Spectrin Concentration Research Articles

Building up spectral libraries for mapping erythrocytes by hyperspectral dark field microscopy

BACKGROUND: Red blood cells (RBC) are obtained by non-invasive methods and widely used for diagnostic tests of health status. Hyperspectral Dark Field Microscopy (HDFM) is a promising technique for nanoscale bio imaging and spectral analysis without additional sample preparation. OBJECTIVE: Develop a protocol for human RBC characterization by HDFM, checking the feasibility of a reference spectral library that can image and afford a new comprehensive descriptor of RBC status. METHOD: A step-by-step protocol for HDFM measurement of human RBC was for the first time established using 5 µl of EDTA-treated whole blood from healthy adults (n = 30). Hyperspectral characteristics of solutions/suspensions at biological concentrations of phospholipids, hemoglobin, spectrin, cholesterol and protoporphyrin IX, as the most relevant RBC components, were also determined. RESULTS: A library made of 8 end-member spectra and classification of their spectral distribution carried out by Single Angle Mapper (SAM) were determined, furnishing a comprehensive mapping and descriptor of healthy human RBC. The spectra of single components allowed some of the RBC spectral bands to be attributed. CONCLUSIONS: This work reports for the first time the hyperspectral optical imaging of the human RBC by a library made of 8 scattering spectra, whose spectral signatures are compared with those of the main RBC molecular components. The percent distribution of the spectral end-members was also achieved, thus giving for the first time the HDFM mapping of human healthy RBCs. The protocol developed herein allows the clinical potential of hyperspectral imaging to be developed for the use of RBC mapping in health and disease.

Developmental mechanism of the periodic membrane skeleton in axons.

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites.

Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone

The transformation of a cut axonal end into a growth cone (GC), after axotomy, is a critical event in the cascade leading to regeneration. In an earlier series of studies we analyzed the cellular cascades that transform a cut axonal end into a competent GC. We found that axotomy of cultured Aplysia neurons leads to a transient elevation of the free intracellular Ca 2+ concentration ([Ca 2+] i), calpain activation and localized proteolysis of submembranal spectrin. These events are associated with the formation of distinct microtubule (MT) based vesicle traps that accumulate anterogradely transported vesicles that fuse with the spectrin free plasma membrane in support of the growth process (Erez, H., Malkinson, G., Prager-Khoutorsky, M., De Zeeuw, C.I., Hoogenraad, C.C., and Spira, M.E. 2007. Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J. Cell Biol. 176: 497–507.; Erez, H., and Spira, M.E. 2008. Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J. Comp. Neurol. 507: spc1.). Here we report that under conditions that limit calcium influx into the cut axonal end, axotomy leads to the formation of endbulbs (EBs) rather than to competent GCs. Under these conditions typical MT based vesicle traps are not formed, and Golgi derived vesicles concentrate at the very tip of the cut axon. Since under these conditions the spectrin barrier is not cleaved, vesicle fusion with the plasma membrane and actin polymerization are retarded and growth processes are impaired. We conclude that the immediate assembly of competent GC or an EB after axotomy is the outcome of autonomous local events that are shaped by the magnitudes of the [Ca 2+] i gradients at the site of injury.

Hereditary Spectrin Deficiency in Golden Retriever Dogs

Spectrin deficiency with increased erythrocyte osmotic fragility (OF) is a hallmark of hereditary spherocytosis, which is the most common congenital hemolytic anemia in humans of northern European ancestry. A radioimmunoassay revealed that erythrocyte spectrin concentration was 50-65% of normal in 5 adult Golden Retriever dogs, which had recovered from hemolytic anemia but whose OF had persistently remained increased. OF also was increased and spectrin concentration was decreased (60-73%) in 10 dogs of an apparently healthy family of 19 Golden Retrievers related to a proband. Pedigree analysis revealed autosomal dominant inheritance. In addition, OF was increased in 23 (17%) of 134 randomly chosen Golden Retrievers with nonhematologic diseases. In these Golden Retrievers, the spectrin concentration was decreased in 5 dogs with increased OF and within the reference range in 6 dogs with normal OF, indicating that in this population spectrin deficiency and increased OF are highly associated (P < .002). Considering these patients a representative sample of the Golden Retriever population in the Netherlands, spectrin deficiency may occur in 11.2-24.6% of Dutch Golden Retrievers (confidence level = 0.95). In blood smears, spherocytes were recognized only in dogs with immune-mediated anemia. At scanning electron microscopy, blood from spectrin-deficient Golden Retrievers showed slight crenation when fixed freshly but abundant echinospherocytes after 24 hours of incubation. We conclude that occult autosomal dominant spectrin deficiency occurs in dogs and is frequent in Dutch Golden Retrievers. It is not clear whether spectrin deficiency in Golden Retrievers may result in hemolytic anemia, as in humans.

High-Performance Liquid Chromatography Analysis of Spectrin Oligomerization

Gel filtration chromatography has been used to analyze the oligomerization of human erythrocyte spectrin. By applying an exponentially modified Gaussian function we have been able to resolve overlapping elution peaks. From these peaks it was possible to calculate the equilibrium composition of each spectrin concentration and thus also the dissociation constants describing the oligomeric process. The determined dissociation constants for tetramer formation (1.3 μM) and for hexamer formation (24 μM) agree well with other measurements.

Binding of a Denatured Heme Protein and ATP to Erythroid Spectrin

Spectrin is a large, worm-like cytoskeletal protein that is abundant in all cell types. The denatured heme enzyme, horseradish peroxidase showed significant decrease in the reactivation yield, after 30 min of refolding, in presence of increasing concentrations of spectrin from that in the absence. This indicated that spectrin could bind denatured HRP and inhibit their refolding. In presence of 1 mM ATP and 10 mM MgCl2 the spectrin binding of denatured HRP is abolished. This activity of decreasing the reactivation yield was found to be ATP-dependent and the denatured enzyme after 30 min refolding in the presence of spectrin, pretreated with Mg/ATP, showed about 40% increase in the reactivation yield compared to the same in absence of spectrin. Fluorescence spectroscopic studies indicated binding of ATP to native spectrin showing concentration-dependent quenching of tryptophan fluorescence by ATP. The apparent dissociation constant of binding of ATP to spectrin was estimated to be 1.1 mM. A high affinity binding of spectrin with denatured HRP has been characterized (Kd = 16 nM). Since these properties are similar to those of established molecular chaperone proteins, these data indicate that spectrin might have a chaperone-like function in erythrocytes.

Assessment of the validity of the Adams and Fujita approximation for the higher oligomers of human spectrin

Analysing the self-association behaviour of human erythrocyte spectrin is complicated by a large degree of nonideality. Adams and Fujita [1] proposed that, as a first order approximation, the logarithm of the activity coefficient of the protomer of a self-associating system can be considered to be linearly dependent on the total concentration of the protein, and that the same second virial coefficient could be considered to apply to all species. As a consequence of the Adams and Fujita approximation, the apparent equilibrium constant is equal to the thermodynamic equilibrium constant. The equilibrium concentrations at 30°C of each oligomer spectrin species up to the 14-mer were determined after electrophoresis at low temperature. An apparent equilibrium constant for forming tetramer ( K 2,4) of (1.2 ± 0.1) × 10 6 l/mol was obtained, a value of (9.4 ± 0.7) × 10 4 l/mol was obtained for K 4,6 and for all reactions forming oligomers higher than the hexamer an average approximate value of (2.7 ± 0.4) × 10 5 l/mol was obtained. The apparent equilibrium constants for the formation of all oligomer species of spectrin up to the tetrakaidecamer (14-mer) remain relatively independent of total spectrin concentration, and indicate that within the precision of the measurements a single virial coefficient is sufficient to account for the nonideality of spectrin self-association over the range 2–42 g/l, thus further justifying the use of the Adams and Fujita approximation for this protein over this concentration range.

The concentration dependence of the activity coefficient of the human spectrin heterodimer. A quantitative test of the Adams-Fujita approximation

The heterodimer and tetramer states of human spectrin, in equilibrium at 30°C, have been resolved by means of sedimentation velocity experiments at low temperature. This has allowed quantification of the concentrations of each oligomer at equilibrium, as a function of the total concentration of spectrin. In separate sedimentation equilibrium experiments, the thermodynamic activity of the heterodimer has been estimated as a function of the total spectrin concentration through the use of the Omega function. Combination of the concentration and thermodynamic activity of the spectrin heterodimer allowed estimation of the activity coefficient of the heterodimer as a function of total spectrin concentration. Over the accessible concentration range of 0–16 g/L, the logarithm of the activity coefficient of the heterodimer was linear in the total concentration, and the apparent equilibrium constant for tetramer formation was only weakly dependent on concentration, implying that a single virial coefficient is sufficient to describe the nonideality of this system over this concentration range.

The self-association of human spectrin at high concentration

The self-association of purified human spectrin has been studied at sedimentation equilibrium over a wide range of concentration (0–20 g/L) at 30°C and pH 7.5. Coincidence of apparent weight average molecular weight and Ω( r) plots as a function of total spectrin concentration indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Under these conditions, no significant dissociation of the heterodimer to component polypeptide chains could be detected. The behavior of spectrin between 0 and 20 g/L can be described reasonably well by a cooperative isodesmic model, in which the protomer for association is the αβ heterodimer. With this model, the equilibrium constant for the heterodimer-tetramer step, K 24, is 2 × 10 6 M −1, and K iso, the equilibrium constant describing all other steps, is approximately 0.2 × 10 6 M −1. The returned value of the second virial coefficient for this model, 1.0 × 10 −7 L mol g −2, is consistent with the lower limit of values calculated for the heterodimer from the charge and Stokes radius of spectrin. On the other hand, the attenuated indefinite association model fails to describe the self-association of spectrin adequately over the range 0–20 g/L. Systematic decreases in the estimates of the second virial coefficient and the equilibrium constants for association beyond the tetramer suggest that the assumption of a single value of the second virial coefficient may not be appropriate for spectrin, and that non-ideality would best be taken into account by consideration of the detailed solution composition.

Störungen des Membranskeletts der Erythrozyten bei hereditärer Sphärozytose und Elliptozytose: Bedeutung des molekularen Defektes für Pathogenese und klinischen Schweregrad*

During recent years an increasing number of inherited variants of erythrocyte membrane proteins and defects of the membrane skeleton could be described. Mostly these defects explain the pathogenesis of hemolytic anemias due to erythrocyte membrane defects. For hereditary spherocytosis and elliptocytosis a close correlation between the clinical severity and the biochemical defect was found; thus biochemical characterization can give valuable information about the expected course of the disease and the need for splenectomy. The erythrocyte membrane skeleton stretches along the inner surface of the membrane; it provides the stability of the erythrocyte under circulatory shear stress. The membrane skeleton consists of spectrin, actin, band 4.1 and band 4.9. Spectrin is the major component. In the membrane mostly all spectrin self-associates to the tetrameric form: one tetramer is formed by two alpha and two beta-chains. By denaturing SDS polyacrylamide gelelectrophoresis the composition of the membrane proteins can be analysed. The portion of tetrameric and dimeric spectrin is determined on native agarose gel electrophoresis. The concentration of spectrin in the membrane can be measured by an enzyme linked immunosorbent assay using monoclonal antibodies against human spectrin. By polymerase chain reaction and DNA sequencing the moleculargenetic cause of singular membrane defects was clarified. Hereditary spherocytosis was mostly due to a more or less diminished concentration of spectrin. Based on hematological, clinical' and biochemical observations, a new classification of spherocytosis (mild, moderate and severe form) is proposed. In addition to routine hematologic determinations and osmotic fragility, erythrocyte spectrin content is taken into account. The disease severity correlates with the diminution of spectrin. In hereditary elliptocytosis the concentration of tetrameric spectrin is reduced in about 30% of the patients. Defects of the N-terminal alpha I 80,000 dalton peptide are predominantly found. The defective alpha chain can be further studied by analysis of "tryptic" peptides after limited tryptic digestion of the spectrin. According to the reduced molecular weight of the anomalous tryptic alpha I peptide the variant spectrin alpha chains are designed as Spectrin alpha I/46, Sp alpha I/50, Sp alpha I/65, Sp alpha I/74 and Sp alpha I/78. In most cases a single amino acid substitution of the alpha chain could be proven. Until now only singular patients with hereditary elliptocytosis due to a shortened spectrin beta chain have been described. The shortening of the beta chain is due to a loss of the C-terminal phosphorylated peptide. The molecular cause is a defect at the 3' end of the beta spectrin gen, resulting in a premature termination of the peptide chain-synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Temperature and pH dependence of the self-association of human spectrin

The self-association of human spectrin between 21 and 35 degrees C and between pH 6.5 and 9.5 has been studied at sedimentation equilibrium. For a given set of solution conditions between pH 6.5 and 8.5, coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Above pH 8.5, however, irreversible aggregation occurred, inferred from a failure of overlap in the omega function and molecular weight distributions. The behavior of spectrin can best be described by a cooperative isodesmic model, in which the promoter for association is the heterodimer and for which K12 is between 10(6) and 10(7) M-1 (depending on pH and temperature) and all other K are approximately 10(6) M-1. The returned values of the second viral coefficient for this model fall within the range calculated from the charge and Stokes radius of spectrin. Association appears to be favored slightly by decreased temperature and by decreased pH. The pH dependence resides only in K12 and is consistent with the presence of a single group, possibly histidine, displaying a slightly higher pKa value in the tetramer than in the dimer. The association reaction appears to be driven by the loss of enthalpy associated with release of strain in the heterodimer. The association sites appear to be conserved in the association reactions, consistent with the images from electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Variable clinical severity of hereditary spherocytosis: Relation to erythrocytic spectrin concentration, osmotic fragility, and autohemolysis

To determine whether stratifying hereditary spherocytosis by degree of severity could provide guidelines regarding which patients would benefit from splenectomy, we evaluated the clinical characteristics of 80 patients (63 children) and 27 healthy relatives. In addition to routine hematologic determinations, osmotic fragility, autohemolysis, erythrocyte spectrin content, and erythrocyte membrane lipid phosphorus were measured and correlated with the disease severity. Four categories were identified: (1) spherocytosis as a trait in symptom-free relatives of patients with recessively inherited disease; (2) mild and (3) moderate spherocytosis, largely observed in patients with dominantly inherited disease; and (4) severe spherocytosis, observed in only two patients, who were characterized by recessive inheritance and transfusion dependence. By the identification of carriers, a recessive mode of inheritance could be demonstrated in 20% of the families with spherocytosis. The erythrocyte spectrin concentration was normal in carriers and patients with mild spherocytosis, and was significantly reduced in the moderate and severe states of the disease. This difference was not accounted for by reduced membrane area of the cells, as measured by the phospholipid concentration per cell. We conclude that patients with mild spherocytosis usually do not require splenectomy during childhood and adolescence; patients with moderate or severe disease should have splenectomy. Patients with severe spherocytosis have a partial response to splenectomy but a considerable degree of increased hemolysis persists. Most patients with less than 80% of normal spectrin content require splenectomy.

A thermodynamic model for the self-association of human spectrin.

The self-association of human spectrin at 28.8 degrees C in 0.11 M salt (pH 7.5) has been studied by means of sedimentation equilibrium. Coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was achieved and that no significant concentration of solute was incapable of participating in the self-association reaction. On the basis of the root-mean-square deviation of the fits and the randomness of the residuals, the behavior can be described equally well, either by a cooperative isodesmic model, in which K12 approximately 2 x 10(6) M-1 and all other K approximately 10(6) M-1, or by an attenuated scheme in which K(i-1)i approximately (3.5 x 10(6)/i M-1. The returned values of the second virial coefficient, B, for both these models fall within the range calculated from the charge and Stokes radius of spectrin. A mechanism for spectrin self-association consistent with both schemes is proposed in which spectrin heterodimers undergo a reversible opening at the self-association interface. These open heterodimers then undergo indefinite self-association to form a series of open-chain oligomers in dynamic equilibrium with closed-loop oligomers.

Drosophila spectrin: the membrane skeleton during embryogenesis.

The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.

Effects of denervation on spectrin concentration in avian skeletal muscle

The effect of denervation on avian muscle alpha-spectrin was examined in fast and slow muscles. Using immunofluorescence, the surgically denervated fast-twitch posterior latissimus dorsi (PLD) exhibited a significant increase in spectrin antigen associated with the sarcolemma and within the sarcoplasm compared with the contralateral innervated control muscle. Using gel electrophoresis followed by immunoblotting, we found a two- to three-fold increase in the levels of spectrin in the denervated PLD over that found in the innervated PLD. These levels were comparable to those found previously in slow and dystrophic muscle. The intrafiber distribution of spectrin is similar between the denervated PLD and the slow-tonic anterior latissimus dorsi (ALD). When spectrin was examined in dystrophic PLD muscle, denervation was found to have no effect. These results support our hypothesis that the concentration of spectrin within muscle fibers reflects the physiological state of those fibers. Changes in spectrin concentration may be a useful probe to study the various alterations in physical parameters found among fast, slow, dystrophic, and denervated fibers.

Effects of denervation on spectrin concentration in avian skeletal muscle

The effect of denervation on avian muscle alpha-spectrin was examined in fast and slow muscles. Using immunofluorescence, the surgically denervated fast-twitch posterior latissimus dorsi (PLD) exhibited a significant increase in spectrin antigen associated with the sarcolemma and within the sarcoplasm compared with the contralateral innervated control muscle. Using gel electrophoresis followed by immunoblotting, we found a two- to three-fold increase in the levels of spectrin in the denervated PLD over that found in the innervated PLD. These levels were comparable to those found previously in slow and dystrophic muscle. The intrafiber distribution of spectrin is similar between the denervated PLD and the slow-tonic anterior latissimus dorsi (ALD). When spectrin was examined in dystrophic PLD muscle, denervation was found to have no effect. These results support our hypothesis that the concentration of spectrin within muscle fibers reflects the physiological state of those fibers. Changes in spectrin concentration may be a useful probe to study the various alterations in physical parameters found among fast, slow, dystrophic, and denervated fibers.

Ultrastructural studies of the interaction of spectrin with phosphatidylserine liposomes

Spectrin was shown previously to interact with phosphatidylserine and phosphatidylethanolamine, which are preferentially localized in the inner half of the membrane lipid bilayer, but this interaction is not well characterized. In the present study we used electron microscopy of rotary-shadowed platinum replicas of spectrin dimer-phosphatidylserine complexes to study the interaction of spectrin with phosphatidylserine vesicles. At a spectrin concentration of 0.6 mg/mL, 60% of spectrin dimers were associated with phosphatidylserine vesicles and at a spectrin concentration of 1.2 mg/mL, some vesicles were crosslinked by spectrin dimers. The length of the protruding segment of spectrin dimer from the liposome edge ranged from 400 to 960A degrees and the contact region to phosphatidylserine extended 272 +/- 144A degrees from either end of the molecule. Therefore, these data are consistent with multiple binding sites to phosphatidylserine throughout the spectrin dimer molecule. Spectrin tetramers, when bound to phosphatidylserine liposomes, extended 1804 +/- 79A degrees from the liposome edge and crosslinked liposomes, suggesting that some of the binding sites to phosphatidylserine vesicles is in the proximity of the tail end of spectrin. The association between spectrin dimers to phosphatidylserine was demonstrated by nondenaturing gel electrophoresis. The complexes were separated into multiple bands with molecular weight of 1.4 X 10(6), 1.8 X 10(6), and 2.3 X 10(6). These bands did not represent self-associated spectrin oligomers, since postincubation treatment with Triton-X-100 dissociated them into spectrin dimers. Furthermore, these spectrin high molecular weight bands, as visualized by Coomassie blue absorbance, closely corresponded to the 14C-phosphatidylserine distribution. These data provide ultrastructural and biochemical evidence that spectrin binds to phosphatidylserine at multiple sites including the tail end region.

Ultrastructural Studies of the Interaction of Spectrin With Phosphatidylserine Liposomes

Abstract Spectrin was shown previously to interact with phosphatidylserine and phosphatidylethanolamine, which are preferentially localized in the inner half of the membrane lipid bilayer, but this interaction is not well characterized. In the present study we used electron microscopy of rotary-shadowed platinum replicas of spectrin dimer-phosphatidylserine complexes to study the interaction of spectrin with phosphatidylserine vesicles. At a spectrin concentration of 0.6 mg/mL, 60% of spectrin dimers were associated with phosphatidylserine vesicles and at a spectrin concentration of 1.2 mg/mL, some vesicles were crosslinked by spectrin dimers. The length of the protruding segment of spectrin dimer from the liposome edge ranged from 400 to 960A degrees and the contact region to phosphatidylserine extended 272 +/- 144A degrees from either end of the molecule. Therefore, these data are consistent with multiple binding sites to phosphatidylserine throughout the spectrin dimer molecule. Spectrin tetramers, when bound to phosphatidylserine liposomes, extended 1804 +/- 79A degrees from the liposome edge and crosslinked liposomes, suggesting that some of the binding sites to phosphatidylserine vesicles is in the proximity of the tail end of spectrin. The association between spectrin dimers to phosphatidylserine was demonstrated by nondenaturing gel electrophoresis. The complexes were separated into multiple bands with molecular weight of 1.4 X 10(6), 1.8 X 10(6), and 2.3 X 10(6). These bands did not represent self- associated spectrin oligomers, since postincubation treatment with Triton-X-100 dissociated them into spectrin dimers. Furthermore, these spectrin high molecular weight bands, as visualized by Coomassie blue absorbance, closely corresponded to the 14C-phosphatidylserine distribution. These data provide ultrastructural and biochemical evidence that spectrin binds to phosphatidylserine at multiple sites including the tail end region.

Increased concentration of spectrin is observed in avian dystrophic muscle.

A significant increase in the concentration of spectrin has been observed in dystrophic chicken pectoralis major muscle when compared to normal fast-twitch muscle. In normal muscle, alpha-spectrin-specific immunofluorescence delineates each myofiber with a network pattern of staining at the sarcolemma with little staining within the cytoplasm. In dystrophic fibers, numerous intensely stained areas occur within the cytoplasm and staining at the sarcolemma is increased, thereby obscuring or eliminating the highly regular network arrangement of spectrin usually seen in this region. When immunofluorescence experiments are performed on microsomal vesicles isolated from normal and dystrophic tissues, only a small fraction of normal vesicles are stained, whereas most of the dystrophic vesicles are associated with spectrin. An increase in spectrin concentration is observed using immunoautoradiography of whole muscle and isolated microsomes, thus supporting the immunofluorescent observations described above. The early-age post-hatching when increases in spectrin concentration can be detected and the simplicity of the immunofluorescent technique make this observation useful as a new diagnostic parameter. This observation also shows that the distribution of spectrin and its concentration within nonerythroid cells can be modified by abnormal physiological states; this modification may contribute to subsequent symptoms, such as increased rigidity and abnormal calcium metabolism, that are observed in dystrophy.

Association of spectrin with desmin intermediate filaments

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.