymerising agent, 1 M guanidine hydrochloride, it breaks down into smaller units of the same stoichiometry. After cross-linking these can be recovered from a gel-filtration column. Complexes prepared starting from G-actin appear to be much more stable than those formed when spectrin and protein 4.1 are bound to F-actin.     

The role of band 4.1 in the association of actin with erythrocyte membranes

Spectrin stimulates the association of F-actin with erythrocyte inside-out vesicles. Although inside-out vesicles are nearly devoid of two of the three major cytoskeletal proteins, spectrin and actin, they retain nearly all of the cytoskeletal protein designated band 4.1. Inside-out vesicles which have been substantially depleted of band 4.1 by extraction in 1 M KCl, 0.4 M urea and then reconstituted with spectrin show a markedly diminished ability to bind actin by comparison with vesicles containing normal amounts of band 4.1. This diminution is not due to an impaired ability of the vesicles to bind spectrin. Addition of purified band 4.1 to vesicles either before or after they have been reconstituted with spectrin restores their actin binding capacity to near normal levels as does addition of a spectrin-band 4.1 complex prepared by sucrose gradient centrifugation. Band 4.1 bound to vesicles in the absence of added spectrin has no effect on actin binding. Our results suggest that a spectrin band 4.1 complex is responsible for binding actin to erythrocyte membranes.     

Hereditary spherocytosis of man. Defective cytoskeletal interactions in the erythrocyte membrane.

Hereditary spherocytosis (HS) is an inherited abnormality of red cell shape and results from defective interactions amongst the components of the cytoskeleton. It is known that spectrin/actin dissociates in low ionic strength media from ghosts and cytoskeletons at a rate which is slower for HS than normal preparations. Hybridization experiments have established that this behaviour is not due to a defective spectrin or actin but resides in a spectrin-binding component of the membrane [Hill, Sawyer, Howlett & Wiley (1981) Biochem. J. 201, 259-266]. In the present study erythrocyte shells have been examined in low ionic strength media and a similar difference in the rate of solubilization has been revealed. Since band 4.1 (but not band 2.1) is a common component of cytoskeletons and shells it is possible that 4.1 may be abnormal in the HS condition. The interaction of band 4.1 with spectrin/actin was examined by low shear falling ball viscometry. The addition of a mixture of band 2.1 and 4.1 to a solution of actin and spectrin tetramer increased the viscosity due to cross-linking of the cytoskeletal elements by band 4.1. When band 2.1/4.1 mixtures were derived from five HS families the viscosity was increased to a greater extent than in the normal controls. This difference was not a result of alterations in the calcium dependence of the spectrin/actin-band 4.1 interaction. The results imply that band 4.1 may be defective in the HS condition.     

Preparation of red-cell-membrane cytoskeletal constituents and characterisation of protein 4.1

A new and rapid method is described for the preparation of protein 4.1, the protein which modulates the interaction between spectrin and actin in the membrane cytoskeleton of the red cell. The method is based on the dissociation of purified membrane cytoskeletons in concentrated Tris at neutral pH, followed by gel filtration in the same medium. This procedure also yields spectrin and actin, as well as the fourth cytoskeletal constituent, protein 4.9, in relatively pure form, and ankyrin. Protein 4.1 is monomeric under our conditions of solvent and protein concentration, with a relative molecular mass, as determined from sedimentation equilibrium, of about 78 000; its sedimentation coefficient and Stokes' radius are those of a globular, though somewhat asymmetric or flexible molecule. It forms a strong complex with F-actin and spectrin. Protein 4.9 is also recovered in active form, and will bind strongly to F-actin.     

Spectrin-actin associations studied by electron microscopy of shadowed preparations

By shadowing specimens dried onto mica sheets we have obtained clear images of actin crosslinked by spectrin, an actin-binding protein found in erythrocytes. We conclude that spectrin dimers possess a single binding site for F actin. Tetramers formed by head-to-head association of two dimers possess two actin binding sites, one at each tail. Polymerizing G actin in the presence of spectrin tetramers or mixing preformed F actin with spectrin tetramer plus band 4.1 results in an extensively crosslinked network of actin filaments. When G actin is polymerized in the presence of spectrin at spectrin:actin mole ratios close to that present on the erythrocyte membrane, large amorphous protein networks are formed. These networks are clusters of spectrin around 25 nm diameter structures which may be actin protofilaments. These networks are similar to the cytoskeletal network seen after erythrocyte membranes are extracted with detergent, and may represent the first in vitro assembly of a cytoskeletal complex resembling that of the native cell both biochemically and structurally.     

The spectrin cytoskeleton is crucial for adherent and invasive bacterial pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.     

Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation.

Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles.     

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.     

The Protein 4.1 family: Hub proteins in animals for organizing membrane proteins

Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and differential mRNA splicing. Finally, the spectrum of interactions of the 4.1 proteins overlaps with that of another membrane-cytoskeleton linker, ankyrin. Both ankyrin and 4.1 link to the actin cytoskeleton via spectrin, and we hypothesize that differential regulation of 4.1 proteins and ankyrins allows highly selective control of cell surface protein accumulation and, hence, function. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Interactions among red cell membrane proteins

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.     

Annexin V binds to the actin-based cytoskeleton at the plasma membrane of activated platelets.

Immunocytochemical studies demonstrate that annexin V relocates to the plasma membranes of intact stimulated blood platelets. Anti-annexin V antibodies label the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed, glass-activated platelets and colocalize with actin. In addition, crosslinking experiments using detergent-solubilized membranes of activated platelets have identified an 85-kDa complex containing annexin V. The 85-kDa complex is also recognized by antibodies against actin, suggesting that annexin V interacts with actin. In addition, annexin V was found to associate with filamentous actin in the presence of millimolar Ca(2+). Annexin V was also shown by immunofluorescence microscopy to be associated with platelet cytoskeletons, colocalizing with actin in the presence of micromolar Ca(2+). These findings provide the first evidence for annexin V binding to the plasma membrane and to the actin-based cytoskeleton in activated platelets and indicate that annexin V may function in both cytoskeletal and membrane domains.     

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin- reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.     

Integral membrane protein interaction with Triton cytoskeletons of erythrocytes

The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.     

Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion

The gastric parietal (oxyntic) cell is presented as a model for studying the dynamic assembly of the skeletal infrastructure of cell membranes. A monoclonal antibody directed to a 95-kD antigen of acid-secreting membranes of rat parietal cells was characterized as a tracer of the membrane movement occurring under physiological stimuli. The membrane rearrangement was followed by immunocytochemistry both at the light and electron microscopic level on semithin and thin frozen sections from resting and stimulated rat gastric mucosa. Double labeling experiments demonstrated that a specific and massive mobilization of actin, and to a lesser extent of spectrin (fodrin), was involved in this process. In the resting state, actin and spectrin were mostly localized beneath the membranes of all cells of the gastric gland, whereas the bulk of acid-secreting membranes appeared diffusely distributed in the cytoplasmic space of parietal cells without any apparent connection with cytoskeletal proteins. In stimulated cells, both acid-secreting material and actin (or spectrin) extensively colocalized at the secretory apical surface of parietal cells, reflecting that acid-secreting membranes were now exposed at the lumen of the secretory canaliculus and that this insertion was stabilized by cortical proteins. The data are compatible with a model depicting the membrane movement occurring in parietal cells as an apically oriented insertion of activated secretory membranes from an intracellular storage pool. The observed redistribution of actin and spectrin argues for a direct control by gastric acid secretagogues of the dynamic equilibrium existing between nonassembled (or preassembled) and assembled forms of cytoskeletal proteins.