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Erythroid Spectrin, Brain Fodrin, and Intestinal Brush Border Proteins (TW260/240) are Related Molecules Containing a Common Calmodulin-Binding Subunit Bound to a Variant Cell Type-Specific Subunit
Abstract
Spectrin, fodrin, and TW-260/240 form a group of structurally and functionally similar but not identical high molecular weight actin-binding proteins from chicken erythrocytes, brain tissue, or intestinal epithelial brush borders. Immunological data and one-dimensional peptide maps of the separated subunits suggest that a common (Mr 240,000) and a variant (Mr 220,000, 235,000, or 260,000) subunit account for the three different heterodimers. These results are in line with the related but distinct morphology of the three proteins observed in micrographs of rotary-shadowed molecules and the finding that the common (Mr 240,000) subunit seems to account for the calcium-dependent calmodulin-binding activity displayed by the three proteins. The possible functions of spectrin-like molecules in nonerythroid cells are discussed.
... In the erythrocyte, spectrin is composed of two nonidentical polypeptides termed a-spectrin (3,/, 240,000) and ~-spectrin (Mr 220,000) and is found on the inner surface of the plasma membrane where, in association with actin and several other polypeptides, it forms a dense protein meshwork thought to be involved in the maintenance of cell shape and the structural integrity of the plasma membrane (reviewed in reference 6). Recent resuits have shown that erythroid spectrin and spectrin analogues found in several nonerythroid cell-types have many biochemical and functional properties in common; both form tetramers (2,12,38), bind actin (2,7,8,11,12) and calmodulin (13,19,31), and are associated with the cytoplasmic surface of the plasma membrane (2, 8, 24, 25, 29, 30, 37; for reviews see references 6,23). ...
... The lens and brain 3"-spectrins, although related, appear to have a few different peptides; both are different from erythrocyte ~-spectrin. This supports the hypothesis that in chickens a-spectrin is highly conserved among different cell types, while polypeptides with which it forms a stoichiometric complex are polymorphic and exhibit cell-type-specific patterns of expression (13,23,29). ...
... The two-dimensional chymotryptic peptide maps of lens and brain a-spectrin revealed that these polypeptides have highly homologous if not identical primary structures equivalent to that of chicken erythrocyte a-spectrin. Similarly, Glenney et al. (13) have shown that a-spectrin from chicken intestinal terminal web (TW240 in their nomenclature) has a one-dimensional peptide map identical to that of chicken erythrocyte a-spectrin. In mammals, however, the peptide maps of erythroid and nonerythroid (brain) a-spectrin are different (2), although the proteins appear to be antigenically related (2,8,24,38). ...
Chicken lens spectrin is composed predominantly of equimolar amounts of two polypeptides with solubility properties similar, but not identical, to erythrocyte spectrin. The larger polypeptide, Mr 240,000 (lens alpha-spectrin), co-migrates with erythrocyte and brain alpha-spectrin on one- and two-dimensional SDS polyacrylamide gels and cross-reacts with antibodies specific for chicken erythrocyte alpha-spectrin; the smaller polypeptide, Mr 235,000 (lens gamma-spectrin), co-migrates with brain gamma-spectrin and does not cross-react with either the alpha-spectrin antibodies specific for chicken erythrocyte beta-spectrin. Minor amounts of polypeptides antigenically related to erythrocyte beta-spectrin with a greater electrophoretic mobility than lens gamma-spectrin are also detected in lens. The equimolar ratio of lens alpha- and gamma-spectrin is invariantly maintained during the extraction of lens plasma membranes under different conditions, or after immunoprecipitation of whole extracts of lens with erythrocyte alpha-spectrin antibodies. Two-dimensional peptide mapping reveals that whereas alpha-spectrins from chicken erythrocytes, brain, and lens are highly homologous, the gamma-spectrins, although related, have some cell-type-specific peptides and are substantially different from erythrocyte beta-spectrin. Thus, the expression of cell-type-specific gamma- and beta-spectrins may be the basis for the assembly of a spectrin-plasma membrane complex whose molecular composition is tailored to the functional requirements of the particular cell-type.
... Immunological assays and peptide mapping data indicate that the 240,000-mol-wt subunit of spectrin, fodrin, and TW 260/240 are very similar (15-17, 23, 24). In addition, all three proteins bind calmodulin and actin (15)(16)(17). ...
... Fluorescein-labeled gpl80 (T-200)patched/capped cells were rendered permeable by methanol treatment (32) and stained with rabbit antibody against pig brain fodrin (anti-fodrin) (17). These cells were then incubated with rhodamine-conjugated goat antibody against rabbit IgG to visualize fodrin distribution. ...
... The polypeptides were transferred to nitrocellulose sheets as described by Burnette (45). Nitrocellulose sheets were subsequently incubated with anti-fodrin (10 ug/ml), followed by incubation with ~251-protein A, and analyzed by previously published autoradiographic procedures (17). ...
A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.
... In nonerythroid chicken tissues, spectrin heterodimers are composed of an asubunit highly homologous to erythroid a spectrin (45), and a variable subunit. In most nonerythroid cell types this variable subunit is the 3,-subunit (23; also referred to as fodrin, reference 36), which has a peptide map distinct from erythroid /3-spectrin (22,43). In skeletal and cardiac muscle, the variable subunit is highly homologous to the/3-subunit of erythroid spectrin (41). ...
... In skeletal and cardiac muscle, the variable subunit is highly homologous to the/3-subunit of erythroid spectrin (41). The third known variable subunit, the TW260 polypeptide (22), is found only in intestinal epithelial cells. Certain neurons in the central nervous system co-express the /3-and 3,-subunits in distinct membrane domains (35), further suggesting that the variable subunits impart specialized properties to membrane skeletons. ...
... Hence, the cDNA demonstrates specificity for/3 spectrin transcripts and the lack of homology with RNAs for other variable subunits that bind a spectrin. The absence of an RNA in fibroblasts or intestine which hybridizes to the /3 spectrin cDNA further suggests that the 3' spectrin and TW260 subunits, with their distinct peptide maps (22), are transcribed from separate genes distinct from the/3 spectrin gene. ...
cDNA probes for three components of the erythroid membrane skeleton, alpha spectrin, beta spectrin, and ankyrin, were obtained by using monospecific antibodies to screen a lambda gt11 expression vector library containing cDNA prepared from chicken erythroid poly(A)+ RNA. Each cDNA appears to hybridize to one gene type in the chicken genome. Qualitatively distinct RNA species in myogenic and erythroid cells are detected for beta spectrin and ankyrin, while alpha spectrin exists as a single species of transcript in all tissues examined. This tissue-specific expression of RNAs is regulated quantitatively during myogenesis in vitro, since all three accumulate only upon myoblast fusion. Furthermore, RNAs for two of the three genes do not accumulate to detectable levels in chicken embryo fibroblasts, demonstrating that their accumulation can be noncoordinate. These observations suggest that independent gene regulation and tissue-specific production of heterogeneous transcripts from the beta spectrin and ankyrin genes underlie the formation of distinct membrane skeletons in erythroid and muscle cells.
... Renewed interest in this function of spectrin has occurred since it has been found that there are nonerythroid forms of this protein (see reviews in references 12, 14, and 18), including studies showing the protein fodrin in lymphocytes and its concurrent redistribution with capping of macromolecules on the surface of T and B lymphocytes (23,25). Fodrin has been recently shown to be one of the forms of spectrin in non-erythroid cells (3,10,(15)(16)(17) and using antiserum directed against chicken erythrocyte a-spectrin, Nelson et al. (28) found a coredistribution of spectrin with surface receptors of lymphocytes. Using freshly isolated splenic and thymic lymphocytes, we have observed that a-spectrin and fodrin antigens occur in a capped configuration in particular subsets oflymphocytes prior to the addition of ligand and show here that these naturally capped cells are morphologically identifiable in situ. ...
... However, at the exposure duration used here, neither antiserum showed cross-reactivity with mouse erythrocyte a-spectrin indicating that the reaction obtained in the lymphocyte lane and by immunofluorescence was not due to contaminating erythrocytes . Although some biochemical differences have been reported between the antigens recognized by antifodrin and anti-a-spectrin (3,10,16,17), the immunoautoradiographic results indicate that in lymphocytes, the two antisera were cross-reacting with the same protein complex. In addition, double immunofluorescence experiments using both antisera on the same capped and noncapped lymphocytes show that the two antisera have identical distributions (not shown) . ...
Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.
... Much interest has been devoted recently to the role of cytoskeletal elements in maintaining distinct surface membrane domains (10,12,27,32,44,45; for a review, see reference 50). An important role in cytoskeleton-cell surface associations has been assigned to the so-called surface lamina structure, which consists of cytoskeletal proteins at the cytoplasmic aspect of the surface membrane and associated cell surface glycoproteins (10,44). ...
... Many recent studies have indicated the presence of a family of polypeptides, immunologically and structurally related to erythroid a-spectrin, in diverse cells and tissues (9,12,26,27,29,31,32,41,45,49,59). In neural cells, a-spectrin-like polypeptides (fodrin) are involved in axonal transport (49) and it has been suggested that they provide mechanical support to cell cortex and plasma membrane in a manner analogous to that of erythroid spectrins (12,41,45,49). ...
Antibodies against different cytoskeletal proteins were used to study the cytoskeletal organization of human spermatozoa. A positive staining with actin antibodies was seen in both the acrosomal cap region and the principal piece region of the tail. However, no staining was obtained with nitrobenzoxadiazol-phallacidin, suggesting that most of the actin was in the nonpolymerized form. Most of the myosin immunoreactivity was confirmed to a narrow band in the neck region of spermatozoa. Tubulin was located to the entire tail, whereas vimentin was only seen in a discrete band-like structure encircling the sperm head, apparently coinciding with the equatorial segment region. Surface staining of the spermatozoa with fluorochrome-coupled Helix pomatia agglutinin revealed a similar band-like structure that co-distributed with the vimentin-specific staining. Instead, other lectin conjugates used labeled either the acrosomal cap region (peanut and soybean agglutinins), both the acrosomal cap and the postacrosomal region of the head (concanavalin A), or the whole sperm cell surface membrane (wheat germ and lens culinaris agglutinins and ricinus communis agglutinin l). In lectin blotting experiments, the Helix pomatia agglutinin-binding was assigned to a 80,000-mol-wt polypeptide which, together with vimentin, also resisted treatment with Triton X-100. Only the acrosomal cap and the principal piece of the tail were decorated with rabbit and hydridoma antibodies against an immunoanalogue of erythrocyte alpha-spectrin (p230). p230 appeared to be the major calmodulin-binding polypeptide in spermatozoa, as shown by a direct overlay assay of electrophoretic blots of spermatozoa with 125I-calmodulin. The results indicate that spermatozoa have a highly specialized cytoskeletal organization and that the distribution of actin, spectrin, and vimentin can be correlated with distinct surface specializations of the sperm cells. This suggest that cytoskeleton may regulate the maintenance of these surface assemblies and, hence, affect the spermatozoan function.
... Recent evidence suggests that this type of membrane association is not restricted to the red cell but represents a universal building block in the architecture of plasma membranes in general (15,30). In this situation actin behaves in a manner suggestively similar to our findings for rhabdoms . ...
... We have not been able to prove the latter possibility directly . However the fine, meandering filaments seen in isolated rhabdoms appear morphologically similar to spectrin-like proteins (15,29,30). Recent demonstrations of actin-binding, spectrin-like proteins in cell types other than erythrocytes (15, 29, 30) including the vertebrate brush-border microvillas, suggest that this molecular component should be sought in the future . ...
Infiltration of compound eyes of crayfish, Cherax destructor, with the thiol protease inhibitor Ep-475 or with trifluoperazine prior to fixation for electron microscopy was found to stabilize an axial filament of 6-12 nm diam within each rhabdomeral microvillus of the photoreceptors. Rhabdoms isolated from retinal homogenates by sucrose gradient centrifugation under conditions that stabilize cytoskeletal material contained large amounts of a 42-kd polypeptide that co-migrated with insect flight muscle actin in one- and two-dimensional PAGE, inhibited pancreatic DNase l, and bound to vertebrate myosin. Vertebrate skeletal muscle actin added to retinal homogenates did not co-purify with rhabdoms, implying that actin was not a contaminant from nonmembranous structures. DNase l inhibition assays of detergent-lysed rhabdoms indicated the presence of large amounts of filamentous actin provided ATP was present. Monomeric actin in such preparations was completely polymerizable only after 90 min incubation with equimolar phalloidin. More than half of the actin present could be liberated from the membrane by sonication, indicating a loose association with the membrane. However, a large proportion of the actin was tightly bound to the rhabdomeral membrane, and washing sonicated membrane fractions with solutions of a range of ionic strengths and nonionic detergents failed to remove it. Antibodies to scallop actin only bound to frozen sections of rhabdoms after gentle permeabilization and very long incubation periods, probably because of steric hindrance and the hydrophobicity of the structure. The F-actin probe nitrobenzoxadiazol phallacidin bound to rhabdoms and labeled F-actin aggregates in other retinal components, but rhabdom fluorescence was not abolished by preincubation with phalloidin. The biochemical data indicate the existence of two distinct actin-based cytoskeletal systems, one being closely membrane associated. The other may possibly constitute the axial filament, although the evidence for this is equivocal.
... These studies have shown that spectrins in different tissues occur as heterodimers and possess a common (c~, Mr from 230 to 260 kD) and a variant (/3 or % Mr from 220 to 260 kD) subunit (Lazarides and Nelson, 1985). The latter show a high degree of variation while the common subunits are much alike in different types of cells (Glenney et al., 1982b). The mammalian erythroid a-chain is, however, a deviant member of the family and diverges from the others by its immunological and structural properties (Glenney and Glenney, 1984a;Harris et al., 1985). ...
... Recently it has been shown that mammalian brain contains two isoforms of spectrin (Lazarides and Nelson, 1983;Lazarides et al., 1984;Riederer et al., 1986;Virtanen et al., 1986), one located primarily in the axons and the other in the cell bodies and dendrites. Another spectrin-like protein that is more thoroughly characterized, was detected in the avian intestinal tissue (Glenney et al., 1982b). This terminal web (TW) 260/240-protein differs from the others by its location in the terminal web of the enterocytes, distant from the plasma membrane. ...
We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.
... CD26 protein is present in the CP epithelial membrane and in the outer layer of brain pia mater particularly in the meningeal macrophages, where it is colocalised with other brain spectrins, known as fordin and ankyrin [29,69]. Similarly, CD26 is expressed in erythrocyte membranes (also having erythrocyte spikes) and is colocalised with membrane proteins, glycophorin and integrin α2β3 [70]. Since SARS-CoV-2 spike (S) glycoprotein binds to erythrocytes, causing clotting defects in the small blood vessels [37,71], we analysed blood smears from young, diabetic (T2DM) and older subjects (ARD). ...
Investigation of CD26, a potential SARS-CoV-2 receptor, as a biomarker of age and pathology
Animesh Alexander Raha1,2, Subhojit Chakraborty1,2, James Henderson1, Elizabeta Mukaetova-Ladinska3, Shahid Zaman4, John Trowsdale5 and Ruma Raha-Chowdhury1,2,4
Objective: In some individuals, coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leads to a variety of serious in ammatory symptoms, including blood clotting and acute respiratory distress. Death due to COVID-19 shows a steep rise in relation to age. Comorbidities such as type 2 diabetes mellitus (T2DM), hypertension, and cardiovascular disease also increase susceptibility. It has been reported that T-cell regulatory dipeptidyl peptidase 4 (DPP4; cluster of differentiation 26 (CD26)) binds to the external spike (S) glycoprotein of SARS-CoV-2 as a receptor, for the viral entry into the host cell. CD26 is expressed on many cells, including T and natural killer (NK) cells of the immune system, as a membrane-anchored form. A soluble form (sCD26) is also found in the blood plasma and cerebrospinal uid (CSF).
Approach and results: To investigate a possible relationship between sCD26 levels, age and pathology, serum samples were collected from control, T2DM and age-related demen- tia (ARD) subjects. A signi cant reduction in serum sCD26 levels was seen in relation to age. ARD and T2DM were also associated with lower levels of sCD26. The analysis of blood smears revealed different cellular morphologies: in controls, CD26 was expressed around the neutrophil membrane, whereas in T2DM, excessive sCD26 was found around the mononucleated cells (MNCs). ARD subjects had abnormal fragmented platelets and haemolysis due to low levels of sCD26.
Conclusions: These ndings may help to explain the heterogeneity of SARS-CoV-2 infec- tion. High serum sCD26 levels could protect from viral infection by competively inhibiting the virus binding to cellular CD26, whereas low sCD26 levels could increase the risk of in- fection. If so measuring serum sCD26 level may help to identify individuals at high risk for the COVID-19 infection.
... CD26 protein is present in the CP epithelial membrane and in the outer layer of brain pia mater particularly in the meningeal macrophages, where it is colocalised with other brain spectrins, known as fordin and ankyrin [29,69]. Similarly, CD26 is expressed in erythrocyte membranes (also having erythrocyte spikes) and is colocalised with membrane proteins, glycophorin and integrin α2β3 [70]. Since SARS-CoV-2 spike (S) glycoprotein binds to erythrocytes, causing clotting defects in the small blood vessels [37,71], we analysed blood smears from young, diabetic (T2DM) and older subjects (ARD). ...
Objective: In some individuals, coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leads to a variety of serious inflammatory symptoms, including blood clotting and acute respiratory distress. Death due to COVID-19 shows a steep rise in relation to age. Comorbidities such as type 2 diabetes mellitus (T2DM), hypertension, and cardiovascular disease also increase susceptibility. It has been reported that T-cell regulatory dipeptidyl peptidase 4 (DPP4; cluster of differentiation 26 (CD26)) binds to the external spike (S) glycoprotein of SARS-CoV-2 as a receptor, for the viral entry into the host cell. CD26 is expressed on many cells, including T and natural killer (NK) cells of the immune system, as a membrane-anchored form. A soluble form (sCD26) is also found in the blood plasma and cerebrospinal fluid (CSF).
Approach and results: To investigate a possible relationship between sCD26 levels, age and pathology, serum samples were collected from control, T2DM and age-related dementia (ARD) subjects. A significant reduction in serum sCD26 levels was seen in relation to age. ARD and T2DM were also associated with lower levels of sCD26. The analysis of blood smears revealed different cellular morphologies: in controls, CD26 was expressed around the neutrophil membrane, whereas in T2DM, excessive sCD26 was found around the mononucleated cells (MNCs). ARD subjects had abnormal fragmented platelets and haemolysis due to low levels of sCD26.
Conclusions: These findings may help to explain the heterogeneity of SARS-CoV-2 infection. High serum sCD26 levels could protect from viral infection by competively inhibiting the virus binding to cellular CD26, whereas low sCD26 levels could increase the risk of infection. If so measuring serum sCD26 level may help to identify individuals at high risk for the COVID-19 infection.
... To our knowledge, no studies performed in vitro have shown an effect of calmodulin on the polymerization state of actin . However, recent studies have demonstrated that the actin-binding proteins fodrin, spectrin, and TW240 all share a conserved calmodulin-binding site (19); these studies, combined with our own observations, suggest that calmodulin might act to regulate stress fibers in intact systems. ...
... To our knowledge, no studies performed in vitro have shown an effect of calmodulin on the polymerization state of actin . However, recent studies have demonstrated that the actin-binding proteins fodrin, spectrin, and TW240 all share a conserved calmodulin-binding site (19); these studies, combined with our own observations, suggest that calmodulin might act to regulate stress fibers in intact systems. ...
The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.
... In vertebrates, there are three major classes of spectrin molecules: erythroid, nonerythroid, and brush border (TW260/240). In chicken, these three classes all share a common ot-spectrin subunit (Glenney et al., 1982a), whereas in mammals diversity is generated by multiple tx-and B-spectrin subnnits (Leto et al., 1988;Birkenmeier et al., 1988). Nonerythroid spectrins (also known as fodrins) have a broad tissue distribution both in vertebrates (Bennett, 1985) and in invertebrates (Pesacreta et al., 1989) and probably play a fundamental role at the plasma membrane. ...
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 fl subunits, is associated with diversity in cell shape and membrane architecture. Here we describe a spectrin isoform from Drosophila that consists of a conventional a spectrin subunit complexed with a novel high molecular weight B subunit (430 kD) that we term/3.. The native otflH molecule binds actin illa-ments 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 B. subunit. /3. is encoded by a different gene than the previously described Drosophila/3-spectrin subunit but shows sequence similarity to B-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 /3-spectrin isoform (BH) than to other members of the spectrin gene family such as ot-spectrin and ot-actinin.
... (30) detected the presence of nonerythroid spectrin in CEF by using antibodies against brain spectrin (fodrin) and immuno-autoradiography on gels. Several laboratories have demonstrated binding of "I-calmodulin to nonerythroid spectrin (fodrin) from various cell types including bovine brain (31,32) . The S and P fractions of CEF were analyzed for the presence of calmodulin-binding proteins that cross-react with bovine brain spectrin (fodrin) antibodies . ...
We recently reported the detection of multiple classes of calmodulin-binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium-modulated proteins. The fact that, in some cases, the same calmodulin-binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium-modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin-binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.
... A recent report also suggests that calmodulin can modify indirectly the polymerization of actin in vitro (Piazza and Wallace, 1985). In fact, calmodulin reportedly binds to a number of actin-binding proteins and to microtubule-associated proteins (Rebhun et al., 1980;Sobue et al., 1981;Glenney et al., 1982;Lee and Wolff, 1984; for review see Kakiuchi and Sobue, 1983). However, the distribution and activity of calmodulin in intact cells is not yet known in detail. ...
We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.
... Recently, the presence of spectrin and spectrin-like proteins has been reported in numerous non-erythroid cells. In these cells the spectrin-like proteins are also found on the cytoplasmic side of the membrane, suggesting that these proteins probably play a similar structural role in non-erythroid cells (2,7,10,13,14,19,25). ...
Friend erythroleukemia cells, grown in the presence of dimethyl sulfoxide for 3 d, synthesize unequal amounts of the two chains (alpha and beta) of spectrin with approximately 15-30% more beta than alpha spectrin. When cells were ruptured by nitrogen cavitation, nascent alpha and beta spectrin were found to be associated with a membranous cell fraction and were not detected in the soluble cytoplasmic cell fraction. Nascent membrane-bound spectrin appeared not to be protected by membranes, since it was susceptible to trypsin degradation in the absence of detergent. On fractionation of cells with 1% Triton X-100, more (1.75-fold) nascent spectrin was found in the Triton-soluble fraction than in the Triton-insoluble fraction (cytoskeleton). In the Triton-soluble fraction, there was 55% more nascent beta spectrin than alpha spectrin, while the cytoskeleton contained nearly equal amounts of alpha and beta spectrin. Cells were pulse-labeled with L-[35S]methionine for 2 min and chase incubated for varying periods of time from 15 to 90 min with nonradioactive L-methionine. Radioactive spectrin accumulated in the Triton-soluble fraction for the first 15 min of chase incubation and then dropped by 25% in the next hour. By contrast, the amount of radioactive spectrin in the Triton-insoluble fraction rose gradually for 1 h of the chase period. This indicates that, in Friend erythroleukemia cells, a pool of membrane-bound spectrin containing an excess of the beta polypeptide is used to form the cytoskeletal system which is composed of equal molar amounts of alpha and beta spectrin. The location of spectrin was determined by immunoelectron microscopy. Small amounts of spectrin were detected in cells not treated with dimethyl sulfoxide and in these cells it was located on the surface membrane and within the cytoplasm. On treatment with dimethyl sulfoxide, complex vacuolar structures containing viruses appeared in the cells. In cells treated with dimethyl sulfoxide for 3 d 30% of the spectrin was near the outer membrane and 25% was associated with vacuolar structures, whereas in cells treated for 5 and 7 d the majority of spectrin (57-61%) was located in the vacuolar areas.
... Moreover, fluorescein-labeled Fab fragments of this antibody were used for fluorescence photobleaching and recovery studies (see below). For tx spectrin immunocytochemistry, we used the antiserum of Glenney et al. (1982). Primary antisera were diluted between 1:150 and 1:250 and added to cells on coverslips in a humidified chamber for 1 h at room temperature. ...
We have used isolated embryonic photoreceptor cells as a model system with which to examine the mechanisms responsible for the development and maintenance of asymmetric Na+,K+-ATPase (ATPase) distribution. Photoreceptor precursors, which appear round and process free at culture onset, develop structural and molecular properties similar to those of photoreceptor cells in vivo. ATPase, recognized by an anti-ATPase antibody, is distributed over the entire surface of round photoreceptor precursors. As the cells develop, ATPase becomes progressively concentrated in the inner segment (where it is found in cells of the intact retina). This phenomenon occurs in cells developing in the absence of intercellular contacts. The development of ATPase polarity correlates with a decrease in the fraction of ATPase molecules that are mobile in the membrane (as determined by fluorescence photobleaching recovery), as well as with an increase in the fraction of ATPase that remains associated with the cells after detergent extraction. The magnitudes of the mobile ATPase fractions agree well with those of the detergent-extractable fractions in both the immature and developed photoreceptors. The distribution of alpha spectrin and ATPase-immunoreactive materials appeared qualitatively similar, and quantitative image analysis showed similar gradients of spectrin and Na+,K+-ATPase immunofluorescence along the long axis of elongated photoreceptors. Moreover, detergent extractability of alpha spectrin and the ATPase showed similar modifications in response to changes in pH or KCl concentration. ATPase detergent-extractable and mobile fractions were not changed in cultures treated with cytoskeletal inhibitors such as nocodazole. These data are consistent with a role for an asymmetrically distributed, spectrin-containing subcortical cytoskeleton in the preferential accumulation of Na+,K+-ATPase in the photoreceptor inner segment.
... In avians, at least three spectrin isoforms exist: a 240/220-kD or 240/230-kD heterodimer found in erythrocytes, brain, and skeletal muscle; a 240/235-kD heterodimer present in brain and most other tissues; and a 240/260-kD found exclusively in intestinal brush borders Nelson and Lazarides, 1983;Glenney and Glenney, 1983a;Lazarides and Nelson, 1985). All of these spectrins share a common alpha (240 kD) subunit (Glenney et al., 1982c) encoded by a single gene (Curtis et al., 1985;Birkenmeier et al., 1985). ...
The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.
... The shape and stability of brush border microvilli are maintained by a cytoskeletal core of actin filaments bundled by the proteins villin (3)(4)(5)(6) and fimbrin (7,8); thisbundle is cross-linked to the membrane by spirally arranged cross-filaments (9), probably composed of a 110,000-M, polypeptide (10) associated with calmodulin (11,12) . The microvillar cores associate with the terminal web filament system, which includes myosin (13,14) and TW 260/240, a fodrin-like protein (15)(16)(17), among other proteins . In recent years several reports on brush border motility have been published (18)(19)(20)(21)(22), thereby extending our knowledge of this system . ...
The shape and stability of intestinal epithelial cell microvilli are maintained by a cytoskeletal core composed of a bundle of actin filaments with several associated proteins. The core filaments are intimately associated with the overlying plasma membrane, in which there occur rapid turnover of proteins and constant incorporation of new membrane. Previous work has shown that starvation or inhibition of protein synthesis results in modulation of microvillar length, which indicates that there may be cytoskeletal protein turnover. We demonstrate herein, by means of in vivo pulse labeling with radioactive amino acids, that turnover of brush border cytoskeletal proteins occurs in mature absorptive cells. Turnover of cytoskeletal proteins appears to be quite slow relative to membrane protein turnover, which suggests that the turnover of these two microvillar compartments is not coupled. We thus conclude that cytoskeletal protein turnover may be a factor used to maintain normal length and stability of microvilli and that the cytoskeleton cannot be considered a static structure.
... Fodrin (brain spectrin) was first described as a high-molecular-weight, axonally transported, spectrinlike protein in mammalian brain, and is also found beneath the plasma membranes of lymphocytes (27)(28)(29)(30)31). The terminal web protein, TW 260/240, another spectrin analogue, has been purified from intestinal brush borders (32,33). ...
Protein 4.1 is a crucial component of the erythrocyte membrane skeleton. Responsible for the amplification of the spectrin-actin interaction, its presence is required for the maintenance of erythrocyte integrity. We have demonstrated a 4.1-like protein in nonerythroid cells. An antibody was raised to erythrocyte protein 4.1 purified by KCl extraction (Tyler, J. M., W. R. Hargreaves, and D. Branton, 1979, Proc. Natl. Acad. Sci. USA, 76:5192-5196), and used to identify a serologically cross-reactive protein in polymorphonuclear leukocytes, platelets, and lymphoid cells. The cross-reactive protein(s) were localized to various regions of the cells by immunofluorescence microscopy. Quantitative adsorption studies indicated that at least 30-60% of the anti-4.1 antibodies reacted with this protein, demonstrating significant homology between the erythroid and nonerythroid species. A homologous peptide doublet was observed on immunopeptide maps, although there was not complete identity between the two proteins. When compared with erythrocyte protein 4.1, the nonerythroid protein(s) displayed a lower molecular weight--68,000 as compared with 78,000-and did not bind spectrin or the nonerythroid actin-binding protein filamin. There was no detectable cross-reactivity between human acumentin or human tropomyosin-binding protein, which are similarly sized actin-associated proteins, and erythrocyte protein 4.1. The possible origin and significance of 4.1-related protein(s) in nonerythroid cells are discussed.
A dot blot is an appropriate hybridoma screening procedure when the antigen is a protein that is available in purified form. The antigen is bound directly to a nitrocellulose sheet and incubated with hybridoma tissue culture supernatant. A dot blot is widely used to determine the productivity of a given hybridoma, and this is described here. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones.
The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.
An important function of the mammalian nonerythroid α-spectrin chain (α-fodrin) that distinguishes it from the closely related erythroid isoform is its ability to bind calmodulin. By analysis of a series of deleted recombinant spectrin fusion proteins, we have identified a region in the nonerythroid α chain involved in calcium-dependent binding of calmodulin. The region is distinctive in that the sequence is absent from the homologous domain of the erythroid α chain and diverges from the normal internal repeat structure observed throughout other spectrins. In order to determine limits of this functional site, a synthetic peptide as small as 24 residues was shown to compete with either recombinant or brain α-spectrin in binding to calmodulin. The active peptide, which was derived from a segment between repeats 11 and 12, was composed of the following sequence: Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn - Ser-Ile-Lys-Glu. Comparison of this sequence with functional sites in other diverse calcium-dependent calmodulin-binding proteins has revealed a structural motif common to all of these proteins, namely clusters of hydrophobic residues interspersed with basic residues. When folded into α-helical conformations, these binding sites are predicted to form amphipathic structures.
The importance of calcium in the regulation of a very wide range of eukaryotic cellular enzymes and processes has become evident over the past two decades. The actions of the metal ion are mediated through a limited number of related specific calcium binding proteins which act as receptors within the cell. One of these proteins, calmodulin, has been the subject of intensive study in recent years, largely as a result of its ubiquitous distribution and the fact that it is involved in the control of the majority of the calcium-regulated processes. However, in spite of the efforts of a large number of researchers, its mode of action is not well understood at the molecular level. On the other hand, the mechanism by which a second calcium binding protein, troponin C, is able to ‘turn off’ or ‘turn on’ striated muscle in response to changes in calcium ion concentration is very well characterised.
The molecular mechanisms that underlie learning and memory have intrigued investigators for over a decade. When formulating a model for memory, an inter-disciplinary approach must be utilized. The specific brain regions that mediate information processing, storage, and retrieval should be identified and their role in each of these steps elucidated. Clearly, many different brain nuclei interact cooperatively in these processes, and a wide variety of experimental models for memory have been developed. Excellent reviews on the neuropsychology of memory (Squire, 1982) and possible biochemical correlates (Thompson et al., 1983) are available.
The prevailing view of membrane structure presented in many undergraduate textbooks is that of proteins floating in the plane of a fluid phospholipid bilayer (Singer and Nicolson, 1972). Many observations in the last 10 years indicate, however, that biological membranes do not behave as simple two-dimensional solutions of proteins. Measurements of the rates of lateral diffusion of membrane proteins in a variety of systems have revealed populations of proteins that are not mobile. The proteins that are mobile move at rates 10- to 100-fold slower than predicted on the basis of membrane viscosity (reviewed by Cherry, 1979). The diffusion constants of these slowly diffusing proteins are increased 200-fold in areas of the cell where the membrane is separated from the cytoplasm (Tank et al., 1982). Furthermore, diffusion of proteins may be nonrandom in some cells (Smith et al., 1979) and can require metabolic energy as in formation of caps of surface-labeled proteins in lymphocytes (Unanue and Karnovsky, 1973). These examples indicate possibilities for long-range interactions and organization in cell membranes and have led to proposals that at least some membrane proteins have direct interactions with underlying cytoplasmic proteins (Singer, 1974; Nicolson, 1974; Edelman, 1976; Bourguignon and Singer, 1977).
The creation of form during embryogenesis is characterized by mitosis, cell and tissue migration, as well as by stabilization of the particular configuration of each single cell (shape) and of the sum of cells (relative positioning). The navigation that cells and tissues seem to perform during migration, and the recognition of the final configuration of cellular topography are processes which will be determined by the encounter between cells.
Intestinal absorptive cells display an asymmetric organization well adapted for their function of transport. The functional polarity of these cells implies the existence of distinct domains of the plasma membrane and an asymmetric distribution of the cytoskeletal network. Their apical surface, facing the external milieu, develops a unique structure named the brush border which provides a greatly expanded absorptive surface facilitating the uptake of nutrients in intestine. It consists of a densely packed array of thousands of microvilli supported by bundles of actin microfilaments. Underneath the microvilli, is a complex mesh-work of microfilaments called the terminal web. The organization of this structure is achieved in the course of terminal differentiation.
To obtain meaningful results from immunocytochemical studies requires the use and production of well-characterized antibodies of defined specificities. The aim in this chapter is to briefly summarize our current understanding of the immune system as much as it concerns the production of specific antibodies for experimental purposes. For a more complete discussion of the immune system and cellular immunology the following reviews and texts are recommended (Paul 1984; Roitt et al. 1985; Harlow and Lane 1988). Detailed practical aspects can be found in Mayer and Walker (1987) and Hudson and Hay (1989). I will then discuss in more detail strategies which may be used for the production and purification of antibodies against defined antigens. Finally, I will point out the various methods available for characterization of antibodies, their respective antigens, and precisely what information can be obtained from these methods.
The protein spectrin was first reported in 1968 in water-soluble extracts from erythrocyte membranes (Marchesi and Steers, 1968). Although for some time it was believed that spectrin represented a specific erythrocyte adaptation, in recent years spectrin and spectrinlike proteins have been found in a wide range of other cell types (e.g., see Goodman et al., 1981). These nonerythroid forms of spectrin all share with erythrocyte spectrin a common set of properties: they are proteins of high molecular mass, comprising two distinct subunits of approximately 250,000 and 230,000 Da, respectively; they bind calmodulin in a calcium-dependent manner, though with greatly differing affinities; they bind to actin filaments; and they are associated with the cell membranes through interaction with other proteins, particularly ankyrin (Bennett, 1985). Certainly in the red blood cell, and probably in other cell types, the major functional role of spectrin is to stabilize the membrane, and to provide a linkage for actin filaments to the membrane. While the red cell may be an inadequate model for other cell types, the organization of the erythrocyte cytoskeleton and its role in maintaining erythrocyte shape and deformability are now reasonably well understood, at least in broad terms, and the findings from the red cell give at least an indication of the organization of such molecules in other cells. The present discussion will be limited to erythrocyte spectrin.
It is now apparent that calcium plays a major role in the regulation of cell growth, the most convincing evidence being that neoplastic transformation generally results in a reduced calcium requirement for the proliferation of various cultured cells of both mesenchymal and epithelial origin (Swierenga et al., 1980; Whitfield et al., 1980; Durham and Walton, 1982). The concentration of this divalent ion in the cytoplasm of mammalian cells is 103 times lower than that present in the extracellular medium, most of it being sequestered in specific organelles such as mitochondria and endoplasmic reticulum. Therefore, a transfer from the medium and/or intracellular storage sites can induce a relatively high transient change in the cytoplasmic calcium concentration and, as in the case of transient changes in cyclic adenosine monophosphate (cAMP) levels, it is generally interpreted as a control signal in the transduction of various external stimuli (Bygrave, 1978; Carafoli and Crompton, 1978; Kretsinger, 1979), including growth factors (McKeehan and MeKeehan, 1981). Moreover, because of its diversified yet crucial role in normal cell functioning, the many facets of the cellular metabolism of calcium have been studied extensively (Carafoli and Crompton, 1978), particularly its interaction with the intracellular transducer protein calmodulin, its relationship to cAMP metabolism, and its role in the activation of distinct protein kinases (Cheung, 1980; Scharff, 1981; Carafoli, 1981; Oldham, 1982; Means et al., 1982a).
Two eukaryotic cell types have been extensively studied as paradigms for the chemotaxis of freely motile cells: the leukocytes such as PMNs and macrophages (Neidel and Cuatrecasas, 1980; Schiffman, 1982; Zigmond, 1978) and the cellular slime molds, particularly the species Dictyostelium discoideum (Devreotes, 1982; Frazier et al., 1982; Gerisch, 1982). Both these systems have inherent complications for studying the mechanism(s) of chemotaxis per se, that is, the process or processes by which the occupancy of chemotactic receptors is sensed by the inner workings of the cell, specifically those components involved in cellular motility, and translated into directional information. The white cells respond to chemoattractants such as complement peptides and N-formylated peptides (e.g., F-Met-Leu-Phe, or FMLP) with a barrage of killer mechanisms such as superoxide and peroxide production, lysosomal enzyme secretion, increased cell-cell adhesiveness, and enhanced phagocytosis (Neidel and Curatrecasas, 1980; Schiffmann, 1982; Zigmond, 1978). Many or all of these responses may have nothing to do with the chemotactic response or effect on cell motility. In general, most chemoattractants also cause the response of chemo-kinesis, or an increase in rate of motility with no imposed directional component.
One of the goals of the cellular neurobiologist is to acquire information on the structure and function of membrane components at a molecular level. Within this area of study, one of the major interests has focused on membrane glycoproteins. Plasma membranes that have most attracted the interests of neuroscientists are membranes that are structurally or functionally distinct to the nervous system, e.g., membranes that make up axons, dendrites, growing neurites, synaptic vesicles, myelin, and synaptic junctions. The brain contains a vast diversity of cell types, both neuronal and nonneuronal; cellular entities that differ on the basis of anatomical location and neurophysiological properties. Thus, studies during the past two decades have concentrated on the identification and characterization of membrane glycoproteins that may be useful in distinguishing, or are unique to, specific classes of neurons, and more recently, different types of synapses. Advances in the areas of biochemistry, cellular and subcellular fractionation, cytochemistry, and immunology have made possible new approaches that have lead to a better understanding of the molecular and functional properties of membrane glycoproteins in the nervous system. One of the ultimate goals of the neurobiologist is to dissect the synapse into its constituent molecules and to elucidate the structural and functional contribution of each, and then in a synthetic approach, to determine intermolecular relationships that will provide a comprehensive understanding of how the synapse works. The development of procedures to isolate subcellular fractions highly enriched in subsynaptic structures such as synaptic junctions and postsynaptic densities has made possible the beginning of this difficult and complex task. The ability to adapt and grow cellular elements of nervous tissues under defined culture conditions has made possible the study of specific physiological and cell-cell interaction properties of individual neurons. The ability to culture embryonic neurons now allows one to study the behavior and molecular properties of neurons as they differentiate and form synaptic connections.
Animal cells employ about 20–35% of their total protein synthesis to construct the cytoskeleton—an intracellular cage of complex structures involved in cellular motility, like intracellular transport, exo- and endocytosis, protoplasmic streaming, locomotion, cellular polarity, anchorage, and cell division. Commonly, the entity of the cytoskeleton is divided into three fibrillar systems: microtubules, microfilaments, and intermediate (10-nm) filaments. In this chapter, we include the coated vesicle system, since it fulfills the criteria most commonly used to define cytoskeletal elements: it is composed of structural proteins that form supramolecular structures via self-assembly, it is involved in transport phenomena, and it associates with cellular membranes as well as with other cytoskeletal systems.
Transduction of extracellular signals requires activation of membrane-bound receptors. The transmission of the external signal across the plasma membrane often leads to an increase in the intracellular level of calcium [1-3]. In neurons, stimulation can increase the level of calcium more than 1000-fold; from < 0.1 μM to 200-300 μM [4]. This internal signal triggers calcium-binding proteins such as calmodulin, calbindin or troponin C, usually by inducing conformation changes that allow interactions with specific target proteins. Whether the target itself is a key protein in a particular cellular process or a regulatory protein, the calcium-dependent interaction with the target will affect the cellular process, either by up-regulating or down-regulating it. One target for calcium signals is spectrin and the spectrin-based membrane skeleton. This membrane skeleton is necessary for proper red cell shape and plasticity as well as lipid asymmetry. In non-erythroid cells, the cellular role of spectrin is probably linked to its ability to connect integral membrane proteins with actin filaments [5-17]. Therefore spectrin may be involved in positioning molecules such as receptors, ion channels and cell adhesion molecules correctly in the plasma membrane [7,18-24]. Since spectrin interacts with several calcium-binding proteins as well as binds calcium directly, it seems very likely that calcium ions are important and even may control some of the properties of spectrin. In this review I will examine the calcium-binding properties of spectrin in detail and discuss possible implications for cellular functions.
Following the discovery of calmodulin (Kakiuchi et al. 1969, 1970; Cheung 1970), it was proposed that the contractile device of smooth muscle and the cytoskeleton of nonmuscle tissues may be regulated by Ca2+/calmodulin. Recently, we have obtained considerable amounts of evidence to support the view that the regulatory actions of Ca2+/calmodulin are mediated by a number of specific calmodulin-binding proteins which also bind to F-actin filaments or to tubulin (Kakiuchi and Sobue 1983). Caldesmon (Sobue et al. 1981a, b) from smooth muscle and tau factor (Sobue et al. 1981c) from brain microtubules are calmodulin-binding proteins which also bind to F-actin or tubulin, respectively. The Ca2+-dependent binding of calmodulin to these proteins obviated the interaction between these proteins and F-actin or tubulin. Therefore, the binding of these proteins to calmodulin and cytoskeletal proteins alternates, depending on the concentration of Ca2+ (Fig. 1). As the binding of the calmodulin-binding proteins to the target cytoskeletal proteins modulates the function of the latter proteins, calmodulin regulates the function of cytoskeletal proteins via these calmodulin-binding proteins. We named this type of regulatory action of calmodulin flip-flop regulation (Kakiuchi and Sobue 1981, see also Kakiuchi and Sobue 1983). This article is a brief description of these calmodulin-binding proteins.
This chapter presents the current state of knowledge concerning the cellular distribution and morphology, biochemistry, and physiology of the integral parts of the neuronal cytoskeleton. It further discusses their roles as mediators of certain neurological disease states. Microtubules in neuronsand in other cells are approximately 25 nm in diameter, have a wall thickness of approximately 5 nm, and possess an electronlucent lumen. Tubulin, the structural protein of the microtubule, is a heterodimer composed of two polypeptides. In adult brain, this protein comprises 10–30% of the soluble protein. Filament proteins from different molecular-weight groups are not related to each other by oligomerization and are not merely derived by proteolytic cleavage from a common precursor. Two characteristic neuronal lesions in the cerebral cortex of patients with Alzheimer's senile dementia are the neuritic plaque formed by degenerating nerve cell processes inscribing a core of amyloid and the neurofibrillary tangle, which is formed of bundles of paired helical filaments.
Eukaryotic cells contain an extensive network of self-associated, interconnected filaments and tubules referred to collectively as the cytoskeleton. The proteins of the cytoskeleton are responsible for the generation of nearly all types of cellular movement, from gross amoeboid migration to subtle intracellular motions of organelles or chromosomes. Cytoskeletons may be defined teleologically as the intracellular filaments or proteins which combine to generate cell movement and to control cell shape. The cytoskeleton can also be defined operationally as the protein residue which remains when cells are extracted with non-ionic detergents such as Triton X-100. Examination of these detergent-resistant cellular residues by electron microscopy shows them to consist of several types of filaments. Each filament type has a distinct diameter and each is distributed within the cell in a characteristic pattern.
The multifunctional capability of the eukaryotic cell is expressed and regulated, to a great extent, by the cell type-specific biophysical properties of the plasma membrane. The plasma membrane is essentially a barrier comprising a phospholipid bilayer structure which acts also as a matrix onto which and into which a variety of specific proteins are attached. It is these proteins which selectively modify the structure and properties of the plasma membrane to create a wide variety of domains of distinctive morphology and function.
Growth factors activate quiescent cells in a stimulus-response coupling process that is initiated by binding the growth factor to its receptor on the cell surface (see Carpenter, 1984). The ultimate result of such activation is DNA synthesis and cell division (mitogenesis). However, such activated cells exhibit a host of earlier responses, some of which begin seconds poststimulation. These “early” cellular responses to growth factors include endocytosis, changes in the cytoskeleton, cell motility, phospholipid turnover, Na+/H+ exchanger activation, fluxes of a variety of ions, calcium transients, protein synthesis, changes in cyclic nucleotide levels, phosphorylation of specific proteins, and gene transcription. The connection, if any, of these early cellular responses with the later DNA synthetic response is obscure at present.
A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.
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.
We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to-head manner to form tetramers 220 nm long. We determined calmodulin-binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin-binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.
A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido- calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.
Further similarity between mammalian erythrocyte spectrin and pig brain spectrin has been demonstrated by (a) formation of hybrid molecules with brain α-chains and erythrocyte β-chains and by (b) identification of an ankyrin protein in brain membranes. Hybrid spectrin molecules prepared from brain α-chains and erythrocyte β-chains were visualized by low-angle rotary shadowing as double-stranded rods (dimers) 100 nM in length. 125I-labeled brain α-chain that was hybridized with erythrocyte β-subunit acquired ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain α-chain bound only to β-subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate (SDS) gels. Thus brain spectrin and mammalian erythrocyte spectrin have shared functional sites involved in association of their subunits. Additional evidence for similarity of brain and erythrocyte membranes is the finding of a 210,000 Mr membrane protein in brain that cross-reacts with erythrocyte ankyrin and has a water-soluble domain of 72,000 Mr that is produced by protease digestion. The 72,000 Mr domain of brain ankyrin has been isolated by affinity chromatography on erythrocyte spectrin-Sepharose, and was demonstrated to bind directly to erythrocyte and brain spectrin. The brain 72,000 Mr fragment has distinct peptide maps from the erythrocyte 72,000 Mr ankyrin fragment and thus is not a result of erythrocyte contamination.
Five different brands of nitrocellulose (NC), each of pore size 0.45 μm and without adsorbed antigen, bound different amounts of two labelled antisera and labelled protein A. Experiments with some non-ionic surface active agents and proteins showed that milk powder and bovine serum albumin were the most effective agents for blocking non-specific binding of labelled protein to NC. With some of the NCs, Nonidet P-40 (NP-40) and Tween 20 were almost as effective as milk powder. The protein-binding capacity of unblocked NC and the level of protein binding after blocking were found to be inversely proportional to the pore size of the NC. A comparison of blocking agents in an immunoassay with pollen proteins adsorbed to NC discs revealed that the highest specific uptakes of antiserum occured with NP-40 and Tween and not with any of the protein blocking agenst such as milk powder. Hence, for the detection of proteins using NC-based assays (but not necessarily following electroblotting), the best choices would appear to be: (1) NC of pore size 0.45 μm; (2) a brand of NC that provides a suitable balance between protein binding capacity and non-specific uptake of protein after blocking; (3) a non-ionic detergent such as NP-40 or Tween 20.
We labeled proteins in the cell bodies of rabbit retinal ganglion cells with [35S]methionine and subsequently observed the appearance of radioactive actin in tissues containing the axons and synaptic terminals of these neurons, i.e., the optic nerve (ON), optic tract (OT), lateral geniculate nucleus (LGN) and the superior colliculus (SC). The temporal sequence of appearance of labeled actin (which was identified by its specific binding to DNase I, its electrophoretic mobility, and its peptide map) in these tissues indicated that actin is an axonally transported protein with a maximum transport velocity of 3.4--4.3 mm/d. The kinetics of labeling actin were similar to the kinetics of labeling two proteins (M1 and M2) which resemble myosin; these myosin-like proteins were previously found to be included in the groups of proteins (groups III and IV) transported with the third and fourth most rapid maximum velocities. The similarity in transport between actin and myosin-like proteins supports the idea that a number of proteins in the third and fourth transport groups may be functionally related by virtue of their involvement in a force-generating mechanism and suggests the possibility that these proteins may be axonally transported as a preformed force-generating unit.
Spectrin, a protein complex which is peripherally attached to the cytoplasmic surface of the human erythrocyte membrane, cannot be detected (by complement fixation with anti-spectrin antibodies) in homogenates of several different human non-muscle cells studied. On the other hand, a protein antigenically identical or similar to human smooth muscle myosin was detected (by complement fixation with antibodies to uterine smooth muscle myosin) in these cells. In the case of human fibroblast line WI38, this smooth muscle myosin like component was shown (by ferritin-antibody experiments in electron microscopy) to be at least partly associated with cytoplasmic surface of the plasma membrane of the cell. It is proposed that the spectrin complex of the erythrocyte membrane and the smooth muscle myosin-like component of the fibroblast membrane play similar roles in regulating the translational mobilities of integral proteins in their respective membranes.
The regulatory component (G/F) of adenylate cyclase has been purified from turkey erythrocyte plasma membranes by adaptation of procedures developed for purification of the rabbit liver protein. The major modifications entail inclusion of high concentrations of NaCl to facilitate extraction and reconstitution of the protein. A typical preparation yields 200 micrograms of protein with a reconstitutive specific activity of 3-4 mumol . min-1 mg-1. Turkey erythrocyte G/F contains two putative subunits of 35,000 and 45,000 daltons. The 52,000-dalton polypeptide that appears to be a component of rabbit liver G/F is lacking. In solution, G/F behaves as a particle with Mr = 81,000. This value is reduced to 50,000 in the presence of activating ligands, suggesting dissociation of subunits. Activation of G/F by guanine nucleotide analogs is markedly accelerated in the presence of high concentrations of Mg2+. Reconstitutive and physical properties of the protein are also affected by fluoride. Cyc- S49 lymphoma membranes reconstituted with turkey erythrocyte G/F acquire properties that are characteristic of the turkey adenylate cyclase system; at least certain differing characteristics of adenylate cyclase systems are thus dictated by the nature of their G/F.
We have demonstrated the existence of a spectrin-like protein in a variety of nonerythroid cultured cells. Indirect immunofluorescence studies with monospecific antispectrin IgG indicated the presence of proteins that have common antigenic determinants to spectrin in embryonic chicken cardiac myocytes, mouse fibroblast lines (3T3, simian virus 4-transformed 3T3), and rat hepatoma lines (HTC, HMOA). Two spectrin-like peptides of 240,000 and 230,000 daltons were immunoprecipitated from octyl glucoside-solubilized embryonic chicken cardiac myocytes, along with associated cytoskeletal proteins. Immunoautoradiographic characterization of the myocyte immunoprecipitate showed that only the spectrin-like 240,000- and 230,000-dalton peptides were stained with monospecific antispectrin IgG and 125I-labeled protein A. One-dimensional partial proteolytic mapping of the myocyte 240,000- and 230,000-dalton peptides showed that these peptides share substantial sequence homology with embryonic chicken erythrocyte spectrin 240,000- and 220,000-dalton peptides.
Isolated microfilament cores of intestinal microvilli are known to contain actin and four major associated proteins among which is calmodulin. Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bound artifactually during the isolation procedure. Identification of the major microvillus calmodulin-binding protein was achieved by the use of an [125I]calmodulin gel overlay technique. Proteins of microvilli or brush borders were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After removal of sodium dodecyl sulfate, direct binding of radiolabeled calmodulin to the separated polypeptides was assayed by autoradiography. Three calmodulin-binding polypeptides are detected in brush borders. Two polypeptides (apparent Mr = 280,000 and 140,000) show Ca2+ -dependent binding, whereas the third polypeptide (Mr = 110,000) can bind calmodulin in the presence or absence of Ca2+. Microvillus core filaments contain only the latter species. Microvillus cores treated with 25 mM Mg2+ retain calmodulin and the 110,000 polypeptide, whereas the other two associated proteins are greatly reduced, consistent with the hypothesis that the 110,000 protein is the major calmodulin-binding protein of the core filament structure. We discuss the currently documentable structure of the core filaments and evaluate the general usefulness of the calmodulin gel overlay technique.
We labeled proteins in the cell bodies of rabbit retinal ganglion cells with [35S]methionine and subsequently observed the appearance of radioactive actin in tissues containing the axons and synaptic terminals of these neurons, i.e., the optic nerve (ON), optic tract (OT), lateral geniculate nucleus (LGN) and the superior colliculus (SC). The temporal sequence of appearance of labeled actin (which was identified by its specific binding to DNase I, its electrophoretic mobility, and its peptide map) in these tissues indicated that actin is an axonally transported protein with a maximum transport velocity of 3.4--4.3 mm/d. The kinetics of labeling actin were similar to the kinetics of labeling two proteins (M1 and M2) which resemble myosin; these myosin-like proteins were previously found to be included in the groups of proteins (groups III and IV) transported with the third and fourth most rapid maximum velocities. The similarity in transport between actin and myosin-like proteins supports the idea that a number of proteins in the third and fourth transport groups may be functionally related by virtue of their involvement in a force-generating mechanism and suggests the possibility that these proteins may be axonally transported as a preformed force-generating unit.
The GAPs (growth associated proteins, axonally transported at elevated levels during periods of axon elongation) and fodrin (polypeptides associated with the outer cytoplasm of neurons and other cells) are novel, rapidly axonally transported proteins. The behavior of the GAPS suggests that the modulation of neuronal gene expression is a critical feature in regulating axon growth during development and regeneration. The properties of fodrin raise the possibility of a relationship between the process of axonal transport and other intracellular transport phenomena.
A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.
Molecules of human erythrocyte spectrin have been examined by electron microscopy after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramere were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the two polypeptide chains, band 1 (240,000 Mr) and band 2 (220,000 Mr), often appeared as two separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appears to be weak, except at both ends of the molecule where there are sites of strong binding. Tetramers are formed by the end-to-end association of two spectrin heterodimer molecules without measurable overlap, and have a mean shadowed length of 194 nm. This association to form tetramers probably involves head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode are not observed. The molecular shape of spectrin is quite distinct from that of myosin, to which it has often been likened.
A HIGHLY sensitive radioimmunoassay has been developed for human spectrin, one of the major components of the erythrocyte membrane1-6. We report here use of this assay on cells of human, monkey and rat origin and show that spectrin is absent from all cell lines studied, with the exception of erythrocytes. These findings seem important when models which invoke spectrin to regulate membrane fluidity,7,8 or membrane-cytoskeletal interaction9,10 are considered.
We describe a convenient technique for shadowing extended, flexible molecules difficult or impossible to visualize after conventional preparative methods. The technique uses glycerol but avoids other harsh conditions; physiological salt concentrations can be maintained until the moment of drying.
Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d--slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins. In this communication, we report the intracellular distribution of fodrin. Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence. Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e.g., skeletal muscle, uterus, intestinal epithelium). Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining). In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro. We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities: (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm; and (c) that fodrin could serve to link other proteins and organelles to a submembrane force-generating system.
The discoveries are reviewed that elucidate how the cytoskeleton of the human erythrocyte membrane is linked to the membrane and make it possible to visualize the molecular interactions among the cytoskeletal components. These discoveries are the result of a coordinated structural and biochemical approach that has revealed a surprisingly complex set of specific interactions whose existence emphasizes the fundamental continuity between the erythrocyte membrane and the erythrocyte cytoskeleton.
Human erythrocyte ankyrin, the membrane attachment protein for spectrin, has been detected by radioimmunoassay in a variety of cells and tissues. This report identifies polypeptides crossreacting with ankyrin in brain and HeLa cells and demonstrates that one function of these ankyrin analogues involves association with microtubules. Ankyrin immunoreactivity was localized by indirect immunofluorescence in a colchicine- and detergent-sensitive cytoplasmic meshwork in interphase cells. There also was specific nuclear staining, localized in a bright spots, which was displaced entirely by ankyrin or by high molecular weight microtubule-associated proteins (MAPs) from brain. In dividing cells, the punctate nuclear staining and the meshwork disappeared. Fluorescence was localized at the spindle pole during metaphase and was redistributed to the cleavage furrow in later stages of mitosis. An immunoreactive Mr 370,000 polypeptide comigrating with MAP1 was identified in brain extracts and copolymerized with microtubules through repeated cycles of polymerization and depolymerization. Finally, erythrocyte ankyrin associated with microtubules prepared from pure tubulin, and this binding was displaced by brain MAPs.
Isolated human erythrocyte spectrin is a dimer of two unique polypeptide chains. The dimer (alpha beta) undergoes reversible salt- and temperature-dependent association to form (alpha beta)2 tetramers. Spectrin also binds with high affinity to a protein receptor on the cytoplasmic surface of erythrocyte membrane vesicles. By cleavage of spectrin at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid, a 50,000-dalton peptide fragment has been isolated which inhibits the binding of spectrin to erythrocyte membrane vesicles. This peptide arises from a terminal region of the beta chain. An 80,000-dalton peptide generated by restricted trypsin digestion binds preferentially to dimeric spectrin. This peptide arises from a terminal portion of the alpha chain. Multiple peptides involved in noncovalent associations between the chains have also been identified. These associations indicate that the two subunits of spectrin are aligned parallel to one another and that the tetramer formation site and the high-affinity membrane binding site are in close proximity to one another.
A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW 260/240), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW 260/240 is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW 260/240 is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW 260/240 indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW 260/240 with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW 260/240 and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW 260/240 (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.
A case of the sick sinus syndrome and constrictive pericarditis with effusion (seroconstrictive pericarditis) as manifestations of lymphomatous involvement of the heart is presented. Autopsy findings documented infiltration of the sinus node by undifferentiated malignant lymphoma cells. The clinical course is described and problems with diagnosis and management of such a complication are discussed. This is one of the few cases in the literature in which an uncommon cause of conduction system disease and pericardial constriction was substantiated by electrocardiogram, angiography, and autopsy.
Previously we have shown that purified spectrin binds calmodulin in the presence of Ca2+ with a Kd value of 3 μM (Sobue, K. et al. (1980) Biochemistry International 1, 561–566). We now provide evidence that the calmodulin-binding activity found in the human erythrocyte cytoskeleton is indeed due to spectrin and no other binding proteins are involved, i.e. the binding activity was purified from the erythrocyte cytoskeleton quantitatively and the purified peak contained spectrin as the only protein constituent. Moreover, Kd value (2.8 μM) and the maximum binding capacity (160,000 – 200,000 calmodulin per cell) obtained from the kinetic analysis of the binding activity in the crude cytoskeleton agreed with the corresponding values reported for purified spectrin. Since the concentration of calmodulin in the erythrocyte cell, which was 2.5 μM or 1.6 × 105 molecules per cell, is close to both the Kd value and the number of the binding sites in the cell, respectively, free calmodulin in the erythrocyte cell may be in a dynamic equilibrium with the spectrin-bound form in vivo depending upon the intracellular concentration of Ca2+.
A new, commercially available oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) was compared with chloramine-T and solid-phase lactoperoxidase in the radioiodination of proteins, glycoproteins, and peptides. A method for performing low-level iodinations is described and was used to determine maximum 125I incorporation. Iodinated proteins were purified on analytical gel filtration columns and peptides by reverse-phase high-performance liquid chromatography. Both methods were designed to analyze the tracers for the presence of aggregate and breakdown products caused by the iodination. All tracers prepared were tested in antibody dilution and dose-response curves in their respective radioimmunoassays. Results indicate that Iodogen can be used for a wide range of proteins and peptides, can permit theoretical iodine incorporation with minimal oxidation damage, and can produce tracer stable for up to 3 months.
The subunits of spectrin from human erythrocytes were separated by ion-exchange chromatography on hydroxyapatite in the presence of urea. When renatured from the urea solution they are found to be monomeric, although the smaller subunit (band 2) is prone to aggregation. In shape, solubility and secondary structure the subunits resemble the native spectrin dimer, indicating that subunit interaction is not essential for maintaining the native conformation. When the subunits are recombined, a dimer with the sedimentation coefficient of the native species is formed. This constitutes direct evidence that native spectrin is a heterodimer, rather than a mixture containing homologous and heterologous species. The interaction of the separated subunits with the chymotryptic fragment of the spectrin-binding protein (protein 2.1, or ankyrin) of the erythrocyte membrane was studied. Only the smaller subunit has the ability to bind, and thus presumably contains the site by which the cytoskeleton is attached to the plasma membrane. On the other hand, the formation of a complex with F-actin and protein 4.1 requires the presence of both subunits. A complex of these proteins with band 2 is formed, however, when traces of an additional, as yet unidentified, protein are present.
Spectrin binds to a population of high-affinity sites on the exposed surfaces of inverted vesicles prepared from human red blood cell ghost membranes. Optimal spectrin binding requires the presence of monovalent salts but does not require calcium or magnesium. The band 2 subunit of spectrin, prepared in SDS, can also bind to vesicles, but isolated band 1 is inactive.
Pre-incubation of inverted vesicles with antibodies directed against the cytoplasmic segment of band 3 or against bands 4.1–4.2 inhibits the binding of spectrin to the same vesicles. Antibodies against the cytoplasmic portion of glycophorin A have no effect.
These results suggest that spectrin binds to a protein acceptor on the cytoplasmic surface of the red cell membrane which is close to the cytoplasmic segments of bands 3 and 4.1 and/or 4.2.
- E Hanski
- P C Sternweis
- J K Northup
- A W Dromerick
- A G Gilman
Hanski, E., Sternweis, P. C., Northup, J. K., Dromerick, A. W.
& Gilman, A. G. (1981) J. Biol Chem. 256, 12913-12919.
- J M Tyler
- D Branton
Tyler, J. M. & Branton, D. (1980)J. Ultrastruct. Res. 71, 95-102.
- J Levine
- P Skene
- M Willard
Levine, J., Skene, P. & Willard, M. (1981) Trends Neurosci. 4,
273-277.
- J R Glenney
- Jr
- P Glenney
- M Osborn
- K Weber
Glenney, J. R., Jr., Glenney, P., Osborn, M. & Weber, K. (1982)
Cell 28, 843-854.
- R Calvert
- P Bennett
- W Gratzer
Calvert, R., Bennett, P. & Gratzer, W. (1980) Eur. J. Biochem.
107, 355-361.
- K Sobue
- Y Muramoto
- M Fujita
- S Kakiuchi
Sobue, K., Muramoto, Y., Fujita, M. & Kakiuchi, S. (1981)
Biochem. Biophys. Res. Commun. 100, 1063-1070.
- J R Glenney
- Jr
- K Weber
Glenney, J. R., Jr., & Weber, K. (1980) J. Biol. Chem. 255,
10551-10554.
- G Hiller
- K Weber
Hiller, G. & Weber, K. (1977) Nature (London) 266, 181-183.
- D Branton
- L M Cohen
- J Tyler
Branton, D., Cohen, L. M. & Tyler, J. (1981) Cell 24, 24-32.