Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins.

Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.

A beta-spectrin isoform from Drosophila (beta H) is similar in size to vertebrate dystrophin.

Spectrins are a major component of the membrane skeleton in many cell types where they are thought to contribute to cell form and membrane organization. Diversity among spectrin isoforms, especially their beta subunits, is associated with diversity in cell shape and membrane architecture. Here we describe a spectrin isoform from Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD) that we term beta H. The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures that are attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit but shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family such as alpha-spectrin and alpha-actinin.

Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells.

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.

Drosophilia spectrin. I. Characterization of the purified protein.

We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.

Golgi spectrin: identification of an erythroid beta-spectrin homolog associated with the Golgi complex.

Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

Decreased membrane deformability in Melanesian ovalocytes from Papua New Guinea.

We examined the ability of Melanesian ovalocytes from Papua New Guinea to be deformed in order to probe the resistance of these cells to invasion by several species of malaria parasite. We found ovalocytes were refractile to drug-induced endocytosis, that they formed abnormal rouleaux, showed reduced deformability when aspirated into 0.6-micron diameter pores in polycarbonate sieves, and failed to crenate when mounted under a glass coverslip. No substantial differences were found between normocytes and ovalocytes in their initial rate of filtration through 4.5-micron pore polycarbonate sieves, their membrane fluidity as measured by the rate of depolarization of fluorescent probes or the rate of extraction of cytoskeletal proteins in low ionic strength buffers. We conclude that the resistance of ovalocytes to undergo localized deformation might be significant in explaining the resistance of these cells to invasion by malarial merozoites.

Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.

Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.

The 22.5 kDa spectrin-binding domain of ankyrinR binds spectrin with high affinity and changes the spectrin distribution in cells in vivo.

It was previously shown that ankyrins play a crucial role in the membrane skeleton arrangement. Purifying ankyrinR obtained from erythrocytes is a time-consuming process. Therefore, cloned and bacterially expressed ankyrinR-spectrin-binding domain (AnkSBD) is a demanded tool for studying spectrin-ankyrin interactions. In this communication, we report on the cloning and purification of AnkSBD and describe the results of binding experiments, in which we showed high-affinity interactions between the AnkSBD construct and isolated erythrocyte or non-erythroid spectrins. pEGFP-AnkSBD-transfected cells co-localised with non-erythroid spectrin in HeLa cells. The functional interactions of the AnkSBD construct in vivo and in vitro open many possibilities to study the structure and function of this domain, which has not yet been as extensively studied when compared to the aminoterminal domain of this protein.

A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.

Defective binding of spectrin to ankyrin in a kindred with recessively inherited hereditary elliptocytosis.

The interaction of spectrin with spectrin-depleted inside-out membrane vesicles was studied in a kindred with an atypical variant of hereditary elliptocytosis inherited in a recessive manner. The probands are characterized by prominent elliptocytosis, decreased erythrocyte thermal stability, an altered limited tryptic peptide pattern of spectrin digested at low ionic strength, and defective spectrin dimer-dimer association. The parents are normal. The spectrin/band 3 ratio determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated membranes of the probands was decreased to approximately 70% of control values, and total erythrocyte spectrin content in one proband was also decreased on SDS-PAGE. When a monospecific antispectrin antibody was used, a faintly labeled fragment of molecular weight approximately 28,000 was detected on immunoblots of whole cell lysates of one proband and a control, but could not account for the decreased erythrocyte spectrin content of the proband on SDS-PAGE. Binding and competitive inhibition studies revealed an alteration in the spectrin-ankyrin interaction due to an abnormality of spectrin in the probands. No defect was found in the mother; the father's spectrin showed decreased binding affinity, although it was not so severe as in the probands. Separation of bound and unbound spectrin dimers from one proband and subsequent conversion to tetramers showed that the self-association defect was detectable only on the bound subpopulation of her spectrin. These findings demonstrate a hitherto undescribed functional abnormality of spectrin in this kindred which could result in decreased stability of the membrane skeleton and contribute to the elliptocytic shape of these erythrocytes.

Diminished spectrin extraction from ATP-depleted human erythrocytes. Evidence relating spectrin to changes in erythrocyte shape and deformability.

We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.

Membrane protein lesions in erythrocytes with Heinz bodies.

We studied Heinz body-containing erythrocytes with three different unstable hemoglobins: Nottingham, Brockton, and unclassified. We demonstrated two classes of membrane protein defects in unstable hemoglobin-containing cells (UH-RBCs), a defect of the spectrin-depleted inside-out vesicle (UH-IOV), and a defect of spectrin (UH-spectrin) itself. The composition of UH-IOVs is the same as control with respect to quantity of ankyrin and proportion inside-out. However, UH-IOVs bind even less spectrin than IOVs derived from sickle erythrocytes (SS-IOVs), suggesting a severe functional defect in the ankyrin of UH-RBCs (UH-ankyrin). Further evidence that UH-ankyrin is abnormal is demonstrated by the virtual absence of ankyrin in isotonic membrane shells of UH-RBCs (UH-shells), and abnormal mobility and decreased binding of the 72-kD (spectrin-binding) alpha-chymotryptic fragment of UH-ankyrin (UH-72-kD) to control spectrin. All UH-RBC membranes were spectrin-deficient (60% of control). In addition, spectrin isolated from UH-RBCs (UH-spectrin) was abnormal in two respects: (a) presence of a fast-moving band on nondenaturing polyacrylamide gels of both 0 degree C and 37 degrees C extracts, and (b) decreased binding to actin in the presence of protein 4.1. UH-spectrin did exhibit normal self-association, binding to IOVs and binding to actin in the absence of protein 4.1. This pattern of normal and abnormal spectrin functions has been described for spectrin subjected to mild diamide oxidation, suggesting the role of oxidation is the pathogenesis of membrane defect(s) of erythrocytes with abnormal hemoglobins.

On the mechanism of vesicle release from ATP-depleted human red blood cells.

The release of spectrin-free vesicles from ATP-depleted human red blood cells (Lutz et al. (1977) J. Cell. Biol. 73, 548) can be considered the final step of a shape change from discocytes to echinocytes. The study of physical and chemical properties of released vesicles suggests that vesicle release is not merely a consequence of charge alterations within either monolayer of the budding membrane. Fresh membranes and released vesicles have within experimental error the same sialic acid content per surface area and the same electrophoretic mobilities. Vesicle release cannot be stimulated by doubling the charge density on the outer monolayer by means of a phospholipase D-treatment, but correlates with a breakdown of polyphosphoinositides to diacylglycerol on the inner monolayer. This breakdown does not lead to a significant change in the negative charge density on the inner monolayer, because an increased phosphatidate content compensates for this alteration. Furthermore, polyphosphoinositide breakdown and diacylglycerol production are not the rate-limiting step in vesicle release from ATP-depleting red blood cells. This is evident from the fact that 10 mM EDTA inhibits vesicle release to 75% without affecting polyphosphoinositide breakdown and diacylglycerol production. Hence, diacylglycerol formation may be sufficient for membrane budding as suggested earlier (Allan et al. (1976) Nature 261, 58), but vesicle release requires a second, as yet unidentified process.

Studies on the structural polymorphism of the alpha-II domain of human erythrocyte spectrin.

Following restricted tryptic digestion at 4 degrees C, a structural polymorphism affecting the alpha-chain of human spectrin, the major erythrocyte membrane skeleton protein, has recently been described in American blacks (Knowles, W.J., Bologna, M.L., Chasis, J.A., Marchesi, S.L. and Marchesi, V.T. (1984) J. Clin. Invest 73, 973-979). Four variants affecting the alpha-II domain or its tryptic products have been characterized, depending on changes in molecular weight and/or isoelectric point. One variant of the alpha-II domain (Type 2) shows an increase in apparent molecular weight and basic shift in pI. It contains a limit chymotryptic peptide showing a change in chromatographic mobility on two-dimensional electrophoresis which is thought to reflect a sequence alteration associated with the increase in apparent molecular weight. We find that this altered limit chymotryptic peptide is not unique to the Type 2 variant, but is also present in a variant (Type 4) showing only the same basic shift in pI as the Type 2 variant. It is not found in a variant (Type 3) showing only an increase in apparent molecular weight. The most likely explanation for these findings is that the altered limit chymotryptic peptide common to both the Type 2 and Type 4 variants is responsible for the change in isoelectric point which is common to both these variants. An as yet unidentified change elsewhere in the polypeptide chain must be responsible for the observed alteration in molecular weight of the Types 2 and 3 variants.

Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.

The temperature-dependent dissociation of spectrin.

Spectrin, isolated from both human and bovine erythrocyte membranes, was found to exist predominantly as a tetramer of 960 000 daltons when prepared at 4 degrees C, but as a dimer of 480 000 daltons when prepared at 37 degrees C. The purified tetramer from both species could be dissociated to dimers by low ionic strength incubation at 37 degrees C.

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.

Human spectrin. I. A classical light scattering study.

Human spectrin heterodimers were analyzed in solutions containing different amounts of salt employing the classical light scattering technique. 1. At 22 degrees C the radius of gyration of isolated human spectrin heterodimers in 0.1 M NaCl aqueous solution (pH 7.3) was found to be about 22 nm. 2. The radius of gyration of isolated human spectrin heterodimers was found to increase to about 40 nm as the ionic strength of the spectrin solution (pH 7.3) was reduced to about 1 mM. 3. The light scattering study indicates that the isolated human spectrin heterodimers were highly expanded and flexible molecules with a contour length exceeding about 140 nm.

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin.

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.

Seeding role of spectrin in polymerization of skeletal muscle actin.

The effect of spectrin on the polymerization of muscle actin has been investigated by hydrodynamic methods and electron microscopy. Spectrin markedly accelerated polymerization of actin. The effect was more easily observed in lower concentrations of KCl (e.g. 24 mM) where spontaneous polymerization was negligibly small. Similarly large acceleration was observed for polymerization in MgCl2 or CaCl2. The rate of polymerization of actin was proportionally increased with the concentration of spectrin added to a fixed concentration of action. The stationary level of specific viscosity also increased with the spectrin concentration, but at larger concentrations it became smaller. The flow birefringence and electron microscope measurements indicated that actin polymers formed under the influence of spectrin were shorter than those of control F-actin filaments. The structural viscosity and electron microscope observations suggested that the interaction between F-actin fibers was not increased by spectrin. These data strongly suggest a seeding role of spectrin in the polymerization of actin. Spectrin accelerates formation of the nuclei for polymerization. The more the nuclei are formed, the larger the number of the grown polymers are and this leads to rapid formation of shorter polymers since the amount of actin is limited. The acceleration activity was found only in freshly prepared spectrin from fresh ghosts taken from freshly drawn blood.