Participation of membrane skeleton proteins in aggregation of epidermal growth factor receptors in A431 cells.

Polyclonal antibody against alpha-spectrin of chicken erythrocytes was prepared. This antibody as well as anti-vinculin and anti-annexin I and II, were used for localization of the antigens in A431 cells during translocation of epidermal growth factor receptors (EGF-Rs) on cell surface. During aggregation of EGF-Rs only spectrin and actin aggregates colocalized with the "capped" receptors in adherent as well as in suspended cells. Physiological implication of spectrin involvement in EGF-Rs redistribution in A431 cells is discussed.     

Immunodetection of spectrin-like proteins in yeasts

Spectrin, a component of the membrane skeleton in erythrocytes and other animal cells, has also been identified in plant and fungal cells. However, its postulated role, i.e., the maintenance of shape and elasticity of the plasma membrane, is probably not exerted in walled cells. To study spectrin in these cells, we chose yeasts because of a high morphological variability of their life cycle. The localization of spectrin in the cells and protoplasts of Saccharomyces cerevisiae and Schizosaccharomyces japonicus var. versatilis was detected by immunoblotting, indirect immunofluorescence, and immunogold electron microscopy techniques with the use of anti-chicken and anti-human erythrocyte spectrin antibodies. A protein band of 220-240 kDa and some bands of lower relative mass were detected in cell and protoplast extracts of both yeast strains. Spectrin-like proteins were revealed by fluorescence microscopy at cell surfaces and in vacuolar membranes. Immunogold-labelling showed spectrin-like proteins in the plasma membrane, endoplasmic reticulum, vacuoles, nuclei, vesicles, mitochondria, and cell walls. The topology of spectrin was not affected by actin depolymerization with Latrunculin B nor was it changed in either act1-1 or cdc42 mutants, under restrictive conditions. Under osmotic stress, both spectrin and actin were delocalized and appeared in the form of large clusters in the cytoplasm. It is concluded that a protein cross-reacting with spectrin antibodies is present in fission and budding yeasts. Generally, it is located in the proximity of the plasma membrane and other intracellular membranes, probably as a part of the membrane skeleton. No evidence of its relationship to either actin or growth zones of the cell can be provided.     

Adducin: Ca++-dependent association with sites of cell-cell contact

Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.     

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.     

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.     

Spatial distribution of cortical proteins in cells of epithelial sheets

Summary In the differentiated pigmented epithelial cells of the retina (RPE) of chick embryos cytoskeletal proteins are found in polygonal rings located in the cell cortex. Within the cortical rings of the RPE cells vinculin and spectrin occupy a characteristic position closest to the plasma membrane; actin is found farther away, while tropomyosin and myosin are located farthest from the plasma membrane. The differences in the distribution of these proteins might reflect the functional specialization of different parts of the cortical ring required to develop and transmit tension from individual cells throughout the entire epithelial sheet.     

Immunofluorescent patterns of spectrin in lymphocyte cell lines

Spectrin, a membrane-associated cytoskeletal protein, has been observed in all of 45 lymphoid and myeloid cell lines examined. For these experiments, formalin-fixed cells from randomly selected lines propagated by using conventional tissue culture procedures were examined by immunofluorescence, using an antibody directed against chicken erythrocyte alpha-spectrin. Two distinct immunofluorescent patterns of spectrin distribution were identified. In most lines examined (16 mouse and 18 human lymphoid or myeloid lines), spectrin was symmetrically distributed near the submembranous region of the plasma membrane. In the remainder of the cell lines examined, a second pattern was observed; in these cultures, the cells contain a polar submembranous aggregate of spectrin with little staining at the rest of the plasma membrane. Long-term T lymphocyte cell lines in which greater than 60% of the cells expressed a polar submembranous aggregate of spectrin (PSA-S) include mouse cell lines EL-4, LBRM-33, CT-6X, NIXT, 22CM-37, and 7ON-2 and human lines JM and PEER. Other established cultures in which PSA-S were observed included the human macrophage-like line U-937 and gibbon T cell line MLA-144. Phorbol myristate acetate or mezerin caused a reversible alteration in the distribution of spectrin in these cell lines. These drugs, which increase membrane fluidity, caused a complete but temporary symmetrical redistribution of the spectrin aggregate. Our results indicate that the pattern of spectrin distribution, either aggregated or evenly dispersed, is a stable characteristic (but one that can be altered) in various cell lines, and that because similar variations in pattern have been noted in situ, it is likely that the pattern present in any given cell line reflects a characteristic associated with a particular stage of a cell's maturation. It is anticipated that these cell lines, positive and negative for the expression of natural polarity of spectrin distribution, will provide useful models for future studies to define further the role of spectrin in lymphocyte plasma membrane functions.     

Relation between the organization of spectrin and of membrane lipids in lymphocytes

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.     

The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.     

Engagement of spectrin and actin in capping of FcgammaRII revealed by studies on permeabilized U937 cells.

Plasma membrane receptors can undergo translocation in the plane of plasma membrane after binding of polyvalent ligands. Ligand/receptor clusters, named patches, can collect into a polar cap, presumably due to their association with the submembrane actin-based cytoskeleton. We found that the assembly of Fcgamma receptor II caps in human monocytic U937 cells was accompanied by the accumulation of spectrin and actin in the cap region. Permeabilization of cells with streptolysin O rendered capping sensitive to inhibition by phalloidin, an actin filament stabilizing agent. A rabbit antibody directed against the chicken erythrocyte alpha-subunit of spectrin, an actin- and membrane-binding protein, also blocked the capping in a dose dependent manner. The inhibition reached approximately 50% after 20 minutes of cell treatment with the antibody. Anti-alpha-spectrin targeted specifically its submembrane antigen, in contrast to unspecific antibodies which remained dispersed in the cell interior and had no influence on the cap assembly. Our results indicate an active engagement of spectrin and actin filaments in the capping of Fcgamma receptor II.     

Spectrin, fodrin and protein 4.1-like proteins in differentiating rat germ cells

The presence and the distribution of proteins of the membrane skeleton in differentiating germ cells of the rat has been investigated. Immunofluorescence and immunoblotting analysis, performed using monoclonal and polyclonal antibodies to human erythroid alpha-spectrin and protein 4.1 and to brain spectrin (fodrin), demonstrated the presence of analogues of spectrin and fodrin in spermatocytes and round spermatids and of protein 4.1-like molecules in spermatocytes, spermatids and spermatozoa. Spectrin and fodrin showed molecular weights comparable to those of their analogues in erythrocytes but a distinct intracellular distribution. Fodrin was localized along the plasma membrane while spectrin appeared associated with the regions of the Golgi apparatus and of the developing acrosome. Antibodies to protein 4.1 recognized molecules with a molecular weight not comparable with that in erythrocytes, and their presence in spermatozoa was confined to specific regions of the head and of the tail.     

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α- and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.     

Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm

The spectrin skeleton of non-erythroid cells is likely to interact with a variety of integral membrane proteins and participate both in stable linkages as well as dynamic structures capable of rapid disassembly and assembly. The basis for diversity of roles for spectrin includes multiple, functionally distinct isoforms of spectrin, ankyrin and other associated proteins, regulation of protein interactions through phosphorylation and calcium/calmodulin, as well as differential expression of accessory proteins that determine the organization and localization of spectrin in cells. Spectrin is highly conserved from Drosophila to man and is likely to be involved in fundamental aspects of membrane structure requiring long range order and organization. Spectrin is a candidate to interact with many integral membrane proteins in roles basic to metazoan cells which must associate into tissues. Organization of cells into tissues requires loss of cell motility, formation of specialized membrane domains and assembly of cell junctions, which are all activities potentially involving spectrin. Future challenges lie in devising direct experiments to evaluate the functions of spectrin in cells and tissues.     

Spectrin loss during in vitro red cell lysis

Spectrin was extracted from washed erythrocyte ghosts in 1 mM EDTA buffer (pH 8.0) and purified to homogeneity by gel filtration. Anti-human spectrin was raised in rabbits. Specificity of the antibody was demonstrated by immunodiffusion, immunoelectrophoresis and immunofluorescent techniques. Membrane-free hemolysate prepared by lysing red cells in 5 mM phosphate buffer (pH 8.0) for variable intervals (5--60 min) at 4 degrees C was found to contain spectrin identifiable by immunodiffusion, immunoelectrophoresis, immunofluorescence and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Spectrin was demonstrable in ultracentrifuged membrane-free hemolysate and, in progressively decreasing amounts, in membrane washes. Membrane-free hemolysate contained more spectrin when erythrocytes were lysed for 60 min than for 5 min. The data indicate that a significant amount of spectrin is detached from the membrane following sysis in hypotonic buffer for different time intervals. Spectrin lost in this manner might be part of spectrin attached to the lipid bilayer.     

Drosophila beta spectrin functions independently of alpha spectrin to polarize the Na,K ATPase in epithelial cells

Spectrin has been proposed to function as a sorting machine that concentrates interacting proteins such as the Na,K ATPase within specialized plasma membrane domains of polarized cells. However, little direct evidence to support this model has been obtained. Here we used a genetic approach to directly test the requirement for the beta subunit of the alphabeta spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. beta Spectrin mutations were lethal during late embryonic/early larval development and they produced subtle defects in midgut morphology and stomach acid secretion. The polarized distributions of alphabeta(H) spectrin and ankyrin were not significantly altered in beta spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independently of one another, and that ankyrin is upstream of alphabeta spectrin in the spectrin assembly pathway. In contrast, beta spectrin mutations had a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for beta spectrin in determining the subcellular distribution of the Na, K ATPase and, unexpectedly, this role is independent of alpha spectrin.     

alpha -Catenin binds directly to spectrin and facilitates spectrin-membrane assembly in vivo

The anchorage of spectrin to biological membranes is mediated by protein and phosphoinositol phospholipid interactions. In epithelial cells, a nascent spectrin skeleton assembles in regions of cadherin-mediated cell-cell contact, and conversely, cytoskeletal assembly is required to complete the cell-adhesion process. The molecular interactions guiding these processes remain incompletely understood. We have examined the interaction of spectrin with alpha-catenin, a component of the adhesion complex. Spectrin (alphaIIbetaII) and alpha-catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and alpha-catenin in vitro. By surface plasmon resonance and in vitro binding assays, we find that alpha-catenin binds alphaIIbetaII spectrin with an apparent K(d) of approximately 20-100 nm. By gel-overlay assay, alpha-catenin binds recombinant betaII-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of alpha-catenin is fully accounted for in recombinant peptides encompassing the NH(2)-terminal 228 amino acid region of alpha-catenin. An in vivo role for the interaction of spectrin with alpha-catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective alpha-catenin. Transfection of these cells with wild-type alpha-catenin reestablishes alpha-catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between alpha-catenin and the amino-terminal domain of beta-spectrin augment the interaction between alpha-catenin and actin, and together they provide a polyvalent linkage directing the topographic assembly of a nascent spectrin-actin skeleton to membrane regions enriched in E-cadherin.     

Spectrin localization in osteoclasts: Immunocytochemistry,cloning, and partial sequencing

The presence of spectrin was demonstrated in chick osteoclasts by Western blotting and light and electron microscopic immunolocalization. Additionally, screening of a chick osteoclast cDNA library revealed the presence of α-spectrin. Light microscope level immunocytochemical staining of osteoclasts in situ revealed spectrin staining throughout the cytoplasm with heavier staining found at the marrow-facing cell margin and around the nuclei. Confocal microscopy of isolated osteoclasts plated onto a glass substrate showed that spectrin encircled the organelle-rich cell center. Nuclei and cytoplasmic inclusions were also stained and the plasma membrane was stained in a nonuniform, patchy distribution corresponding to regions of apparent membrane ruffling. Ultracytochemical localization showed spectrin to be found at the plasma membrane and distributed throughout the cytoplasm with especially intense staining of the nuclear membrane and filaments within the nuclear compartment. J. Cell. Biochem. 71:204–215, 1998. © 1998 Wiley-Liss, Inc.     

Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβ_H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβ_H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.     

Diversity in protein associations of the spectrin-based membrane skeleton of nonerythroid cells

Summary The purpose of this review is to summarize recent progress in understanding interactions of spectrin with membranes from brain and other tissues. Spectrin has at least two choices in linkages with the membrane, one through ankyrin, which in turn is associated with integral membrane proteins, and another linkage directly with integral membrane sites identified recently in brain membranes. Some of the integral membrane protein sites in brain bind preferentially with one spectrin isoform, while some can interact with both erythroid and the general isoform of spectrin. Ankyrin also has different isoforms, and these exhibit specificity in binding to spectrin isoforms and associate with distinct integral membrane proteins. The membrane binding sites for ankyrin include several integral membrane proteins, which are differentially expressed in different cells: the anion exchanger of intercalated cells of mammalian kidney, the sodium/potassium ATPase of kidney, and the voltage-dependent sodium channel of neurons. Ankyrin is present in many other cell types and it is likely that additional ankyrin-binding proteins will be identified. Each of the proteins that now are candidates for ankyrin binding proteins are ion channels or transporters and are localized in specialized cellular domains. The polarized localization of the ankyrin-associated membrane proteins is an essential aspect of their function at a physiological level. Spectrin and ankyrin thus exhibit an unsuspected diversity in protein linkages and have the potential for cell domain-specific interactions with a variety of membrane proteins.     

Presence of spectrin in untreated friend erythroleukemic cells. Its accumulation upon treatment of the cells with dimethyl sulfoxide

Friend leukemia cells (FLC) are nucleated erythroid precursors, and are markedly stimulated towards more advanced stages of differentiation by treatment with dimethyl sulfoxide (DMSO). The presence of spectrin, an erythrocyte membrane protein, has been investigated in untreated and in DMSO-treated FLC by indirect immunofluorescence and by analysis in SDS-polyacrylamide gel electrophoresis of low-ionic-strength cell extracts immuno-precipitated with a monospecific anti-spectrin serum. Spectrin is detectable in significant amounts in the “inducible” clones prior to DMSO stimulation, and accumulates 4- to 5-fold upon addition of this compound to the cultures. Spectrin accumulation occurs rather early (24 hours after cell seeding) and reaches its peak on the third day, to decline thereafter. Semiquantitative determinations of spectrin amounts present in DMSO-stimulated 745A and A°1 cells on the third day after treatment were 2.4 × 10⁵ and 3.0 × 10⁵ molecules/cell, respectively. Spectrin is also detectable in very low amounts in an “uninducible” line of FLC, and is not accumulated upon DMSO treatment thereof, whereas treatment with hemin does cause a significant increase of spectrin-positive cells. These data indicate that spectrin is a convenient “early” marker for in vitro studies of erythropoiesis.