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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. This
work was supported by a grant from the State Committee
for Scientific Research KBN 0082/P2/94/07 and by a
grant from the State Committee for Scientific Research to
the Nencki Institute of Experimental Biology.
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