Cell Motility and the Cytoskeleton 36:253–265 (1997) Local Accumulation of a-Spectrin-Related Protein Under Plasma Membrane During Capping and Phagocytosis in Acanthamoeba Katarzyna Kwiatkowska and Andrzej Sobota* Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Poland During capping and phagocytosis the interaction between clustered cell surface receptors and the submembraneous actin-based skeleton may be mediated by spectrin-like proteins. To test this possibility we examined the localization of an a-spectrin immunoanalogue, that had been previously identified in whole extracts of Acanthamoeba, during capping of Con A receptors and during phagocytosis of Con A-coated yeast. During capping a-spectrin and filamentous actin co-migrated with the Con A receptors and accumulated in the region of cap formation, as demonstrated by double immunofluorescence studies. Immunoelectron microscopy revealed submembraneous location of a-spectrin in cells exposed to Con A, both at the time of initial cross-linking and during accumulation of a-spectrin in the region of the cap. Phagocytosis studies showed that a-spectrin and actin filaments were concentrated around phagocytic cups that enclosed Con A-coated yeast upon internalization. The proteins also surrounded nascent phagosomes present in the vicinity of the plasma membrane but were absent at the later time point of phagosome maturation. These data demonstrate a correlation between clustering of cell surface receptors and submembraneous localization of a-spectrin, suggesting an involvement of spectrin-like proteins in mediating the interaction of receptor clusters with the actin cytoskeleton. Cell Motil. Cytoskeleton 36:253–265, 1997. r 1997 Wiley-Liss, Inc. Key words: concanavalin A receptors; receptor clustering; actin filaments; spectrin-like proteins INTRODUCTION Spectrin-like proteins are a family of actin crosslinking proteins present in various eukaryotic cells including protozoa [Goodman et al., 1981; Levine and Willard, 1981; Repasky et al., 1982; Glenney et al., 1982; Pollard, 1984; Kwiatkowska and Sobota, 1990, 1992]. Erythroid spectrin, the most extensively characterized member of the family, is a heterodimer composed of 240-kDa a- and 220-kDa b-subunits which self-associate into oligomers. In erythrocytes, spectrin binds actin filaments forming a submembraneous network and participates in linking of the network with integral membrane proteins [Byers and Branton, 1985; Ursitti et al., 1991]. The spectrin/actin scaffolding defines planar organization of the erythrocyte membrane constituents and stabilizes the membrane, r 1997 Wiley-Liss, Inc. providing the basis of the membrane skeleton structure and function [for review see Bennett, 1990]. Participation of the membrane skeleton in spatial organization of the plasma membrane in motile cells is of special interest; however, details of membrane-skeleton interactions in such dynamic systems remain obscure Contract grant sponsor: State Committee for Scientific Research; Contract grant number KBN 0082/P2/94/07; Contract grant sponsor: State Committee for Scientific Research to the Nencki Institute of Experimental Biology. *Correspondence to: Andrzej Sobota, Nencki Institute of Experimental Biology, Department of Cell Biology, 3 Pasteur Str., 02-093 Warsaw, Poland. Received 22 July 1996; accepted 22 October 1996. 254 Kwiatkowska and Sobota [Edidin et al., 1991; Edidin, 1992; Kusumi et al., 1993]. Several lines of evidence point to a correlation between clustering of cell surface receptors and their anchoring within the actin-based cytoskeleton and indicate that the skeleton promotes grouping of the receptors in the plane of the plasma membrane [Bourguignon et al., 1988; Bloch and Morrow, 1989; Apgar, 1990; Schwartz, 1992]. Capping of cell surface receptors is a model example of this type of ‘‘clustering-anchoring’’ mechanism. At the first stage of capping the receptors become clustered upon binding of external polyvalent ligands (antibodies, lectins). Clustering of the receptors triggers their association with the actin-based cytoskeleton, which in turn actively assembles the clusters into one compact structure—a cap [Bourguignon and Singer, 1977; Bourguignon and Bourguignon, 1984]. In the past two decades, the ‘‘clusteringanchoring’’ mechanism of capping has been confirmed by many cytochemical and biochemical data as well as by particle tracking studies, and is now commonly accepted [e.g., Braun et al., 1982; Bourguignon and Bourguignon, 1984; Turner and Shotton, 1987; Sheetz et al., 1989; Holifield et al., 1990; de Brabander et al., 1991]. Certain integral membrane proteins were found to interact with members of the spectrin family and their accessory proteins, suggesting that spectrin-like proteins could be involved in anchoring of the clustered receptors within the actin cytoskeleton [Bourguignon et al., 1985, 1986; Kalomiris and Bourguignon, 1988; Lokeshwar and Bourguignon, 1992]. To study this possibility we examined the localization of the a-spectrin immunoanalogue and actin filaments in Acanthamoeba cells during capping of concanavalin A (Con A) receptors as well as during phagocytosis of Con A–coated yeast particles. The nature of Con A–binding molecules in Acanthamoeba plasma membrane is complex and likely to involve glycoproteins as well as lipophosphonoglycan [Bailey and Bowers, 1981; Clarke et al., 1988]. These molecules, despite their heterogeneity, are all referred to as Con A receptors. We found that a-spectrin and actin filaments co-migrated with the lectin receptors and accumulated under the plasma membrane in the cap region. The proteins were concentrated also at early stages of the Con A–coated yeast engulfment. These results suggest that during capping and phagocytosis a relation between clustering of cell surface receptors and spectrin distribution exists which is consistent with the involvement of spectrin in the ‘‘clustering-anchoring’’ mechanism. MATERIALS AND METHODS Cell Culture Acanthamoeba castellanii, Neff strain, was grown axenically in the medium containing proteose peptone, yeast extract, and glucose, without aeration, in the dark, as described previously [Sobota et al., 1984]. A 4-day-old culture was plated on precleaned coverslips, placed in 12-well Costar dishes, and grown overnight. Coverslips were precleaned by boiling with a solution containing 5 mM EDTA and 0.2 N acetic acid, followed by extensive washing. For capping experiments, 2 3 105 cells in 1 ml of the growth medium were plated per well, giving approximately 4 3 104 cells/cm2 on the coverslip. For studies of phagocytosis a culture twice as dense was prepared. Capping of Con A Receptors Cells grown on coverslips were rinsed twice with the 100 mM NaCl/10 mM Hepes buffer, pH 7.0 (‘‘NaCl/ Hepes’’), at 20°C and once with ice-cold buffer and were submerged in the same cold buffer supplemented with 100 µg/ml Con A (80% conjugated to FITC; Sigma, St. Louis, MO) to cross-link the lectin receptors. The crosslinking was conducted for 30 min at 0°C. The unbound lectin was washed out with cold NaCl/Hepes. To induce capping of the Con A receptors, the washed samples were transferred into NaCl/Hepes prewarmed to 20°C and incubated for 30 min. Every 5 min a part of the samples was removed, fixed with 3% formaldehyde, and processed for fluorescence microscopy as described later. A similar method was applied for labeling of Con A receptors in suspended cells except that after every step of the procedure the cells were pelleted by centrifugation (1,000g, 2 min). In control experiments, as well as in samples for electron microscopy the cells were treated with non-conjugated Con A (100 µg/ml). Phagocytosis To induce phagocytosis, cells attached to coverslips were exposed to Con A–coated, lipid-extracted bakers yeast. Yeast were coated with the lectin by incubation with 100 µg/ml Con A in 100 mM NaCl and 0.01 mM MgCl2, for 1 h at room temperature, with stirring. Con A–coated yeast were washed twice and resuspended in 100 mM NaCl/0.01 mM MnCl2 at a concentration of 2 3 109/ml. For the experiments, amoeba cultures grown in 12-well dishes were washed twice with the phagocytosis buffer (100 mM NaCl, 2 mM MgCl2, 20 mM Hepes, pH 7.0) at room temperature, once with the buffer at 0°C, and placed on ice in 0.5 ml of the buffer per well. Yeast (1.5 3 107 ) were added to each well at a ratio of about 20 yeast per amoeba. The samples were incubated for 10 min at 0°C to promote binding of the yeast to the cells. Surface unbound yeast were washed out with cold buffer. The dishes were next rinsed briefly with the buffer warmed to 28°C and transferred to water bath at 28°C to Spectrin in Capping and Phagocytosis start phagocytosis. After 1 and 3 min the cells were fixed with 3% formaldehyde. Immunofluorescence Microscopy Con A receptors were cross-linked on the surface of living Acanthamoeba cells using FITC-conjugated or non-conjugated lectin according to the procedure described above. The cells labeled with Con A as well as cells collected after phagocytosis were processed for fluorescence microscopy to determine the localization of the a-spectrin immunoanalogue and filamentous actin. For this purpose, the cells were fixed at various stages of capping and phagocytosis using 3% formaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgCl2, pH 6.9; protease inhibitors: 2 mM PMSF, 100 µg/ml leupeptin, 10 µg/ml pepstain A, 2 µg/ml aprotinin [Sigma]). After 30 min of fixation (room temperature) the cells were quenched with 50 mM NH4Cl and permeabilized with 0.2% Triton X-100 in PHEM buffer, followed by acetone extraction as described previously [Kwiatkowska and Sobota, 1990]. The permeabilized cells were incubated for 30 min with 3% goat serum in PBS to block nonspecific binding of antibodies. After blocking, affinity-purified anti-a-spectrin antibody, characterized elsewhere [Kwiatkowska and Sobota, 1992], was applied for 1 h at 1 µg/ml in PBS containing 1% goat serum. One hour later the cells were extensively washed with PBS and exposed for another hour to goat F(ab) 2 anti-rabbit IgG conjugated with Texas Red (1:150 in PBS with 1% goat serum) (Jackson ImmunoResearch, West Grove, PA). The samples were washed again and mounted in Mowiol containing 2.5% DABCO. Filamentous actin was stained in permeabilized cells using 0.5 µg/ml phalloidin-TRITC or phalloidin-FITC (Sigma). The samples were examined under a Nikon microscope and photographed using Kodak T-MAX 400 Asa film. Control experiments included: (1) omitting anti-a-spectrin antibody, (2) applying nonimmunized serum instead of the antibody, and (3) labeling of non-permeabilized cells. Electron Microscopy Suspended Acanthamoeba cells were washed three times with NaCl/Hepes buffer at 20°C and resuspended at a density of 4 3 106 cells/ml in ice-cold buffer containing 100 µg/ml Con A for cross-linking of the cell surface receptors. After 30 min the cells were washed with NaCl/Hepes (0°C) and transferred into NaCl/Hepes warmed to 20°C. At various time points of the incubation, cell samples were withdrawn and fixed with 3% formaldehyde in PHEM buffer (30 min, room temperature). The fixed cells were treated with 50 mM NH4Cl in PHEM (10 min, room temperature), permeabilized with methanol (5 255 min, 220°C) and incubated in the blocking buffer containing 1% BSA in PHEM (30 min, room temperature). After each step of the procedure, the cells were washed with PHEM buffer. In addition, the cells after blocking were washed once with PBS supplemented with 1 mM CaCl2 (‘‘PBC/Ca’’) and subsequently resuspended in PBS/Ca containing 0.02% peroxidase, a glycosylated enzyme which binds to Con A [Straus, 1983]. One hour later the cells were washed three times with PBS/Ca and incubated for 5 min in the presence of 0.5% diaminobenzidine (DAB) and 0.02% H2O2 in 50 mM Tris, pH 7.8. The enzymatic reaction was stopped by washing the cells with PBS. In order to localize the a-spectrin immunoanalogue, the cells were incubated for 2 h in the presence of affinity-purified anti-a-spectrin (1 µg/ml) and then for 1 h with goat anti-rabbit IgG conjugated with 10 nm gold particles (1:20) (Sigma). The antibodies were diluted into PBS containing 0.2% BSA. After either incubation the cells were washed with 5 changes of PBS (10 min each). The cells were postfixed with 2.5% glutaraldehyde in 50 mM cacodylate buffer (2 h, room temperature) followed by 1% OsO4 in the same buffer (1 h, room temperature). After dehydration in graded ethanol series and in propylene oxide the samples were embedded in Epon 812. Ultrathin sections of the samples were stained with uranyl acetate and lead citrate and examined under a JEM 100B electron microscope. In control samples, the same procedure was carried out except that the incubations with peroxidase and anti-a-spectrin antibody were omitted. Immunoblotting To identify the immunoanalogue of a-spectrin in Acanthamoeba cells pretreated with Con A, the cells were collected by centrifugation, washed with 100 mM NaCl, and resuspended in ice-cold NaCl/Hepes buffer containing 100 µg/ml Con A. After 20 min, the cells were shifted to 20°C and incubated for an additional 15 min. Unbound Con A was removed by washing the cells twice with NaCl/Hepes. The pelleted cells were resuspended in ice cold buffer composed of 50 mM NaCl, 10 mM EGTA, 20 mM Tris, pH 7.5, 2 mM PMSF, 100 µg/ml leupeptin, 10 µg/ml pepstain A, 2 µg/ml aprotinin (Sigma), then disrupted by passage through a 26G needle at least 20 times and centrifuged for 5 min at 5,000g. The clarified supernatant was collected for protein determination and electrophoresis. The whole homogenization procedure was carried out at 0°C. As a positive control for the detection of a-spectrin, a total cell lysate was prepared from rat brain. Rat brain was cut into small pieces and solubilized in a boiling buffer consisting of 3% SDS, 5 mM EDTA, 5 mM EGTA, 20 mM Tris, pH 7.6, for 5 min. The lysed tissue was 256 Kwiatkowska and Sobota additionally disrupted by passage through a 26G needle, and centrifuged as described above. The protein concentration of the lysates was determined by the micro BCA method (Pierce, Rockford, IL). The proteins were separated on 8% SDS-polyacrylamide gels [Laemmli, 1970] and transferred onto nitrocellulose sheets [Towbin et al., 1979] (Bio-Rad, Richmond, CA). The blots were developed according to a routine procedures including overnight blocking in 5% non-fat milk solution in TBS/0.05% Tween-20 and 2 h incubation with anti-a-spectrin (1 µg/ml) followed by 2 h with anti-rabbit antibodies conjugated with peroxidase (1:2,000) (Boehringer Mannheim, Indianapolis, IN). The antibodies were prepared in blocking buffer. Immunoreactive bands were visualized with the Enhanced Chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL). RESULTS Redistribution of a-Spectrin and Actin During Capping of Con A Receptors To cross-link cell surface receptors of Con A in Acanthamoeba, the cells grown on coverslips were incubated with FITC-conjugated lectin at 0°C. On exposure to low temperature, the cells rounded up and formed numerous thin retraction fibers at the edges (Figs. 1C, 2C). At the moment of initial cross-linking, the Con A receptors were diffusively distributed over the cell surface (Figs. 1A, 2A). The cell periphery usually displayed stronger fluorescence of Con A-FITC, presumably due to membrane folding around the retracted cell body (Figs. 1A, 2A). Distinct staining of the a-spectrin immunoanalogue colocalized with staining of Con A-FITC/receptor complexes apparent at the cell periphery, suggesting submembraneous localization of the antigen (Fig. 1B). Filamentous actin was uniformly distributed throughout the cells as revealed by phalloidin staining (Fig. 2B). To induce capping of Con A receptors the cells were shifted to 20°C. During the first 15–20 min of incubation, Con A receptors were still dispersed over the whole cell surface occasionally forming small aggregates which colocalized with a-spectrin and actin (not shown). In this time the retraction fibers diminished, the cells polarized and started to move (Fig. 1D–F). Cell migration was accompanied by an accumulation of cross-linked Con A receptors in the posterior part of the crawling cells (Fig. 1D, arrow). Within 5–10 min the receptors were cleared from the cell surface and assembled in the uropod into a single compact aggregate—a cap (Figs. 1G, 2D, arrows). The caps were easily distinguished under phase contrast (Figs. 1I, 2F, arrows). The distribution of the a-spectrin immunoanalogue and actin filaments followed the redis- tribution of Con A receptors (compare Fig. 1D,G and 1E,H; Fig. 2D and E). Figures 1D and E show a-spectrin colocalized with the receptors gradually collecting in the posterior region of the cell. The a-spectrin immunoanalogue and actin seen in Figures 1H and 2E, respectively, are accumulated under the plasma membrane in those places where caps of Con A receptors have finally formed. On the other hand, a-spectrin and actin filaments also concentrated at the leading edge, particularly in acanthopodia, of the moving cells (Fig. 1E,H,K, and Fig. 2E,H, arrowheads). Spectrin distinctly delineated the anterior edge of the cells (Fig. 1E,H,K, arrowheads). In contrast, complexes of Con A-FITC/receptors were no longer present at the leading edge of the cells (Fig. 1D,G,J, and Fig. 2D,G). Shortly after their formation, caps of Con A receptors disintegrated and were internalized into large vacuoles detectable in the uropod (Fig. 1J–L and Fig. 2G–I, arrows). Both the a-spectrin immunoanalogue and filamentous actin were seen in the vicinity of these vacuoles (Fig. 1J,K, and Fig. 2G,H, arrows). Concomitantly with internalization of Con A/receptor complexes, FITC fluorescence appeared around the nuclear envelope (Fig. 1J). Ultrastructural Studies of a-Spectrin Redistribution During Capping Ultrastructural localization of the a-spectrin immunoanalogue and Con A receptors was investigated in Acanthamoeba cells grown in suspension. Preliminary immunofluorescence studies showed that in suspended cells, similarly as in adherent ones, a-spectrin and filamentous actin co-cap with Con A receptors (not shown). Fig. 1. Redistribution of the a-spectrin immunoanalogue during capping of Con A receptors. Con A receptors were cross-linked on the Acanthamoeba surface upon exposure to Con A-FITC at 0°C and induced to accumulate into a cap by subsequent incubation of the cells at 20°C. The a-spectrin immunoanalogue was localized in the cells with anti-a-spectrin/anti-rabbit-Texas Red antibodies. A,D,G,J: Distribution of Con A receptors; B,E,H,K: staining of a-spectrin; C,F,I,L: phase contrast images of the cells. In A–C a cell fixed after crosslinking of the Con A receptors (0°C) is shown. The receptors and a-spectrin display a homogeneous distribution over the plasma membrane; however, their fluorescence labeling is more pronounced at the cell edges. D–L show cells incubated at 20°C for 20 min (D–F), 25 min (G–I), 30 min (J–L). Accumulation of Con A receptors into a cap (arrows in D,G) was accompanied by accumulation of a-spectrin (arrows in E,H). Arrow in I points to the cap visible under phase contrast. Upon the cap disintegration (J–L) a-spectrin was detected around vacuoles enclosing the Con A/receptor complexes (arrows in J,K). Note the presence of a-spectrin at the leading edge of moving cells (arrowheads in E,H,K). Bar 5 10 µm. Spectrin in Capping and Phagocytosis Figure 1. 257 258 Kwiatkowska and Sobota Fig. 2. Actin filament distribution during capping of Con A receptors. The cells were exposed to Con A-FITC to induce redistribution of the lectin receptors (A,D,G), and double-labeled with phalloidin-TRITC to visualize actin filaments (B,E,H). C,F,I show phase contrast images of the cells. A–C: A cell after cross-linking of Con A receptors (0°C). D–I: Cells shifted to 20°C for 25 min (D–F), and 30 min (G–I). Actin filaments co-capped with Con A receptors (arrows in D,E) and were present in the vicinity of vacuoles into which Con A/receptor complexes were internalized (arrows in G,H). The fully assembled cap could be distinguished under phase contrast (arrow in F). Actin filaments were concentrated also at the leading edge of moving cells (arrowheads in E,H). Bar 5 10 µm. To visualize complexes of Con A/receptors, the lectin-treated cells were additionally incubated with peroxidase, a Con A-binding enzyme, and stained for its activity with DAB. Upon exposure to osmium tetroxide, the reaction product formed electron dense precipitates. In the cells fixed immediately after Con A binding (0°C) a delicate surface labeling was only occasionally detectable, probably due to dispersed localization of the receptors (see also Figs. 1A, 2A). In these cells, a-spectrin was remarkably homogeneously distributed over the cytoplas- Spectrin in Capping and Phagocytosis mic surface of the plasma membrane (Fig. 3A). Most gold particles identifying the a-spectrin antigen were seen at the plasma membrane and only a few remained scattered in the cortical cytoplasm (Fig. 3A). During induced receptor redistribution (20°C), the DAB-based deposits clearly decorated a distinct region of the cell surface, reflecting concentration of the receptors in the cap (Fig. 3B). Magnification of the cap region revealed the presence of multilayered systems of membranes (Fig. 3C,D). High amounts of a-spectrin were associated with these membranes and accumulated in the adjoining cytoplasm (Fig. 3C,D, arrowheads). The a-spectrin immunoanalogue was found also along borders of large vacuoles localized beneath the cap, into which complexes of Con A/receptors were internalized (Fig. 3C,D, circles; compare Fig. 1J,K, arrows). In contrast, there was little staining of a-spectrin outside the cap region (Fig. 3E). Traces of the antigen could be detected at the plasma membrane away from the cap. The membranes of neighbouring vacuoles were devoid of the label (Fig. 3E). Accumulation of a-Spectrin and Actin at Early Stages of Phagocytosis Con A-coated yeast were avidly internalized by Acanthamoeba. Phagocytosis was coincident with arrested cell movement and often was followed by detachment of the cells from the substratum (Fig. 4, note the non-polarized profile of the cells). As revealed by phalloidin staining, phagocytic cups embracing the yeast were surrounded by highly concentrated actin filaments (Fig. 4B, arrow). The a-spectrin immunoanalogue was also present at the phagocytic cups and appeared to underlie the cytoplasmic surface of the invaginated membrane (Fig. 4A, arrow). While the engulfment proceeded, a-spectrin and actin were visible around nascent phagosomes (Fig. 4C–F, arrows). Double-labeling demonstrated colocalization of the proteins at the phagosome membrane and in the neighbouring cytoplasm (Fig. 4G,H, arrows). On the contrary, neither the a-spectrin immunoanalogue nor filamentous actin were detected around maturing phagosomes which enclosed the yeast displaced deeper into the cell interior (Fig. 4D–F, asterisks). Only a fraction of the total cellular a-spectrin and actin was involved in the yeast internalization. Actin filaments, in addition to being accumulated at the sites of phagocytosis, were present in the remaining parts of cell cortex (Fig. 4B,D,H); a-spectrin displayed a more diffusive pattern of staining (Fig. 4A,C,E,G). 259 Immunocytochemical Staining Was Specific for a-Spectrin Immunoanalogue To confirm the results of immunocytochemical studies, we reexamined the immunoreactivity of our anti-a-spectrin antibody [Kwiatkowska and Sobota, 1990, 1992]. ECL immunoblot analysis showed that among proteins of the whole Acanthamoeba extract, the antibody recognized specifically only one polypeptide (Fig. 5A). The polypeptide co-migrated with the 240-kDa polypeptide of rat brain homogenate, as expected for the a-spectrin immunoanalogue (Fig. 5B). Several sets of control experiments, described under Materials and Methods, confirmed that the immunocytochemical staining of Acanthamoeba with anti-aspectrin was specific and confined to the cell interior (not shown). The possible overlapping between the fluorescence generated by FITC and Texas Red conjugated to Con A and secondary antibodies, respectively, was also considered. To rule out this possibility the cells were treated with Con A non-conjugated with FITC to induce capping of the receptors and subsequently processed according to the standard immunofluorescence procedure to visualize the a-spectrin immunoanalogue. In these cells polar accumulation of a-spectrin was easily distinguished (Fig. 5C, arrow). This spectrin accumulation corresponded to the cap region which was also detectable under phase contrast (Fig. 5D, compare Fig. 3B). DISCUSSION In this report, we analyzed the localization of the a-spectrin immunoanalogue in Acanthamoeba cells during capping of Con A receptors and phagocytosis of Con A–coated yeast. We found that a-spectrin co-capped with Con A receptors and accumulated at early stages of the yeast engulfment. The local enrichment of a-spectrin was correlated with accumulation of translocated and/or newly polymerized actin filaments. Although the structure and function of spectrin-like protein in Acanthamoeba remain unclear [Pollard, 1984; Kwiatkowska and Sobota, 1990], our observations point to its actin-binding ability. We have previously established that three different patterns of a-spectrin distribution can be distinguished in non-treated Acanthamoeba cells: (1) plasma membraneassociated, (2) diffusive cytoplasmic, (3) cytoplasmic aggregates [Kwiatkowska and Sobota, 1990]. This heterogeneity stands in contrast with the almost exclusively submembraneous location of a-spectrin found in the Con A–treated cells when cross-linking of the lectin receptors occurred (Fig. 3A). In the cap region, where co-migrating receptors and a-spectrin were collected, a substantial fraction of a-spectrin was still associated with mem- 260 Kwiatkowska and Sobota Figure 3. Spectrin in Capping and Phagocytosis branes (Fig. 3C,D). These membranes, bearing surfacelabeled Con A/receptor complexes, were likely to originate from the plasma membrane [see also Suchard et al., 1988]. We assume that recruitment of spectrin to the plasma membrane was triggered by clustering of Con A receptors upon the lectin binding. A similar redistribution of spectrin occurs in T cell lines in response to activation signals including cross-linking of T cell receptors [Lee et al., 1988; Gregorio et al., 1993]. During capping, plasma membrane-associated spectrin may be involved in anchoring of clustered membrane receptors within the submembraneous actin-based cytoskeleton and may participate in their further lateral translocations according to the ‘‘clustering-anchoring’’ model of the process [Bourguignon and Singer, 1977; Bourguignon and Bourguignon, 1984]. This could explain simultaneous clearing of plasma membrane remaining outside the cap region of both Con A/receptor complexes and a-spectrin (Figs. 1D–I, 3). In addition to Acanthamoeba, spectrin-related proteins were found to co-cap with several membrane proteins in lymphocytes, EGF receptors in A431 cells, and Con A receptors in Dictyostelium [Levine and Willard, 1983; Nelson et al., 1983; Bennett and Condeelis, 1988; Kwiatkowska et al., 1991]. In view of bifunctional properties of spectrin-like proteins as membrane- and actin-binding molecules, their potential role in the linkage of clustered receptors with actin cytoskeleton was proposed [Bourguignon and Bourguignon, 1984; Bourguignon et al., 1985; Kwiatkowska et al., 1991]. In contrast, the deficiency of a-spectrin in murine erythroleukemia cells was shown to cause rapid capping of glycoproteins, suggesting that the spectrin-based submembraneous network could constrain the mobility of integral membrane proteins rather than promote their redistribution [Dahl et al., Fig. 3. Pre-embedding immunoelectron microscopy localization of a-spectrin in Acanthamoeba during capping of Con A receptors. Cells exposed to Con A were subsequently labeled with anti-a-spectrin and gold-conjugated anti-rabbit antibodies to localize the antigen. Con A/receptor complexes, as peroxidase-binding sites, were visualized by electron dense DAB-based products of the enzyme activity. A: A cell fixed after cross-linking of Con A receptors (0°C). Note submembraneous localization of the a-spectrin immunoanalogue (arrowheads). B: A cell shifted to 20°C to induce capping of Con A receptors and fixed 20 min later. The cap revealed by electron dense deposits of the peroxidase activity product is clearly seen (arrow). Two parts of the cap region outlined by (—) and (– · –) are magnified in C and D, respectively. The outlined region of the cell placed opposite the cap is magnified in E. C,D: The cap area displays a heavy decoration of the a-spectrin immunoanalogue localized often in the vicinity of membrane folds (arrowheads). Gold labels of the antigen are also detectable around vacuoles adjoining the cap (circles). In contrast, small amounts of a-spectrin can be found outside the cap (E). Bar 5 0.25 µm in A, C–E; 1 µm in B. 261 1994]. It is possible, however, that the hindering of capping by spectrin observed in the erythroleukemia cells could reflect a specific organization and stabilizing function of the spectrin/actin membrane skeleton in cells of erythropoietic origin. In mature erythrocytes capping can not be induced [Loor et al., 1972]. On the other hand, in nonerythroid cells spectrin-like proteins often display a more dynamic nature, being shifted between the cytoplasmic and submembraneous locations depending on extraor intracellular signals [for review see Bennet, 1990; Hitt and Luna, 1994]. In addition, the presence of src homology 3 (SH3) and pleckstrin (PH) domains broadens the range of possible intermolecular interactions mediated by the non-erythroid spectrins [Roadway et al., 1989; Macias et al., 1994]. Thus, members of the spectrin family might be actively engaged in hindering the lateral diffusion as well as in redistribution of integral membrane proteins. There is now increasing evidence indicating that the ‘‘clustering-anchoring’’ scheme, with participation of spectrin-like proteins, is active in developing epithelial cells and muscles [Bloch and Morrow, 1989; Nelson and Hammerton, 1989; Nelson et al., 1990]. Our observations of spectrin and actin redistribution during uptake of Con A–coated yeast suggest that in motile cells this mechanism may be involved also in phagocytosis. Phagocytosis is believed to be driven by the actin-based cytoskeleton since the uptake of particles is accompanied by local polymerization of actin and the process is sensitive to actin filament-disrupting agents [Axline and Reaven, 1974; Sheterline et al., 1984; Greenberg et al., 1991]. The mechanism underlying the interaction between the receptors that mediate phagocytosis and the submembraneous actin skeleton remains unknown. We found that the a-spectrin immunoanalogue was accumulated at early stages of phagocytosis of Con A–coated yeast (Fig. 4A,C,E,G, arrows). Only a fraction of the cellular a-spectrin was translocated toward the sites of phagocytosis, reflecting the local character of the cytoskeletal rearrangement during uptake of the particles. The a-spectrin immunoanalogue was seen also around vacuoles enclosing Con A/receptor complexes after capping (Figs. 1K, 3C,D). On the other hand, the protein was no longer detected at maturing phagosomes during the yeast engulfment when sorting and degradation of internalized material took place (Fig. 4E). Submembraneous accumulation of actin filaments and a-spectrin at the sites of phagocytosis resembles accumulation of the proteins induced by capping of Con A receptors. It is tempting to speculate that reorganization of the cytoskeleton upon the onset of phagocytosis may be triggered by clustering of receptors mediating the uptake of particles. The receptors could be Fig. 4. Accumulation of the a-spectrin immunoanalogue (A,C,E,G) and actin filaments (B,D,H) at early stages of Con A-yeast phagocytosis. F: Phase contrast image of the cell from D. a-Spectrin and actin filaments were concentrated at phagocytic cups encompassing the yeast (arrows in A,B). The proteins surrounded nascent phagosomes captured in the vicinity of plasma membrane (arrows in C–F). Double-labeling revealed colocalization of a-spectrin and filamentous actin at the nascent phagosomes (arrows in G,H). Maturing phagosomes seen in the cytoplasm were devoid of the a-spectrin and actin filaments labeling (asterisks in D–F). Bar 5 10 µm. Spectrin in Capping and Phagocytosis 263 Fig. 5. Analysis of specificity of a-spectrin labeling. A,B: Immunoblotting examination of the anti-a-spectrin antibody reactivity with proteins of Acanthamoeba cell extract, 200 µg (A) and rat brain tissue homogenate, 15 µg (B). The antibody recognized selectively the polypeptide of approximately 240 kDa in both samples (arrowhead). C,D: Polar accumulation of a-spectrin observed in Acanthamoeba cells treated with non-labeled Con A. The cells were incubated with Con A at 0°C for 30 min to cross-link the lectin receptors and shifted to 20°C for 20 min to induce capping of the receptors. After fixation the cells were stained by immunofluorescence for a-spectrin. Note the polar concentration of a-spectrin (arrow in C) which corresponds to the cap visible also under phase contrast (arrow in D). Bar 5 10 µm. clustered during their interaction with certain ligands dispersed on the surface of the particles [Swanson and Bear, 1995]. Subsequently, the clustering could trigger the local accumulation of spectrin engaged in anchoring of the clusters within the actin cytoskeleton in a way analogous to the capping process. Clustering of Con A receptors in Chinese hamster ovary (CHO) cells, socalled ‘‘non-professional phagocytes,’’ seems to be essential for engulfment of Con A–coated zymosan particles since the particles coated with monovalent, succinylated Con A were not internalized [Veras et al., 1994]. Microinjection of anti-spectrin antibodies inhibited phagocytic activity of Amoeba proteus, pointing to direct involvement of the protein [Choi and Jeon, 1992]. We observed that a-spectrin accompanied also the internalization of uncoated yeast, mediated probably by mannose receptors [Allen and Dawidowicz, 1990] (our unpublished observations). The function of spectrin-related proteins in phagocytosis may be elucidated by studies of ‘‘professional phagocytes’’ currently being carried out that employ specific Fc receptors for internalization of immunological complexes. Phagocytic cups are transiently differentiated protrusions of the cell surface, similar in many ways to pseudopods at the leading edge of motile cells. Both structures emerge in response to local receptor-ligand interactions and their expansion is believed to be driven by the actin-based cytoskeleton controlled by several common actin-binding proteins [Stendahl et al., 1980; Greenberg et al., 1990; Stossel, 1993; Allen and Aderem, 1995; Maniak et al., 1995]. From this point of view, localization of a-spectrin in phagocytic cups and at the leading edge of adherent crawling Acanthamoeba cells is of special interest (Figs. 1E,H,K,4A). Phagocytosis was found to arrest the cell movement and diminish the cell adhesion. This suggests a possible competition between formation of phagocytic cups and cell migration as proposed before [Maniak et al., 1995]. On the contrary, capping of Con A receptors was closely related to crawling of the cells. This phenomenon is consistent with the cortical flow hypothesis considering capping as part of the cell movement machinery [Bray and White, 1988]. ACKNOWLEDGMENTS We thank Drs I.C. Baines and E.D. Korn for a critical reading of the manuscript. We also thank Kazimiera Mrozinska for excellent technical assistance. 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