Widespread occurrence of avian spectrin in nonerythroid cells

Cell, Vol. 29, 821-833,

July 1982,

Copyright

0 1982

by MIT

Widespread Occurrence in Nonerythroid Cells Elizabeth A. Repasky,* Bruce L. Granger Elias Lazarides Division of Biology California Institute of Technology Pasadena, California 91125

of Avian Spectrin

and

Summary We have prepared an antibody against chicken erythrocyte (Y spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with (r spectrin in chicken erythrocytes and crossreacts with (Y spectrin in erythrocytes from various mammals. lmmunofluorescence reveals that this antiserum reacts with a plasma membrane component in erythrocytes as well as in most nonerythroid avian and mammalian cells. Intense staining is seen at or near the plasma membrane in neurons, lens cells, endothelial and epithelial cells of the gastrointestinal and respiratory tracts, skeletal and cardiac muscle, as well as skeletal myotubes grown in tissue culture. Immunoautoradiography indicates that the crossreactive antigen in these nonerythroid tissues has the same molecular weight and isoelectric point as the chicken erythrocyte antigen. Smooth muscle, tracheal cilia, myelin and mature sperm stain weakly or not at all. These results suggest that spectrin is more extensively distributed than previously recognized, and that the functions of spectrin elucidated for erythrocytes may apply to other cell types as well. Introduction Spectrin is the major extrinsic protein of the plasma membrane of circulating erythrocytes (Marchesi and Steers, 1968). It is composed of two nonidentical polypeptide chains termed band 1 or (Y spectrin (molecular weight 240,000) and band 2 or p spectrin (molecular weight 220,000) (for reviews see Lux, 1979; Marchesi, 1979; Branton et al., 1981). In adult mammalian erythrocytes, spectrin is found on the inner surface of the plasma membrane, where it forms, in association with actin and several other proteins, a network believed to be responsible for maintaining the shape of, and conferring structural integrity to, mature erythrocytes (for a review see Branton et al., 1981). Spectrin also appears to interact with the plasma membrane through its association with ankyrin, a protein that binds to a subset of the transmembrane anion channels (Bennett and Stenbuck, 1979a, 1979b). Immunologically crossreactive forms of ankyrin have been observed in nonerythroid cells, giving rise to the l Present Memorial

address: Institute,

Department of Radiation Medicine, Buffalo, New York 14263.

Roswell

Park

suggestion that, like ankyrin, the distribution of spectrin may also be widespread (Bennett, 1979). Thus far, however, spectrin has been considered to be limited to erythrocytes. This assumption has stemmed from a failure to detect immunoreactive forms of spectrin in nonerythroid cells (Painter et al., 1975; Hiller and Weber, 1977), limiting generalization regarding the function of spectrin to the terminally differentiated and highly specialized red blood cell. Although most investigations of spectrin have focused on the protein in mammals, spectrin has been tentatively identified in the erythrocytes of other vertebrates such as birds (Blanche& 1974; Jackson, 1975; Caldwell, 1976; Weise and Ingram, 1976; Chan, 1977; Pinder et al., 1978; Watts and Wheeler, 1980; Granger et al., 1982) and amphibians (Pinder et al., 1978) as well as in certain invertebrates (Pinder et al., 1978). We show that antibodies raised against avian erythrocyte (Y spectrin react with both avian and mammalian (Y spectrin, and exhibit a wide crossreactivity in immunofluorescence with a plasma-membrane-associated polypeptide in a variety of avian and mammalian cell types. The crossreactive antigen in these nonerythroid cells has the same isoelectric point and electrophoretic mobility as the avian erythrocyte antigen. These results demonstrate the widespread occurrence of an antigen highly homologous to, if not identical with, avian erythrocyte (Yspectrin, suggesting that spectrin may mediate cytoplasmic filamentmembrane interactions and confer form or structural integrity to the plasma membranes of diverse cell types. Results Solubility Properties of Chicken Erythrocyte Spectrin The solubility properties of chicken spectrin were examined by subjecting chicken erythrocyte plasma membranes to a variety of extraction procedures. Figure 1 is a one-dimensional gel showing some of the extracts and residues obtained. Human erythrocyte ghosts were run for reference (lane 1). Lane 2 shows whole chicken erythrocyte membranes, the starting material for subsequent extractions. Brief treatment with distilled water at 0°C releases much of the intermediate filament proteins, vimentin and synemin (Granger et al., 1982) but releases little of the spectrin (lane 3). Synemin is barely visible at this loading; it runs immediately beneath (Y spectrin. Longer extraction of the membranes with water at O’C releases more spectrin (and actin) from the membranes (lane 4) but this still represents a minority of the spectrin present (residue is shown in lane 5). Similar results are obtained if reducing agents or divalent cation chelators are present in low (l-l 0 mM) concentrations (not shown). If such extractions are performed at 37°C instead of 0°C spectrin is released more

Cell 022

Figure 1. Analysis Properties

&~kJctrin P

4Vimentin &Actin

rapidly, but after 1 hr the solubilized spectrin still represents only a small fraction of the total (not shown). Extraction of the membranes with a solution containing 1 M KCI, Triton X-100, EDTA and dithiothreitol (DTT) leaves an insoluble residue composed primarily of spectrin, actin and vimentin (lane 6). The prominent cluster of proteins at the 100,000 dalton position (including a protein presumably analogous to the mammalian erythrocyte anion channel, or band 3) is largely solubilized under these conditions, as is goblin, a membrane phosphoprotein (Beam et al., 1979). High pH solubilizes a set of proteins (lane 7) similar to that left insoluble in the detergent solution (the most obvious exception being goblin). The corresponding high pH residue (lane 8) consists of the lipid bilayer and its most tightly associated proteins (Steck and Yu, 1973). Treatment with 6 M urea (lane 11) leaves a similar set of proteins in the residue (except for goblin; compare with lane 8) but only if the membranes have not become oxidatively crosslinked (Steck, 1972b; Haest et al., 1977, 1981). Pretreatment of the membranes with the thiol-oxidizer diamide (Kosower et al., 1969) renders the spectrin insoluble in urea (lane 9, residue; lane 10, extract), but has little effect on the solubilities of actin and vimentin. This crosslinking can be reversed by inclusion of DTT in the urea solution (lane 11, residue; lane 12, extract). If membranes are washed free of reducing agents but not pretreated with diamide, the urea (without DTT) residue and extract are similar to those in lanes 11 and 12, showing that oxidative crosslinking of spectrin witn no added oxidizer is insignificant. Identical results are obtained with sodium tetrathionate (Phil and Lange, 1962) in place of diamide (Haest et al., 1977, 1981); this agent has been used as a protease inhibitor (Liu. 1967) in many studies of avian erythrocytes

of Avian Spectrin

Solubility

(Lane 1) Human erythrocyte ghosts. (Lanes 2-12) Extracts and residues of chicken erythrocyte ghost plasma membranes. All extractions were performed at 0°C. (Lane 2) Whole membranes; (lane 3) 1 hr distilled water extract; (lane 4) 70 hr distilled water extract: (lane 5) residue: (lane 6) 1 M KCI, 1% Triton X-i 00 residue; (lane 7) 0.1 M NaOH extract; (lane 8) residue. (Lanes 9-12) Diamidetreated membranes; (lane 9) 6 M urea residue; (lane 10) extract: (lane 11) 6 M urea, 20 mM DTT residue; (lane 12) extract.

(Jackson, 1975, 1976; Weise and Chan, 1977; Weise and Chan, 1978).

Ingram,

1976;

Immunological Characterization of Chicken (Y Spectrin For the preparation of antibodies, chicken erythrocyte cytoskeletal proteins were resolved by two-dimensional, isoelectric focusing and SDS-polyacrylamide gel electrophoresis (O’Farrell, 1975; see Experimental Procedures, Figure 9). The polypeptide designated (Y was excised from gels and used as the immunogen. The specificity of the resulting antiserum was determined by immunoautoradiography, a technique in which antibodies are used to detect antigens in polyacrylamide gels (Burridge, 1978). Figure 2a shows a Coomassie-blue-stained, two-dimensional gel of chicken erythrocyte ghosts. This gel was processed for immunoautoradiography with the presumptive antia-spectrin antiserum, followed by radioiodinated protein A, and the resulting autoradiogram is shown in Figure 2b. The antiserum stains only the immunogen, a! spectrin, and does not crossreact with any other proteins displayed by this gel system. Overexposure of the autoradiogram, as in this figure, reveals a set of labeled polypeptides smaller than the parent molecule; these probably represent the breakdown products of (Y spectrin that result from the action of endogenous proteases during preparation of the sample for electrophoresis (see Granger et al., 1982). A streak of unfocused protein trailing back to the origin (basic end) of the isoelectric focusing gel is also evident. Similar results are obtained with two-dimensional gels of both whole erythrocytes and erythrocytes extracted with Triton X-l 00 (not shown). Figure 3a shows a one-dimensional Coomassieblue-stained gel of various spectrin-containing

Spectrin 823

Figure

in Nonerythroid

Cells

2. lmmunoautoradiographic

Characterization

of Anti-n-Spectrin

Antiserum

(a) Coomassie-blue-stained. two-dimensional gel of chicken erythrocyte ghosts. This gel was incubated sequentially with the antiserum and radioiodinated protein A before staining. (A) actin; (V) vimentin; (a and P, a spectrin and B spectrin; pH gradient from 4 (left) to 7 (right). (b) Corresoonding autoradiogram showing specific labeling of a spectrin (and minor amounts of presumed degradabon products).

a+

A+

Figure 3. lmmunoautoradiographic man Erythrocyte Spectrins

Comparison

of Chicken

and Hu-

(a) Stained gel; (b) corresponding autoradiogram. (Lane 1) High pH extract of human erythrocyte ghosts; (lane 2) human erythrocyte ghosts; (lane 3) chicken erythrocyte ghosts; (lane 4) chicken erythrocyte cytoskeletons; (lane 5) chicken erythrocyte plasma membranes. In each of these lanes, (Y spectrin is specifically labeled. The autoradiogram of the human samples (b, lanes 1 and 2) was exposed approximately five times longer than that of the chicken samples (b, lanes 3-5) indicating a relatively weak crossreactivity of human rx spectrin with this antiserum. Two proteolytic fragments of a spectrin that form during the isolation and storage of the plasma membranes (see text) are evident in lane 5.

chicken erythrocyte fractions (lanes erythrocyte fractions (lanes 1 and 2) ing immunoautoradiograms. In each rum reacts only with (r spectrin. The

3-5) and human and their resultcase this antiseexposure of the

mammalian erythrocyte autoradiogram is five times longer than that used for the avian erythrocyte gel, indicating that the crossreaction of this antiserum with ’ the mammalian polypeptide is weaker than the reaction with the avian protein. The polypeptide doublet near the 150,000 dalton position that labels with the antiserum (most evident in lane 5) appears to be degradation products of (Y spectrin; these polypeptides increase in relative amount during storage and processing of the samples (see also Clarke, 1971; Jackson, 1975) and increase dramatically at the expense of (Y spectrin during limited proteolytic digestion of chicken erythrocyte membranes with Staphylococcal protease V8 (unpublished observations). Figure 4a shows a one-dimensional gel of various chicken tissues as well as chicken erythrocytes (which provide a molecular weight marker for OLspectrin). The resulting anti-a-spectrin immunoautoradiogram is shown in Figure 4b. A protein that comigrates with avian (Y spectrin crossreacts to varying extents with the antiserum in all of the tissues examined (lens, small intestine, cerebellum, spinal cord, liver and muscle tissue from the gizzard, heart and leg). The crossreaction of the antiserum with lens (lane 2) which is avascular, indicates that the observed labeling is not due to erythrocyte contamination. Electrophoresis of the other tissue samples on a less porous gel, which resolved lower molecular weight polypeptides, revealed no hemoglobin, demonstrating that the level of erythrocyte contamination was extremely low (data not shown). In several of the lanes in Figure 4b, minor amounts of the two presumptive proteolytic fragments seen in Figure 3 are also evident. The basis of the

Cell 624

a-

Figure

4. Anti-c-Spectrin

lmmunoautoradiography

of Chicken

and Mouse

Tissues

Stained gel (a) and corresponding autoradiogram (b) of various chicken tissues and a spectrin; (lane 2) lens; (lane 3) small intestine; (lane 4) cerebellum: (lane 5) spinal cardiac muscle; (lane 9) leg muscle. In each lane, a crossreactive polypeptide with evident. (c) Stained gel of mouse cerebellum (lane 1) and mouse erythrocytes crossreactivity of a 240,000 dalton polypeptide in the cerebellum. Under the same a spectrin is barely detectable, but is evident upon longer autoradiographic exposure

immunoautoradiographic labeling of the stacking gels evident in Figures 3 and 4 has not been determined, but may be a function of the quantity of crosslinked or otherwise poorly solubilized antigen in the sample. Examination of mouse cerebellum by immunoautoradiography shows the presence of a 240,000 dalton polypeptide that crossreacts with this antiserum (Figures 4c and 4d; lane 1). Comparison of the crossreactivity of mouse cerebellum with mouse erythrocytes reveals that the latter (Figures 4c and 4d; lane 2) is much weaker. Clear visualization of a 240,000 dalton band in the mouse erythrocyte lane requires an exposure approximately four times as long as that for cerebellum (not shown; compare with Figure 3). Chicken cerebellum was further examined for crossreactivity by two-dimensional immunoautoradiography. The Coomassie-blue-stained gel of whole cerebellum and the resulting immunoautoradiogram are shown in Figures 5a and 5b. A band that appears to have similar electrophoretic mobility and isoelectric point as erythrocyte a spectrin (compare with Figure 2) labels specifically with the anti-a-spectrin antiserum. No other proteins are seen to crossreact. Similar results are obtained when mouse cerebellum is used (not shown). On two-dimensional gels of chicken skeletal muscle and mouse kidney, a band in the position of chicken erythrocyte a spectrin is barely detectable by Coomassie blue staining (shown for mouse kidney in Figure 5~). This band labels specifically with the antiserum, as shown in Figure 5d. Since mouse erythrocyte spectrin is not detectably labeled on similar

and Erythrocytes erythrocytes. (Lanes 1 and 10) Whole erythrocyte controls for cord: (lane 6) liver; (lane 7) gizzard smooth muscle; (lane 6) the same electrophoretic mobility as erythrocyte (II spectrin is (lane 2). and corresponding autoradiogram (d), showing exposure conditions as in (b), labeling of mouse erythrocyte (compare with human erythrocytes in Figure 3).

gels under these autoradiographic exposure conditions (see Figure 4d), the immunolabeling of the mouse tissue samples cannot be attributed to contaminating erythrocytes. Mouse erythrocyte a spectrin has a slightly more acidic isoelectric point than the mouse nonerythroid immunoreactive polypeptide, when subjected to electrophoresis on similar gels (not shown). This slight difference may be the basis of the observed quantitative difference in immunoreactivity. lmmunofluorescence Indirect immunofluorescence was used to examine the distribution of a spectrin in erythroid and nonerythroid cells. The circulating erythroid cell population of a 5-day chicken embryo consists of nearly equal numbers of mature and immature primitive series erythrocytes (Bruns and Ingram, 1973). Figures 6a and 6b show the immunofluorescence pattern of such a cell population stained with the anti-a-spectrin antiserum. The large, immature cells are stained much less intensely than the smaller, more mature cells. Adult chicken erythrocytes show strong staining (Figures 6c and 6d). Patches of plasma membrane left attached to coverslips after sonication of adherent erythrocytes (Granger et al., 1982; see below) also show strong staining (Figures 6e and 6f). Staining with the preimmune serum gives negligible fluorescence. Incubation of the antiserum with purified chicken a spectrin (eluted from preparative slab gels) abolishes most of the fluorescence, giving levels comparable to that of the preimmune serum; no diminution

Spectrin 825

in Nonerythroid

Figure

5. Two-Dimensional

Cells

lmmunoautoradiographic

Detection

of (Y Spectrin

in Chicken

Cerebellum

and Mouse

Kidney

(a) Stained gel of whole chicken cerebellum: (b) corresponding autoradiogram showing polypeptide (designated by arrow in a) that labels specifically with the cr-spectrin antiserum. Similar results were obtained with mouse cerebellum. (c) Gel of whole mouse kidney; arrow points to a polypeptide barely detectable by Coomassie blue staining that specifically labels with the antiserum, as shown by the immunoautoradiogram in (d). The immunoreactive polypeptide in the chicken (and mouse) cerebellum and in the mouse kidney has an isoelectric point and electrophoretic mobility similar to that of chicken erythrocyte a spectrin (compare with Figures 2 and 4). (A) actin; (V) vimantin; (T) a and p tubulin.

of staining occurs after parallel incubation of the antiserum with vimentin purified in the same way, showing that the spectrin adsorption is specific (data not shown). Several lines of immunofluorescence evidence have suggested that the antiserum is labeling a cytoskeletal component on the inner surface of the plasma membrane. First, extraction of the cells with Triton X-l 00 before fixation does not alter the intensity or distribution of the immunofluorescence as compared to whole cells or ghosts. If whole cells or resealed ghosts, either fixed or unfixed, are incubated with the antise-‘ rum, there is no staining; staining requires permeabil-

ization of the plasma membrane, with either detergent or organic solvents, demonstrating that the antigen is not exposed on the outer surface of the cell. Finally, coverslips coated with adherent patches of plasma membrane, from which the rest of the erythrocyte has been removed by sonication, label intensely with the antiserum (Figure 6e). In conjunction with electron micrographs that show the cytoplasmic surfaces of these membrane patches exposed (Granger et al., 1982), this result demonstrates that the antigen is localized to the inner surface of the membrane. This is consistent with the immunoelectron microscopic localization of spectrin on the inner side of the mam-

Cell

828

Figure 6. lmmunofluorescence Spectrin

of Chicken

Erythrocytes

with Anti-u-

Fluorescence (a, c. e) and corresponding phase contrast (b. d. f) micrographs. (a) Erythrocytes from a Way embryo. Large, immature cells (arrow) stain less intensely than the smaller, more mature cells. (c) Adult chicken erythrocytes showing bright, uniform labeling of their plasma membranes. (e) Isolated patches of plasma membrane produced by sonication of adult chicken erythrocytes adhering to glass coverslips (see Granger et al., 1982) also react with the antiserum. Nucleated cells (nuclei visible in 0 have an overlying layer of plasma membrane in addition to the patch of membrane attached to the coverslip; this results in more intense labeling than in the single layers of membrane. Bars = 20 pm.

malian erythrocyte plasma membrane (Nicolson et al., 1971). Frozen sections of various tissues were examined by immunofluorescence to study the nonerythroid distribution of the crossreactive antigen. Figure 7 shows some of the staining patterns given by this antiserum in chicken tissues. Positive staining is seen in nerve tissue in both the central and peripheral systems. In cross sections of sciatic nerve and spinal cord (Figures 7a, 7b and 7c), the positive staining is at or near the axolemma. Faint staining is frequently seen at the periphery of the Schwann cell, but the myelin sheath is negative (Figure 7a). Intense staining of many cell

bodies is also evident. All epithelial tissues thus far examined show fluorescence at or near the plasma membrane. The lumenal, basal and lateral regions of the plasma membrane are labeled in epithelial cells of the gastrointestinal tract; this is evident in sections both perpendicular and parallel to the epithelial sheet in the small intestine (Figure 7d) and the proventriculus (Figure 7e). In tracheal epithelium, the cilia are negative (Figures 79, 7h and 7i). Since the plasma membrane between rows of cilia is positive, a pattern of alternating positive and negative regions results cells are viewed en face (Figure 79). when these Cortical cells of the lens were found to be positive as well (Figure 70, and the crossreactive antigen appears to be associated with the plasma membrane; this pattern is similar to that recently described for actin (Kibbelar et al., 1980). In striated muscle, the plasma membrane stains, but not uniformly; rather, intersecting horizontal and longitudinal bands of fluorescence can be visualized (Figure 71). The circularly disposed bands have the same periodicity as the sarcomeres, and the longitudinal bands seem to correspond to intermyofibrillar spaces. Cross sections of muscle fibers show no cytoplasmic staining, but show positive staining of the sarcolemma and of capillaries and nerves between the fibers (Figures 7j and 7k). Differentiating skeletal muscle myotubes grown in tissue culture exhibit labeling of a sarcolemmal network (Figure 7n) that is not as regular as in adult muscle. The preimmune serum gives negligible staining of these tissues. Smooth muscle was completely negative in all organs examined. Figures 70 and 7s depict arteries in which the smooth muscle of the media is negative; the endothelial cells are positive: however, as are erythrocytes inside the artery. Smooth muscle layers of the gastrointestinal tract are negative, but invading connective tissue cells, nerves and vascular elements are positive (Figures 7q and 7r); these positive elements are probably responsible for the faint positive band in the autoradiogram of whole gizzard tissue seen in Figure 4a. Other cells, such as cultured chicken embryo fibroblasts and mature sperm (Figure 7t), appear to stain weakly or not at all. The mouse tissues examined appear to have a staining distribution and intensity comparable to that seen in avian tissues. Figure 8c is a section through mouse small intestine; the epithelium stains positively with this antiserum, whereas the longitudinal and circular layers of smooth muscle are negative. Positive cords of vascular and nervous elements can be seen interspersed among the smooth muscle cells. However, mature mouse erythrocytes (Figure 8b) stain significantly less intensely than chicken erythrocytes (Figure 8a) or even mouse small intestine (Figure 8~). Similarly, human erythrocytes stain very weakly (not shown). This is consistent with the weak crossreactivity of human and mouse erythrocyte (Y spectrin seen with immunoautoradiography (Figures 3 and 4).

Spectrin 827

in Nonerythroid

Cells

The intensity of the immunofluorescence of chicken erythrocytes, cerebrum and small intestine, as well as mouse small intestine, is reduced to the level of the preimmune serum when the antiserum is preadsorbed with the putative LI spectrin eluted from two-dimensional gels of chicken cerebellum (as in Figure 5a). Parallel adsorption with vimentin from the same gels did not diminish the intensity of fluorescence, showing that the results obtained with this adsorption protocol were not due to technical artifacts (data not 8hOWtQ. Discussion Avian erythrocyte plasma membranes possess a protein that, by several biochemical parameters, appears to be analogous to mammalian erythrocyte spectrin. The avian protein is similarly composed of two nonidentical high molecular weight polypeptides; one has an electrophoretic mobility similar to that of human (Y spectrin, the other has a mobility slightly greater than human B spectrin (/3 spectrin also differs between closely related avian species [Granger et al., 19821, and both (Yand /? spectrin vary slightly among various mammals in our gel system [unpublished observations]). The appearance of those polypeptides at an early stage of erythropoiesis (Ghan, 1977) is also a characteristic of mammalian spectrin (Koch et al., 1975; Chang et al., 1976; Geiduschek and Singer, 19791, and is supported by the immunofluorescence data on avian erythrocytes in this paper (Figure 6a). Mammalian spectrin can be selectively solubilized from erythrocyte plasma membranes by treatment with alkaline solutions (Steck and Yu, 19731, low ionic strength solutions containing divalent cation chelators (Marchesi and Steers, 1968; Fairbanks et al., 1971) and high concentrations of chaotropic agents such as guanidine-HCI (Steck, 1972a; Maddy and Kelly, 1971) and urea (Juliano and Rothstein, 1971). We have shown that the corresponding chicken protein has similar solubility properties. A notable exception is the extent to which these polypeptides are released from the membranes in low ionic strength solutions. Their extractability from chicken erythrocytes is low in comparison to that from most mammalian erythrocytes (see also Watts and Wheeler, 1980), but similar to that from the elliptical erythrocytes of the Camellidae (Ralston, 1975; Smith et al., 1980). However, experimental manipulations may also be relevant (see Lux et al., 1978) and difficult to control for when comparing nucleated avian erythrocytes and anucleate mammalian erythrocytes. The spectrin network of mammalian erythrocytes remains insoluble in solutions of Triton X-l 00 (Vu et al., 1973); a similar phenomenon is observed in avian erythrocytes. Mammalian spectrin can be crosslinked in situ by oxidation of thiols (Steck, 1972b; Haest et al., 1977, 1981); we show the same to be true for the avian counterpart. All these observations suggest that

the avian protein can be regarded as spectrin, but is not identical with mammalian erythrocyte spectrin (see also below). Both mammalian and avian cu spectrin subunits focus in the same acidic isoelectric range, whereas the p spectrin subunits do not focus discretely in the system used in this study (data not shown for mammals; compare with Harell and Morrison, 1979). The ability of chicken LYspectrin to migrate as a relatively compact band on two-dimensional gels allowed its isolation in pure form, free of other polypeptides with similar molecular weights (for example, synemin; Granger et al., 1982), solubility properties or charge characteristics. When the polypeptide purified in this manner was used as an immunogen, it elicited an antiserum that reacts exclusively with (11spectrin, as shown by one- and two-dimensional immunoautoradiography. In immunofluorescence, this antiserum reacts with erythrocytes, but also has revealed the presence of a crossreactive component associated with the plasma membranes of a variety of avian cell types, including neurons, skeletal and cardiac muscle cells; epithelial cells, endothelial cell8 and lens cells. The crossreactive polypeptide in these tissues has been shown by one- and two-dimensional immunoautoradiography to be highly homologous to its erythrocyte counterpart. It is not a trace component in most nonerythroid cells, but is present in substantial quantities (for example, see cerebellum, Figure 5). These results demonstrate the widespread distribution in chickens of a protein similar to or identical with erythrocyte (Y spectrin. The anti-o-spectrin antiserum crossreacts with the same subset of cell types in mammalian tissues as it does in avian tissues, supporting the proposition of a widespread, nonerythroid distribution of (Y spectrin, as well as indicating some degree of antigenic conservation of the polypeptide among different vertebrate classes. The crossreactivity of this antiserum with mammalian erythrocyte (Y spectrin also strengthens the identification of the avian protein as a nonmammalian form of spectrin. The slight difference in the isoelectric points of mammalian erythrocyte and nonerythroid spectrin is one indication of their nonidentity; however, no electrophoretic or immunologic differences among the spectrins from mou8e and chicken nonerythroid cells and chicken erythrocytes were detected in this study, although subtle differences seem likely to exist. The antigenic heterogeneity of avian spectrins has not been investigated. Though reaction of this antiserum with mouse erythrocytes is considerably weaker than with chicken erythrocytes or other chicken or mouse tissues, we have observed considerable variation in the intensity of immunofluorescence staining among different mammalian erythrocytes. For example, erythrocytes from dogs and sheep appear to stain significantly more intensely than erythrocytes from humans, baboons, goats and mice (unpublished observations);

Cell 828

Figure

7. Antia-Spectrin

lmmunofluorescence

of Frozen

Sections

of Chicken

Tissues

Fluorescence micrographs (a, c. d, e. f, g, h, j, I, n, 0, q, s, t) and phase contrast micrographs (b, i, k, m, p, r, u) of spinal cord (a and b; arrows point to periphery of myelin sheath), sciatic nerve (c), epithelia of adjacent villi (lumen left of center) in the small intestine (d), epithelium of the proventriculus (e), epithelial cells of the lens (0, different views of the tracheal epithelium (g. h. i), striated muscle associated with the trachea (j, k), posterior latissimus dorsi muscle (I, m; photographed in different planes of focus to show relationship of sarcolemmal staining to underlying myofibrils), a cultured chicken myotube (n). an artery of the heart (0. p), gizzard smooth muscle tissue (q, r), periphery of the spinal cord with an associated artery (s) and sperm (t. u). See text for descriptions. Bars = 20 pm (10 Am in I).

the weak crossreactivity of mouse and human erythrocytes is evident by both immunofluorescence and immunoautoradiography. These results are in accordante with the observations of Tillack et al. (1970), who showed that human erythrocyte spectrin shares antigenic determinants with erythrocyte spectrin of other mammalian species, but that it also contains unique antigenic determinants. Similarly, peptide mapping of several mammalian spectrins has revealed the presence of both common and different peptides, suggesting that mammalian spectrins are homologous but not identical molecules (Tillack et al., 1970).

These results, together with the immunological results presented here, suggest that spectrins from avian and mammalian cells exhibit both homologous and nonhomologous domains. The failure of previous studies to detect spectrin in nonerythroid cells (Painter et al., 1975; ljiller and Weber, 1977) may have been because these antisera were elicited in mammals against native human erythrocyte spectrin, which might have resulted in specificity for antigenic determinants unique to this form of the protein. Furthermore, the cells examined were cell lines in culture, lymphocytes and platelets, all of which

Spectrin 829

in Nonerythroid

Cells

may be low in spectrin. However, a spectrin-like protein has been detected in cultured cell lines as well as in embryonic chicken cardiac myocytes (Goodman et al., 1981). The common belief that spectrin is found only in erythrocytes (see reviews referenced in Introduction) is therefore based on limited and misleading evidence. The realization that the distribution of spectrin may be widespread suggests that the functions of spectrin hypothesized for erythrocytes may apply to

other cell types as well. Since not all nonerythroid cell types crossreact with the cu-spectrin antiserum, the expression of (Y spectrin in nonerythroid cells may be related to certain physiological or morphological characteristics of their plasma membranes. Of the cells examined thus far, those that do not stain or stain weakly with this antiserum are smooth muscle cells, cultured chicken embryo fibroblasts and sperm; tracheal cilia and myelin also appear negative. Perhaps

Ce!l 830

E

Figure 8. Comparison Patterns and Intensities

that it was not a nonerythroid form of spectrin. However, the observation that spectrin from erythrocytes of different mammalian species can be electrophoretically and antigenically distinct (Tillack et al., 1970, and our unpublished observations), coupled with the different immunoreactivities of mammalian erythrocyte and nonerythroid spectrins (this paper) and the observation that a spectrin can split into a doublet in different SDS-polyacrylamide gel electrophoresis systems (unpublished observations), supports the possibility that a spectrin and fodrin are highly related or similar polypeptides. Preliminary comparisons of these two molecules indicate that this is the case. Experlmental of Chicken and Mouse with AntiaSpectrin

Procedures

lmmunofluorescence

Fluorescence micrographs of erythrocytes from chicken (a) and mouse fb), prepared under the same conditions and photographed and printed with the same exposure times. (c) Frozen section of mouse small intestine showing staining patterns of epithelium (upper right) and smooth muscle that are similar to those seen in chicken (Figure 7). Bars = 10 sm.

the presence of spectrin is a function of how dynamic a cell’s form is; plasma membranes of relatively static cells might rely on a spectrin-based cytoskeletal scaffold for maintenance of their overall shape or rigidity, whereas this would be a hindrance to more mobile or mutable cells (see Hardy and Schrier, 1978; Greenquist et al., 1978; Tokuyasu et al., 1979). Spectrin might also play a role in controlling the mobility of membrane proteins or receptors (Nicolson and Painter, 1973; Elgsaeter and Branton, 1974), or in mediating actin filament-membrane interactions. Actin filaments interact with the plasma membrane in a variety of cell types, but the molecular details of such interactions (other than in mammalian erythrocytes) have not been elucidated. The presence of a spectrin (this study) and ankyrin (Bennett, 1979) in certain nonerythroid cells may provide a more generalized mechanism for the interaction of actin filaments with membrane proteins. Nevertheless, the apparent absence of a spectrin from certain cell types implies that other mechanisms must also be operative. It will be of interest to isolate and characterize spectrin from nonerythroid cells, and to determine whether the distribution of p spectrin, which is the ankyrin-binding subunit (Litman et al., 1980; Calvert et al., 1980; Morrow et al., 19801, and which exhibits considerable variation in electrophoretic mobility among different species, parallels the widespread distribution of (Y spectrin. Many similarities are evident in the cell and tissue immunofluorescence patterns of a spectrin (this paper) and fodrin, a recently characterized, axonally transported, high molecular weight pair of polypeptides (Levine and Willard, 1981). Guinea pig fodrin was found to be electrophoretically and antigenically distinct from human erythrocyte spectrin, suggesting

Pdyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis was based on the diecontinuous Tris-glycine system of Laemmli (1970). as modified and described previously (Hubbard and Lazarides. 1979). Gels contained 12.5% acrylamide and 0.1% N,N’-methylene bisacrylamide. Sample buffer consisted of 1% SDS, 125 mM Tris-Cl (pH 8.8). 10% glycerol, 1% P-mercaptoethanol. 1 mM EDTA and 0.004% bromophenol blue. Two-dimensional gel electrophoresis involved isoelectric focusing followed by SDS-polyacrylamide eel electrophoresis. according to the method of O’Farrell (I 975), as modified and described (Hubbard and Lazarides. 1979). except that Nonidet-P40 was omitted from all gels and samples (Granger et al., 1982), and no stacking gel (other than that provided by the equilibrated isoelectric focusing gel and agarose embedding matrix) was used in any of the second-dimension gels except for the one in Figure 9. Erythrocyte Collection Erythrocytes were harvested and processed essentially as described (Granger et al., 1982). Blood was collected from embryonic, adult or 2-3 week old chickens in 0.01% heparin, 155 mM choline chloride and 5 mM HEPES (pH 7.4). Erythrocytes were washed and purified by three to five centrifugations in the same buffer (without heparin), and the supernatant and buffy coat were removed each time by aspiration. Extraction Propsrties of Chicken Erythrocyte Spectrln Spectrin extractability was studied under a variety of conditions with isolated plasma membranes prepared by hypotonic lysis and mechanical enucleation of chicken erythrocytes in 5 mM MgCI,. 5 mM NaNa. 1 mM EGTA, 1 mM DTT. 0.5 mM phenylmethyl sulfonyl fluoride (PMSF). 5 mM Tris-Cl (pH 7.5) at 0°C (Granger et al., 1982). Membranes were stored on ice or in liquid nitrogen as a suspension in this solution (storage had no obvious effect on spectrin extractability), and were washed with 2 mM EDTA, 10 mM Tris-Cl (pH 7.4) at O’C, prior to further use in order to rmove M&, which interfered with the extractions. Aliquots of washed, packed membranes were suspended in approximately 500 volumes of distilled water, or 0.1 M NaOH. or 1 M KCI, 1% Triton X-l 00,l mM DTT. 2 mM EDTA. 10 mM Tris-Cl (pH 7.4). or 2 mM diamide for 2 mM Na&O& 2 mM EDTA, 10 mM Tris-Cl (pH 7.4). or 8 M urea, 1 mM EDTA, 0.1 M Tris-Cl (pH 7.2) with or without 20 mM DTT. Incubations were carried out on ice for 1 hr unless noted otherwise. After centrifugation for 10 min at 20.000 x Q. pellets were either boiled in 50 volumes of SOS sample buffer (pellets containing Triton X-l 00 or diamide or Na2S40e were first washed by resuspension and repelleting in 2 mM EDTA, 10 mM Tris-Cl [pH 7.41). or subsequently extracted with another of these solutions (see Results). Supernatants were lyophilized directly or were dialyzed against distilled water and then lyophilized. before being boiled in SDS sample buffer. Each extract and residue appearing on the electropherogram of Figure 1 is derived from the same quantity of membranes (approximately 2 Al packed membranes), except for the human erythrocyte ghost membranes (I pl). The latter

Spectrin 831

in Nonerythroid

Cells

antiserum and removal of radioiodinated protein A. Exposure times with (+) or without (-) an intensifying screen were as follows: Figure 2b (+) 14 hr; Figure 3b. lanes 1 and 2 (+) 8 days; lanes 3-5 (+) 40 hr; Figure 4b (-) 48 hr; Figure 4d (+) 60 hr: Figure 5b (+) 30 hr; Figure 56 (+) 30 hr.

i

Figure

9. Typical

Gel Used for Final Step of a-Spectrin

Purificahon

This and similar twodimensional gels of cytoskeletons of adult chicken erythrocytes prepared by extraction with Triton X-l 00 were used for obtaining pure a spectrin. The band marked u, bounded by the arrows, was excised and used as antigen. (A) actin; (V) vimentin; (8) /3 spectrin. were prepared from outdated bank blood in 5 mM sodium phosphate (pti 8.0). 0.1 mM DTT. 2 mM EDTA and 10 PM PMSF (a gift from J. Falke). Preparation of Antigen and Immunization Chicken erythrocytes were lysed by suspension in ice-cold 10 mM Tris-Cl (pH 7.5). 0.5% Triton X-l 00. 130 mM NaCI, 5 mM KCI. 5 mM NaN3. 5 mM MgCk, 1 mM EGTA (buffer TM); 10 mM p-tosyl-L-arginine methyl ester (TAME) was added before use. After approximately 1 min, the resultant cytoskeletons were pelleted by centrifugation and then washed one to three times in ice-cold buffer TM without Triton X-l 00. The final pellet was prepared for two-dimensional gel electrophoresis by solubiliration in 10 M urea, 1% P-mercaptoethanol. The major isoelectric variant of u spectrin was cut from Coomassie-bluestained gels (as in Figure 9) and neutralized in 0.15 M sodium phosphate (pH 7.4). The gel slices were homogenized in a motordriven Teflon and glass homogenizer(Potter-Elvehjem) and emulsified with Freund’s complete adjuvant for immunization of a rabbit, as described previously (Granger and Laxarides, 1979); booster injections contained no adjuvant. Injections made on day 0 consisted of material from 7 gels, on day 20 from 8 gels, and on day 43 from 16 gels. Blood was collected 8 days after the second and third injections. The IgG fraction was partially purified by fractionation with ammonium sulfate at 50% saturation at 0°C. Serum from the second bleed was used in this study. It yielded a single precipitin line after double immunodiffusion with urea extracts of avian erythrocyte cytoskeletons. lmmunoautoradiography Various tissues were removed from a 12-day chickens, minced, washed extensively with ice-cold PBS (137 mM NaCI, 3 mM KCI, 2 mM KH2P0. and 8 mM Na2HP0, [pH 7.41) to remove most of the blood, extracted with 20 volumes of 100% ethanol, dried under vacuum and boiled in SDS sample buffer for one-dimensional gels or solud/ized in 10 M urea and 1% P-mercaptoethanol for isoelectric focusing. Whole chicken red cells and cytoskeletons were also extracted briefly with ethanol before solubilization. Human spectrin was obtained by stripping erythrocyte ghosts with 5 mM sodium phosphate, 0.1 mM DTT and 2 mM EDTA (pH 12) (a gift from J. Falke). This sample was neutralized, lyophilized and dissolved in SDS sample buffer for one-dimensional gels or in 10 M urea and 1% P-mercaptoethanol for isoelectric focusing. lmmunoautoradiography (Burridge. 1978) was performed as described previously (Granger and Lazarides, 1980). except that only 1 day of washing followed removal of

lmmunoffuorescsnce For frozen sections, organs from chickens l-3 weeks old were placed in O.C.T. compound (Tissue Tek) and frozen at -20°C. Frozen sections were melted onto glass coverslips and immediately placed in a solution of 2% formaldehyde, 130 mM KCI, 16.9 mM K2HPOd, 3.1 mM KH2P04. 5 mM NaCI. 1 mM NaN3. 5 mM MgC12 and 1 mM EGTA (pH 7.5) at room temperature for 1 hr. Erythrocytes were allowed to settle onto glass coverslips that had been pretreated with Alcian blue to promote adhesion (Sommer. 1977; Granger et al., 1982). Adherent cells were hypotonically lysed and either fixed as described above or sonicated as described in Granger et al. (1982). In all cases, subsequent washes and incubations with antisera were in buffer TM. The primary antiserum was used at approximately 1:50 serum concentration. Fluoresceinconjugated goat antirabbit IgG (Miles-Yeda) was diluted 150-fold for use. Incubation periods were for 30-60 min at room temperature or 37’C. Coverslips were mounted in 90% glycerol in PBS or in Elvanol (Rodriguez and Deinhardt, 1960). and were photographed with Kodak Tri-X film with a Leitx phase/epifluorescence microscope with filter modules K and H.

lmmunoadsorption Coomassie-blue-stained bands containing approximately equal amounts of a spectrin and vimentin were excised from preparative slab gels of chicken erythrocyte ghosts and two-dimensional gels of chicken cerebellum. Each gel slice was neutralized and then homogenized in 1% SDS, 130 mM NaCI. 5 mM KCI, 10 mM NaN, and 10 mM Tris-Cl (pH 7.5). Protein was eluted from the gel homogenate by diffusion into this buffer, dialyzed against distilled water for 2 days and lyophilized. These electrophoretically pure samples of u spectrin and vimentin were resuspended in equal volumes of diluted anti-aspectrin antiserum and were incubated at room temperature for 2 hr. The mixture was centrifuged for 10 min at 100,000 X g, and the supernatant was used for immunofluorescence. Acknowledgments We thank John Ngai for his helpful comments on the manuscript, and llga Lielausis and Adriana Cortenbach for their technical assistance and cell culture work. This work was supported by grants from the National Institutes of Health, the National Science Foundation and the Muscular Dystrophy Association of America, and by a Biomedical Research Support Grant to the Division of Biology, California Institute of Technology. E. A. Ft. was also supported by a postdoctoral fellowship from the National Institutes of Health. B. L. G. was supported by a predoctoral training grant from the National Institutes of Health. E. L. is the recipient of a Research Career Development Award from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

25. 1982;

revised

April 16. 1982

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