Contributions of the beta-subunit to spectrin structure and function

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.     

Comparison of spectrin isolated from erythroid and non-erythroid sources

Spectrin from erythrocytes and two other tissues (brain and intestine) were isolated from two distant species, pig and chicken; some structural and functional properties were compared. A quantitative antibody inhibition assay was used to determine that antibodies to mammalian red cell spectrin cross-react very poorly, if at all, with their non-erythroid (brain) counterpart and similarly antibodies to pig brain spectrin (fodrin) cross-react very weakly with erythroid spectrin. By contrast, antibodies which were directed against the 240000-Mr subunit of avian fodrin were completely inhibited with avian spectrin and vice versa. To analyze the structural relatedness of these molecules further we compared the chymotryptic iodinated peptide maps generated from each individual subunit. Consistent with the antibody results, we find little (less than 10%) homology between peptides derived from mammalian fodrin and spectrin, but complete homology (100%) of the peptides derived from the 240000-Mr subunits of chicken fodrin, spectrin and another related molecule from intestine, TW260/240. Whereas the peptide maps of fodrin (brain spectrin) revealed striking similarity between divergent species, suggesting a high degree of structural conservation, the peptide maps of erythrocyte spectrin was highly variable between species, indicating that it has diverged considerably in mammalian evolution. In addition we have compared a functional activity of mammalian spectrins, the ability to bind calmodulin, using two different assays. Both results show that, whereas fodrin-calmodulin interaction can be readily demonstrated, the binding to mammalian erythroid spectrin is negligible. This suggests that the high-affinity calmodulin site present on fodrin has been lost from spectrin in mammalian evolution.     

Separation of fodrin subunits by affinity chromatography on calmodulin-Sepharose

Spectrin is composed of two nonidentical subunits, with the 240-kDa subunit of nonerythroid spectrin (fodrin) able to bind calmodulin (CaM) Ca2+-dependently. It was found that in the presence of chaotropic salts this binding site was still expressed, although the subunits of fodrin were dissociated. This has been exploited for separating the fodrin subunits rapidly and quantitatively by affinity chromatography on calmodulin-Sepharose. When bovine fodrin was dissolved in 2 M KI + 1 mM Ca2+ and applied to CaM-Sepharose the beta subunit (235-kDa) passed through unretarded whereas the alpha subunit (240-kDa) bound and could be eluted with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. These subunits would reform the intact molecule when mixed and dialyzed.     

An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin

A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW $\text{260}\text{240}$), 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 $\text{260}\text{240}$ 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 $\text{260}\text{240}$ 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 $\text{260}\text{240}$ indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW $\text{260}\text{240}$ with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW $\text{260}\text{240}$ 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 $\text{260}\text{240}$ (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.     

Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.     

Developmental organization of the intestinal brush-border cytoskeleton

At the terminal web of chicken intestinal epithelial cell, the actin bundles are cross-linked by a fine filamentous network of actin-associated cross-linkers. Myosin, fodrin, and TW 260/240 have been identified as major components of the cross-linkers. We studied the development of the cross-linkers by quick-freeze, deep-etch electron microscopy, and the expression of cross-linker proteins (myosin, fodrin 240, and TW 260) by immunofluorescence and immunoblotting analysis during the embryogenesis. Microvilli start to form at 5-7 days, and the rootlets begin to elongate at 10 days. At an early stage of the development of the terminal web (13 days), fodrin 240 and a small amount of myosin are expressed, and a few actin-associated cross-linkers are present between the rootlets. However, TW 260 is not expressed at this stage. At an intermediate stage (19 days), the amount of myosin increases, and TW 260 begins to be expressed. The number of cross-linkers associated with the unit length of the rootlets is 24/microns. At the final stage of the terminal web formation (2 days after hatching), the amount of fodrin 240, myosin, and TW 260 is similar to the adult level, and the number of the actin-associated cross-linkers per unit length of the rootlet is 27/microns (approximately 85% of the adult). These results suggest that the synthesis of cross-linker proteins may be intricately regulated to achieve the desired density of cross-linkages at each developmental stage: at early and intermediate stages, sufficient and not an excess of cross-linkages are formed; and at a final stage, a higher complexity of cross-linkages is achieved. In addition, there is a differential expression of the components of the actin-associated cross-linkers: myosin and fodrin could be early components of the cross-linkers involved in the basic stabilization of the terminal web structure, whereas TW 260/240 becomes incorporated later, possibly involved in the stabilization preparatory to the rapid elongation of microvilli, which occurs after the formation of the terminal web.     

Expression and assembly of the erythroid membrane-skeletal proteins ankyrin (goblin) and spectrin in the morphogenesis of chicken neurons

The membrane-skeleton of adult chicken neurons in the cerebellum and optic system is composed of polypeptides structurally and functionally related to the erythroid proteins spectrin and ankyrin, respectively. Neuronal spectrin comprises two distinct complexes that share a common alpha subunit (Mr 240,000) but which have structurally distinct polymorphic subunits (beta' beta spectrin; Mr 220/225,000; gamma spectrin, Mr 235,000); the brain-specific form (alpha gamma spectrin or fodrin) and an erythrocyte-specific form (alpha beta' beta spectrin). Two structurally related isoforms of ankyrin have also been identified and are termed alpha (Mr 260,000) and beta (Mr 237,000) ankyrin. Immunofluorescence demonstrates that the variants of spectrin and ankyrin, respectively, have different distributions within neurons. On the one hand, alpha gamma spectrin and beta ankyrin are present throughout the neuron, in the perikaryon, dendrites, and axon, whereas alpha beta' spectrin and alpha ankyrin are localized exclusively in the perikaryon and dendrites where they are actively segregated from alpha gamma spectrin and other components of axonal transport. This asymmetric distribution of spectrin and ankyrin isoforms is established in distinct stages during neuronal morphogenesis. Early in cerebellar and retinal development, alpha gamma spectrin is expressed in mitotic cells. Subsequently beta ankyrin and alpha gamma spectrin are coexpressed in postmitotic cells and gradually accumulate on the plasma membrane in a uniform pattern throughout the neuron during the phase of cell growth. At the onset of synaptogenesis and the cessation of cell growth, their levels of synthesis decline sharply while the assembled proteins remained as stable membrane components. Concomitantly, there is a dramatic induction in the accumulation of alpha ankyrin and alpha beta' spectrin, whose assembly is limited to the plasma membrane of the perikarya and dendrites. These results demonstrate that two successive, developmentally regulated programs of ankyrin and spectrin expression and patterning on the plasma membrane are involved in the assembly of the spectrin-based asymmetry in the neuronal membrane-skeleton, and that their asymmetric distribution is actively maintained throughout the life of the neuron.     

Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions

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.     

Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.     

A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.     

Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G- spectrin, although alpha-spectrin was still present. Beta G-spectrin- deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin- associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E- cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)- ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.     

Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.     

Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures

The intracellular precipitation of nonerythrocyte spectrin has been achieved by the microinjection into cells of either a monoclonal antibody (IgM) directed against the alpha chain of nonerythrocyte spectrin or an affinity-purified polyclonal antibody raised against bovine brain spectrin (fodrin). This antibody-induced precipitation of spectrin was observed in fibroblastic and epithelial cell types, including embryonic bovine tracheal fibroblasts, a bovine kidney epithelial cell line (MDBK), Hela cells, gerbil fibroma cells, and fibroblast lines of human and mouse origins. The precipitation of the spectrin was specific and two proteins with a similar distribution to the nonerythrocyte spectrin were not induced to co-precipitate in the spectrin aggregates. Comparing the two types of antibody microinjected, the affinity-purified polyclonal antibody resulted in more compact aggregates of spectrin and these were frequently aligned with microfilament bundles. The rate at which the spectrin aggregates were cleared into presumptive lysosomes varied with different cell types: in some such as the bovine kidney epithelial cells, this appeared complete within 3 h after microinjection, whereas in some of the fibroblasts the spectrin aggregates were prominent in the cytoplasm at 24 and even 48 h after microinjection. Microfilament bundles appeared unaffected by the aggregation of spectrin. We conclude that the integrity of the actin microfilament bundles does not require nonerythrocyte spectrin and that most probably these structures are linked at their termini to the membrane through proteins other than nonerythrocyte spectrin. No effect of the intracellular spectrin precipitation was observed on cell shape, or on the distribution of coated vesicles or microtubules. The aggregation of the nonerythrocyte spectrin, however, did affect the distribution of the vimentin type of intermediate filaments in most of the cell types studied. These filaments became more distorted and condensed, but generally did not collapse around the nucleus as occurs following microtubule disruption induced by colchicine treatment. The clumped intermediate filaments were frequently seen to coincide with regions of aggregated spectrin. This aggregation of intermediate filaments was not induced by microinjection of irrelevant antibodies, nor was it induced by the monoclonal antibody against spectrin in cells with which it did not cross-react.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption

We have previously shown that the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet-intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non-erythroid spectrin). Here we demonstrate that Pet has affinity for alpha-fodrin (formally named alphaII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST-fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain. Site-directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet-treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST-fodrin. This is the first report showing cleavage of alpha-fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.     

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

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

Biogenesis of Polarized Epithelial Cells During Kidney Development In Situ: Roles of E-Cadherin–mediated Cell–Cell Adhesion and Membrane Cytoskeleton Organization

Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based on studies with cultured kidney cells, we have hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves E-cadherin–mediated cell–cell adhesion and integration of Na/K-ATPase into the Triton X-100–insoluble ankyrin- and spectrin-based membrane cytoskeleton. In this study, we examined the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo during mouse kidney development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, we demonstrate the following. First, expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase, E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase. Second, expression of the 190-kDa slice variant of ankyrin-3 correlates with maximal accumulation of Na/K-ATPase. Third, Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all epithelial cells in which they are expressed and during all stages of nephrogenesis. Fourth, the relative immunofluorescence staining intensities of Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells in vivo regulate the accumulation of E-cadherin–mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized distributions of these proteins in vivo and in vitro involve cell–cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration and retention of Na/K-ATPase in this complex.     

Immunolocalization of a spectrin-like protein (fodrin) in pancreatic acinar cells

A spectrin-like protein (fodrin) was localized in porcine pancreas using an immunoperoxidase procedure with antibodies raised against erythrocyte spectrin. Fodrin was primarily associated with the cell plasma membrane although some was also detectable in the cytoplasm of the acinar cells. The membrane labelling of the acinar cells was uneven such that the lateral and basal membranes were strongly labelled by anti-spectrin antibodies whereas the apical membranes were poorly labelled. The implications of the results to secretion and to the occurrence of specific membrane domains are discussed.     

Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae

We have used a polyclonal affinity-purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae of Dictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod-shaped protein 118 +/- 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes' radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd from Dictyostelium myosin II heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one-dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.