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INTRODUCTION |
Dynamic tissue rearrangements are fundamental to both embryonic
morphogenesis and adult processes such as stem cell development, immune
responses, and cancer progression (1). These rearrangements involve the
coordination of cell-cell adhesion with cytoskeleton-based cell
migration and cell shape changes. Thus, cytoskeleton interactions with
adhesion complexes are probably of critical importance. We have used
Dictyostelium as a model system to examine interactions between adhesion complexes and the actin cytoskeleton during
multicellular development. Dictyostelium has a simple and
well defined life cycle that permits biochemical and genetic analyses
of cell-cell interactions (2). During the aggregation stage of
development, Dictyostelium undergoes a transition from
individual cells to multicellular streams. The process begins with the
chemotactic migration of single cells toward a central source of cAMP
(2). Cell-cell contacts are initially established through the
interdigitation of filopodia (3). The contacts break and reform
continually as the cells migrate to the aggregation center (4). Even
within large migratory multicellular streams, individual cells form
only transient cell-cell contacts and continually exchange partners (5).
In general, cell-cell adhesion is mediated by large adhesion complexes
composed of a core of adhesion molecules and peripheral attachments to
the cytoskeleton (6). Within the complexes, cis- and
trans-oligomers of adhesion molecules can assemble into zippers and lattices (7-9), structures that can convey strong cell-cell adhesion through enhanced binding avidity (10). The adhesion
receptors are typically connected to the cytoskeleton via adapter
proteins, such as the catenins that link cadherin complexes to the
actin cytoskeleton (11). In Dictyostelium, cell-cell
contacts are first formed by the Ca2+-dependent
adhesion molecule DdCAD-1 (12-14) and distinct
Mg2+-dependent adhesion sites (15). The
Ca2+/Mg2+-independent adhesion molecule gp80 is
expressed at the onset of the chemotactic migration stage (16) and
mediates strong cell-cell adhesion (17, 18) via
trans-homophilic interaction (19, 20). The expression of the
next major cell adhesion molecule gp150 is activated at the cell
aggregation stage (21), and rapid accumulation of gp150 occurs in the
postaggregation stage (22).
gp80 is phospholipid-anchored (23, 24), as are many other adhesion
molecules such as NCAM-120, axonin-1/TAX-1/TAG-1 (25), and T-cadherin
(26). Since these receptors lack direct access to the cytoplasm, the
mechanism by which they engage the cytoskeleton is unclear. Many
glycosylphosphatidylinositol
(GPI)1-anchored proteins
preferentially partition into rafts, which are membrane microdomains
formed from the close packing of sterols and saturated lipids into
liquid ordered structures (27). Rafts can be isolated from cells as
Triton X-100-insoluble floating fractions (TIFF) that contain the raft
lipids, as well as GPI-anchored proteins, acylated proteins, and some
transmembrane proteins (28-30).
Raftlike membrane fragments have been isolated from
Dictyostelium as both TIFF and a low density plasma membrane
fraction after sonication. Both types of membrane fractions were highly enriched in sterols and contained many of the proteins found in the
bulk plasma membrane, suggesting that raftlike domains exist in the
Dictyostelium plasma membrane (31, 32). Moreover,
immunoprecipitation experiments have shown that the major proteins in
the complex sediment together, indicating that they are components of
the same membrane domains (32). At the aggregation stage of
development, gp80 is the main component of these complexes, suggesting
that they participate in gp80-mediated adhesion. Indeed, large
sterol-rich domains have been localized to gp80-mediated cell-cell
contacts, and sterol sequestration weakens cell-cell adhesion (31). It is also evident that raftlike domains facilitate gp80 oligomerization and adhesion complex formation (32), but the mechanism of cytoskeleton attachment to these complexes remains to be elucidated. During Dictyostelium aggregation, TIFF does become associated with
cytoskeleton complexes and TIFF itself contains a number of
cytoskeletal proteins, including actin, comitin, regulatory myosin
light chain, and ponticulin (31, 32).
We hypothesized that ponticulin is the primary link between gp80
adhesion complexes and the actin cytoskeleton, since it is a major high
affinity actin-binding protein in the plasma membrane of
Dictyostelium cells (33). To address this hypothesis and assess how such connections form and function, we monitored
interactions among gp80, ponticulin, and actin through subcellular
fractionation and localization studies in gene deletion and
drug-treated cells during development. We found that gp80 is involved
in establishing TIFF-cytoskeleton interactions during development. A
major proportion of cellular ponticulin is associated with TIFF and the
cytoskeleton and is required to mediate their interaction. gp80
apparently regulates ponticulin function by recruiting it into TIFF and
cell-cell contacts. Ponticulin also has an effect on the size of gp80
adhesion complexes within multicellular streams.
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EXPERIMENTAL PROCEDURES |
Cell Growth and Development--
Dictyostelium
discoideum strains were cultured in association with
Klebsiella aerogenes (34). In most experiments, the
csaA
, gp80-null strain (GT10) (17) was
compared with its parental strain AX2, and the
ponA
, ponticulin-null strain (Tf24.1)
(33) was compared with its parental strain KAX3. For development in
suspension, cells were collected, washed free of bacteria, and
resuspended at 2 × 107 cells/ml in 17 mM
sodium phosphate buffer (pH 6.1) and then shaken at 180 rpm. Cells were
pulsed with cAMP every 7 min at a final concentration of 2 × 10
8 M beginning at 2 h of development.
Alternatively, cells were developed on poly-L-lysine-coated
coverslips for 12 h as described previously (4).
Pharmacological Studies--
Cells were developed in suspension
in 17 mM sodium phosphate buffer (pH 6.1). To perturb the
actin cytoskeleton or raftlike domains, cells were treated for 30 min
with 5 µM latrunculin B (Sigma), 0.001% (w/v) digitonin
(S. B. Penick and Co., New York, NY), or 0.0005% (w/v) filipin (Sigma).
Detergent Extraction and Immunoprecipitation--
For detergent
extractions, cells were resuspended at 5 × 107
cells/ml in cold extraction buffer (40 mM sodium
pyrophosphate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 mM EDTA, 1 mM EGTA, 3 mM sodium
azide, 10 mM Tris-HCl, pH 7.6). Triton X-100 was added to a
final concentration of 0.2% (v/v), and the suspension was shaken at
180 rpm for 2 min at 4 °C. The samples were centrifuged at
14,500 × g for 10 min at 4 °C. The supernatants
were collected, and the pellets were washed once with cold extraction
buffer without detergent. To compare the partitioning of proteins
between the fractions, the pellets were resuspended in volumes
equivalent to the supernatant, and equal proportions were used for
further analysis.
For immunoprecipitation, gp80 mAb was added to supernatant fractions
resulting from the above protocol and incubated for 1 h at
4 °C. Protein A-Sepharose beads (Amersham Biosciences) were added to
5 mg/ml and incubated for 1 h at 4 °C. The samples were centrifuged at 1,000 × g for 10 s. The
supernatants were collected, and the beads were washed three times with
cold extraction buffer without detergent. To compare the partitioning
of proteins between these fractions, the beads were resuspended in
volumes equal to the supernatant, and equal proportions of these
samples were used for further analysis.
Isolation of TIFF, Contact Regions, and Cytoskeleton
Complexes--
TIFF and Triton-insoluble contact regions were isolated
as previously described (31). Briefly, cells were resuspended at 5 × 107 cells/ml in cold extraction buffer (40 mM sodium pyrophosphate, 0.4 mM dithiothreitol,
0.1 mg/ml phenylmethylsulfonyl fluoride, 2 mM EDTA, 1 mM EGTA, 3 mM sodium azide, 10 mM
Tris-HCl, pH 7.6). Triton X-100 was added to a final concentration of
0.2% (v/v), and the suspension was shaken at 180 rpm for 1 min at
4 °C. The insoluble material was collected by centrifugation and
washed. For TIFF isolation, the resulting pellet was increased to 45% (w/w) sucrose in 1.5 ml, deposited on a 0.5-ml 60% (w/w) cushion in a
centrifuge tube, and overlaid with a step gradient of 1 ml of 20% and
2.5 ml of 38% (w/w) sucrose. The gradients were centrifuged at
120,000 × g for 15-17 h at 2 °C using a Beckman
SW50.1 rotor. TIFF was collected from the interface between 20 and 38%
sucrose. The initial steps of the contact region isolation were similar to the TIFF isolation protocol, and the contact regions were isolated from complexes that sedimented to the interface between 45 and 60%
sucrose with centrifugation. The material was washed and then dialyzed
in a cytoskeleton depolymerizing solution containing 0.1 mM
EDTA, 0.2 mM sodium phosphate buffer, pH 7.6 (35). Then the
sample was layered on top of a discontinuous gradient of 2.5 ml of 28%
and 2.5 ml of 38% (w/w) sucrose in a centrifuge tube and centrifuged
at 120,000 × g for 3 h at 2 °C. The contact
regions were collected from the interface between 28 and 38% sucrose. The depolymerized cytoskeleton components, released during the contact
region isolation protocol after 1 day of dialysis, were collected as
supernatant after centrifugation at 14,500 × g for 20 min at 2 °C. The cytoskeleton samples were concentrated using a
Centricon YM10 centrifugal filtration device (Millipore Corp., Bedford,
MA) for further analysis.
Gel Electrophoresis--
Protein samples were separated by
SDS-PAGE (36). For immunoblot analysis, bound antibodies were detected
using horseradish peroxidase-conjugated secondary antibodies and the
enhanced chemiluminescence kit (Amersham Biosciences). Immunoblots were
imaged and quantified using the Fluor-S Max multi-imager system
(Bio-Rad).
Protein Identification and Quantification and Lipid
Analysis--
For protein identification by mass spectrometry,
silver-stained gel bands were excised, macerated, reduced, alkylated,
and then digested with trypsin as previously described (31). Peptide masses were determined using a PerSeptive Biosystems Voyager Elite MALDI-TOF mass spectrometer (PerSeptive Biosystems, Inc., Foster City,
CA). Peptide masses were submitted to the ProFound search engine
(available on the World Wide Web) for matches. Search parameters were
held constant and included all Dictyostelium proteins in the
NCBI nonredundant data bases, tolerance for peptide mass error of 1 Da,
and no missed cut sites per peptide.
Protein concentrations were determined using the bicinchoninic acid
protein assay kit (Pierce), using bovine serum albumin as a standard.
For lipid analysis, membrane samples were subjected to gas-liquid
chromatography as described previously (31).
Immunofluorescence Staining--
Cells were collected from
suspension cultures and deposited on poly-L-lysine-coated
coverslips for 10 min. Alternatively, cells were developed on
poly-L-lysine-coated for 12 h as described (4). Then
samples were fixed, stained, and mounted on slides. For double staining
of gp80 and F-actin, the cells were fixed in 3.7% (v/v) formaldehyde
for 15 min at room temperature followed by 1% (v/v) formaldehyde in
ethanol for 5 min at
20 °C. They were incubated with gp80 mAb and
then Alexa 488-phalloidin (Molecular Probes, Inc., Eugene, OR) at a
1:10 dilution. For double staining of gp80 and ponticulin, the cells
were fixed in 1% (v/v) formaldehyde in methanol for 5 min at
20 °C and then incubated in gp80 mAb and ponticulin polyclonal
antibodies (37). For staining of gp80 alone, the cells were fixed in
methanol for 5 min at
20 °C. Samples were incubated with Alexa
488/568-conjugated secondary antibodies (Molecular Probes) at 1:300
dilution. Laser-scanning confocal microscopy was performed using a
Leica DM IRBE inverted microscope equipped with a Leica TCS SP confocal
system. Detection was maintained within the range of the gray scale to
prevent signal saturation. The images were processed using Adobe
Photoshop software (Mountain View, CA).
Cell Adhesion Assays--
The maintenance of cell aggregation
was assayed by depositing 1 ml of intact aggregates in suspension at
~2 × 107 cells/ml in 17 mM sodium
phosphate buffer (pH 6.1) in a 50-ml conical tube. The samples were
then shaken at 180 rpm for 30 min, and samples were taken for cell
counting. Singlets, doublets, and triplets were counted as dissociated
cells, and the percentage of cell aggregation was calculated relative
to the total number of cells present. Assays for cell reassociation
were also performed (38). Additionally, cell dissociation was assessed
under high shear force by counting dissociated cells following 30 s of fast shaking with a Vortex Genie 2 at setting 8 (31).
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RESULTS |
Colocalization of gp80 and Actin during Cell-Cell
Interactions--
To investigate the relationship between gp80 and the
actin cytoskeleton during cell aggregation, cells were developed on
coverslips for 12 h, fixed, and stained for gp80 and F-actin. At
noncontact regions of the cell membrane, gp80 staining was low and
displayed a more or less even distribution. However, at both initial
and mature cell-cell contact regions, extensive areas of strong gp80 staining were observed (Fig.
1A). For F-actin, strong
staining was detected at filopodia emanating from noncontact regions,
cellular protrusions mediating initial cell-cell contacts, and smooth
membrane interfaces at mature contacts (Fig. 1B). When
compared at noncontact regions, no clear relationship was evident
between gp80 and F-actin, since only low levels of gp80 were present at
the sites with high levels of F-actin (Fig. 1C). In
contrast, high levels of gp80 and F-action colocalized in cell-cell
contact regions, although their distributions lacked complete overlap,
especially at initial cell-cell contacts where F-actin-rich protrusions
appeared to extend beyond the established contact (Fig. 1C).
Thus, high levels of F-actin alone are not sufficient to organize
detectable gp80 complexes at noncontact regions, but rather cell-cell
contact is required to instigate their assembly.

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Fig. 1.
Colocalization of gp80 and actin filaments
specifically at regions of cell-cell contact. The distributions of
gp80 (red) and F-actin (green) were imaged by
confocal microscopy in wild-type cells developed on a coverslip
for 12 h. An overlay of the distributions is shown at the bottom.
Colocalization is specific for cell-cell contacts. gp80 staining is low
at F-actin-rich protrusions at noncontact regions (arrows)
and at similar protrusions observed to extend past sites of adhesion
complex assembly (arrowheads). Bar, 5 µm.
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gp80 Induces TIFF-Cytoskeleton Interactions during
Development--
A biochemical approach was taken to address the
involvement of raftlike membranes and gp80 in the establishment of
cytoskeleton connections to gp80 adhesion complexes. We have shown
previously that TIFF co-fractionates with the Triton X-100-insoluble
cytoskeleton in a form termed "contact regions" (31). These
complexes are rich in sterols and contain many of the proteins found in
TIFF. They also have the morphology of stacked adherent membranes and co-fractionate with the cytoskeleton at 12 h of development but not at the single cell stage at 0 h (35). The contact regions can
be released from the cytoskeleton as a low density membrane fraction
after the depolymerization of the cytoskeleton (Fig. 2A). To characterize the
cytoskeletal components, the depolymerized complexes were separated
from the membranes by centrifugation and then concentrated. The
concentration procedure led to polymerization of the fraction into a
gel, consistent with the nature of an actin and myosin mixture. After
separation by SDS-PAGE, all silver-stained bands were excised and
analyzed by MALDI-TOF mass spectrometry. Actin and components of the
myosin II hexamer (myosin II heavy chain, regulatory myosin light
chain, and essential myosin light chain) were identified as major
species, and components of the Arp 2/3 complex (Arp 3 and p21-Arc) as
minor species (Fig. 2B). Components of the tubulin
cytoskeleton were not detected. The data indicate that TIFF interacts
with an actin-myosin complex in the contact region fraction.

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Fig. 2.
Requirement of gp80 for the recovery of
cytoskeleton-associated Triton X-100-insoluble contact regions.
A, a schematic showing the fractionation of cells into the
Triton X-100-soluble supernatant (S) and the Triton
X-100-insoluble pellet (P). The pellet fraction was further
fractionated by equilibrium density centrifugation into a TIFF
that floated to a 28/38% (w/w) sucrose interface and a TISF that
banded at a 45/60% (w/w) sucrose interface. A dialysis procedure
released the depolymerized cytoskeleton fraction and allowed for the
isolation of low density contact regions through sedimentation to a
28/38% (w/w) sucrose interface by equilibrium density centrifugation.
B, protein components of the depolymerized cytoskeleton were
separated by 12% SDS-PAGE, silver-stained, and prepared for analysis
by MALDI-TOF mass spectrometry. Protein identifications and
corresponding Z scores are indicated at the left.
C and D, relative amounts of protein recovered in
TIFF (C) and the contact region fraction (D) were
determined at 0, 4, and 8 h of development. Equal cell numbers
were used at each time point, and protein levels were normalized in
each experiment to the values at 8 h, which were taken to be 1.0. Values represent the mean ± S.D. (n = 6 for TIFF
and n = 4 for the contact regions). E and
F, relative amounts of protein recovered in TIFF
(E) and the contact region fraction (F) were
compared between equal numbers of wild-type and gp80-null
(csaA ) cells at 12 h. For each
experiment, the protein levels were normalized to the values of the
wild type, which were taken to be 1.0. Values represent the mean ± S.D. (n = 3). G, equal numbers of
wild-type and csaA cells at 12 h were
separated by 12% SDS-PAGE, blotted, and probed for ponticulin and
actin.
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Since the expression of at least three adhesion molecules is induced
between 0 and 12 h of development, cells were fractionated at 0, 4, and 8 h to determine which of them contribute to the formation
of the detergent-insoluble complexes. TIFF protein displayed a
~2-fold increase over the time period (Fig. 2C). For the
contact regions, however, only background levels of protein were
recovered up to 4 h, a stage at which the EDTA-sensitive adhesion
molecules mediate aggregation. At 8 h, recovery of contact regions
increased by 10-fold (Fig. 2D). Since high levels of gp80
were expressed by 8 h, we examined the role of gp80 in the
interaction between TIFF and the cytoskeleton by comparing wild-type
and gp80-null (csaA
) cells at 12 h of
development. The level of TIFF protein was reduced by ~50% in
gp80-null cells relative to wild-type cells (Fig. 2E).
However, protein recovery in the contact region fraction was reduced to
background levels in gp80-null cells (Fig. 2F). Since stable
contact regions could only be recovered with the expression of gp80,
they probably represent gp80 adhesion complexes.
The lower levels of TIFF protein detected in the gp80-null cells could
be due partly to the physical absence of gp80, since it is the major
protein component of TIFF (31). Indeed, lipid analyses revealed that
similar levels of TIFF lipids were recovered from the wild-type and
gp80-null cells (Table I). TIFF isolated from gp80-null cells was highly enriched in sterols. It contained mainly stigmasterol (84%), with lower levels of campesterol (8%) and
sitosterol (8%). Similar results were obtained for wild-type cells,
consistent with our previous observation (31). Additionally, similar
levels of both ponticulin and actin were expressed in these two cell
strains (Fig. 2G).
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Table I
Recovery of lipids in TIFF isolated from wild-type and csaA
cells
Values represent the mean ± S.D. (n = 3).
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Ponticulin Mediates the TIFF-Cytoskeleton Interactions--
Since
ponticulin interacts with the actin cytoskeleton and is a TIFF
component, could it mediate interactions between the gp80 adhesion
complex and the cytoskeleton? To address this question, we first
examined the distribution of gp80 between the Triton X-100-insoluble
complexes. Total detergent-insoluble material was separated using
single step gradients into TIFF, which floated to a 28/38% sucrose
interface, and a Triton-insoluble sedimenting fraction (TISF), which
banded at a 45/60% sucrose interface. The TISF contained both the
Triton-insoluble cytoskeleton and the gp80 adhesion complexes (see Fig.
2A). To determine whether the partitioning of gp80 complexes
into TISF required an intact actin cytoskeleton, cell aggregates were
pretreated with latrunculin B (Fig.
3A). In control samples, both
gp80 and ponticulin were evenly distributed between TIFF and TISF,
suggesting that about half of the complexes were associated with the
cytoskeleton. Actin was mainly detected in TISF and was present at a
much lower level in TIFF. Following latrunculin B treatment, both gp80
and ponticulin showed reduced levels in TISF and were predominantly
associated with TIFF. The treatment dramatically reduced the level of
actin in TISF and eliminated actin from TIFF.

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Fig. 3.
Ponticulin mediates the interactions between
the gp80 adhesion complex and the actin cytoskeleton. To assess
the bases for the co-fractionation of gp80 adhesion complexes with the
detergent-insoluble cytoskeleton, 10-h cell aggregates were extracted
with cold 0.2% Triton X-100, and the resulting total Triton
X-100-insoluble material was separated into TIFF and TISF using single
discontinuous gradients. One-ml fractions were collected, and
equivalent volumes were separated by 12% SDS-PAGE, blotted, and probed
for gp80, ponticulin, and actin. A, samples collected after
pretreating wild-type cells with 5 µM latrunculin B are
compared with the ethanol carrier control. The results are
representative of three experiments. B, samples from
ponticulin-null (ponA ) cells are compared with
those of the parental strain. The results are representative of three
experiments. Note the loss of gp80 from TISF after latrunculin B
treatment and in the ponA cells.
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To test whether ponticulin mediates the interactions between the gp80
complexes and the actin cytoskeleton, we analyzed wild-type and
ponticulin-null (ponA
) cells (Fig.
3B). In wild-type cells, gp80 and actin displayed distribution patterns similar to the controls described above (see Fig.
3A). For ponticulin-null cells, however, gp80 was lost from
TISF and became predominantly found in TIFF, similar to the results of
latrunculin B treatment of wild-type cells. Analyses of actin revealed
its complete loss from the TIFF of null cells, suggesting that
ponticulin was required for the binding of actin to TIFF. A partial
loss of actin was also observed in TISF, but to a lesser extent than
that resulting from the latrunculin B treatment, suggesting that the
actin cytoskeleton remained largely intact. Taken together, these
results suggest that ponticulin is required for the gp80 adhesion
complex to establish cytoskeleton connections.
Ponticulin Associates Primarily with TIFF and the
Cytoskeleton--
Next, we assessed the proportion of total cellular
ponticulin that may be involved specifically in the TIFF-cytoskeleton
interactions. Cell aggregates were collected at 10 h of
development, extracted with cold 0.2% Triton X-100, and fractionated
into detergent-soluble supernatants and detergent-insoluble pellets.
SDS-PAGE and protein blot analyses were performed to assess the
partitioning of ponticulin between the two fractions (Fig.
4). To address the bases for detergent insolubility, we pretreated the cells with either latrunculin B, which
induces actin depolymerization, or low levels of digitonin and filipin,
which perturb rafts by sequestering membrane sterols. In carrier
controls, ~75% of total cellular ponticulin partitioned into the
detergent-insoluble pellets (Fig. 4). gp80 displayed a slightly greater
degree of solubilization, consistent with our previous observation
(31). With latrunculin B pretreatment, ponticulin displayed
enhanced solubilization, resulting in even partitioning between the
pellet and supernatant fractions (Fig. 4). After digitonin or filipin
treatment, the detergent solubility of ponticulin increased to ~50
and ~40%, respectively (Fig. 4). Pretreatment with latrunculin B in
combination with either digitonin or filipin further increased the
detergent solubility of ponticulin to ~75 and ~60%, respectively,
suggestive of additive effects (Fig. 4). Thus, the insolubility of
ponticulin in Triton X-100 is probably due its association with both
TIFF and the actin cytoskeleton.

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Fig. 4.
Insolubility of ponticulin in cold Triton
X-100 due to interactions with the actin cytoskeleton and TIFF. To
assess the degree to which gp80 and ponticulin associate with the actin
cytoskeleton and TIFF, 10-h cells were extracted with cold 0.2% Triton
X-100 and separated into supernatants (S) and pellets
(P) by centrifugation. An equal proportion of each fraction
was separated by 12% SDS-PAGE, blotted, and probed for gp80 and
ponticulin. Drug pretreatments were performed to assess the bases for
detergent insolubility. Latrunculin B binds actin monomers and depletes
F-actin, whereas digitonin and filipin sequester sterols and disrupt
raft structure. Cells pretreated with 5 µM latrunculin B
were compared with control samples exposed to the ethanol carrier.
Cells pretreated with 0.001% (w/v) digitonin alone, 0.001% (w/v)
digitonin plus 5 µM latrunculin B, 0.0005% (w/v) filipin
alone, or 0.0005% (w/v) filipin plus 5 µM latrunculin B
were compared with control samples exposed to the ethanol and
Me2SO carriers. For each of the treatments, the
partitioning of ponticulin between the two fractions was quantified as
a percentage of their total sum. Values represent means ± S.D.
(n = 3 for latrunculin B experiments, n = 2 for digitonin and filipin experiments).
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In contrast to ponticulin, gp80 was not significantly affected by the
drug treatments (Fig. 4). Low levels of filipin and digitonin were used
in this study to maintain cell structure, and they were 10-fold lower
than those required to block TIFF recovery (27). In comparison with
other TIFF components, ponticulin appears to be more sensitive to the
sequestration of membrane sterols.
Lack of Direct Interactions between Ponticulin and gp80--
We
have shown previously that gp80 and ponticulin are components of the
same raftlike domain (32). The above results also indicate that
ponticulin is a major link between the gp80 adhesion complexes and the
cytoskeleton, but do these proteins interact directly? Since
substantial amounts of both ponticulin and gp80 can be solubilized
under fairly gentle conditions of 0.2% cold Triton X-100 after
treatment with latrunculin B or the sterol sequestering agents, we
determined whether these proteins could be co-immunoprecipitated from
the resulting supernatants (Fig. 5).
Under six different conditions, including those with drug pretreatments and the untreated controls, ~50% of gp80 could be specifically immunoprecipitated. However, ponticulin remained in the
unbound fraction in all of the conditions. Thus, direct gp80-ponticulin
interactions were not apparent.

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Fig. 5.
Ponticulin does not co-immunoprecipitate with
gp80. To examine whether gp80 and ponticulin have direct
protein-protein interactions, immunoprecipitation of gp80 was carried
out following solubilization of 10 h cells with cold 0.2% Triton
X-100. Insoluble complexes were removed by centrifugation, and gp80 mAb
was added to the supernatants. Protein A-Sepharose beads were then
added, and equal proportions of unbound material (U) and
bound material (B) were separated by 12% SDS-PAGE, blotted
on nitrocellulose, and probed for gp80 and ponticulin. The procedure
was performed in the absence of gp80 mAb to control for nonspecific
binding to the beads. The experiments were performed with untreated
cells and after pretreatment with 5 µM latrunculin B
alone, 0.001% (w/v) digitonin alone, 0.0005% (w/v) filipin alone,
0.001% (w/v) digitonin plus 5 µM latrunculin B, or
0.0005% (w/v) filipin plus 5 µM latrunculin B. In each
case, ~50% of detergent-solubilized gp80 was immunoprecipitated in
the bound fraction. Ponticulin remained entirely in the unbound
fractions.
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gp80 and Membrane Sterols Act Together to Recruit Ponticulin into
TIFF--
Since direct interactions between gp80 and ponticulin could
not be detected in Triton X-100-soluble complexes, we assessed whether
membrane sterols played a role in the co-fractionation of gp80 and
ponticulin in TIFF and TISF. Pretreatment with low levels of digitonin
or filipin did not affect the partitioning of gp80 into TIFF (Fig.
6A). In contrast, the amount
of ponticulin present in TIFF was markedly reduced, suggesting that its
co-fractionation with gp80 was dependent upon membrane sterols. The
levels of gp80 and ponticulin associated with TISF were not affected
(Fig. 6A), suggesting that fully assembled adhesion
complexes might resist the sterol sequestration.

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Fig. 6.
Ponticulin displays reduced partitioning into
TIFF after sterol sequestration and also in gp80-null cells.
A, to assess whether membrane sterols are involved in
mediating interactions between gp80 and ponticulin, 10-h wild-type
cells were treated with 0.001% (w/v) digitonin, 0.0005% (w/v)
filipin, or Me2SO carrier and fractionated into TIFF and
TISF using single discontinuous gradients. One-ml fractions were
collected, and equivalent volumes were separated by 12% SDS-PAGE,
blotted, and probed for gp80, ponticulin, and actin. Sterol
sequestration resulted in a loss of ponticulin from TIFF, whereas the
pattern for gp80 remained unchanged. The results are representative of
two experiments. B, to examine the effects of gp80 on the
partitioning of ponticulin, samples of TIFF and TISF were prepared from
equivalent numbers of wild-type and csaA cells
at 10 h of development and examined as above. The results are
representative of three separate experiments.
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The role of gp80 in the partitioning of ponticulin into TIFF and TISF
was examined using wild-type and gp80-null cells. In the gp80-null
cells, the level of ponticulin in TIFF was markedly reduced, whereas
its level in TISF remained relatively high (Fig. 6B). These
results were similar to those obtained by sequestering membrane
sterols, suggesting that gp80 may act through sterols to recruit
ponticulin into raftlike domains in the plasma membrane.
Requirement of gp80 for Ponticulin Recruitment to Cell-Cell
Contacts--
To determine whether gp80 acts upstream of ponticulin
during the assembly of adhesion complexes, the subcellular
distributions of gp80 and ponticulin were examined by confocal
microscopy in wild-type and mutant cell aggregates after 10 h of
development in suspension. In wild-type cell aggregates, high levels of
gp80 and ponticulin colocalized at cell-cell contacts, and both
displayed lower staining intensity at noncontact regions (Fig.
7). In ponticulin-null aggregates, strong
gp80 staining was also detected along cell-cell contacts, suggesting
that gp80 was able to assemble into large adhesion complexes in the
absence of ponticulin. In gp80-null cells, the ponticulin antibodies
detected low levels of even staining along the plasma membrane and some
punctate cytoplasmic staining. However, ponticulin enrichment at
cell-cell contacts was not detected (Fig. 7). Since gp80 can assemble
into cell-cell contacts in the absence of ponticulin and is required to
recruit ponticulin to contact regions, gp80 appears to act upstream of
ponticulin during the assembly of adhesion complexes.

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Fig. 7.
gp80 recruits ponticulin to cell-cell
contacts. Wild-type and mutant cells were developed in suspension
for 10 h and then processed for confocal microscopy. Top
panels, the distributions of gp80 and ponticulin are compared in a
wild-type aggregate. Bottom left, the distribution of gp80
in a ponA cell aggregate is shown.
Bottom right, the distribution of ponticulin in a
csaA cell aggregate is shown. Bars,
5 µm.
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Effects of Latrunculin B on gp80-mediated Adhesion in Wild-type and
Mutant Strains--
To investigate the relationship between
gp80-mediated cell-cell adhesion and the cytoskeleton, 10-h cell
aggregates were exposed to latrunculin B and then monitored for
dissociation (Fig. 8A). Wild-type cell aggregates were able to withstand the latrunculin B
treatment, and ~85% of cells remained in aggregates in both treated
and control samples. gp80-null cells dissociated readily with the
latrunculin B treatment that reduced their aggregation from 70 to 25%.
However, ponticulin-null cell aggregates were able to withstand the
latrunculin B treatment and exhibited minimal dissociation in both
treated and control cells, similar to wild-type cells. Ponticulin-null
cells and latrunculin B-treated cells were also subjected to two
additional adhesion assays: a cell dissociation assay to test the
degree aggregate dissociation under high shear forces and a cell
reassociation assay to test the reaggregation of dissociated cells.
However, no differences in gp80-mediated adhesion were observed
relative to controls (data not shown). These results show that gp80 is
unique among the adhesion systems expressed in early development, since
it can mediate cell-cell adhesion despite F-actin perturbation or
ablation of its principle link to the cytoskeleton.

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Fig. 8.
gp80 can mediate cell-cell adhesion after
latrunculin B treatment and in ponticulin-null cells that show altered
cytoskeleton structure. A, wild-type,
csaA , and ponA cells
were developed in suspension for 10 h, treated at 1.5 × 107 cells/ml for 30 min at 180 rpm with ethanol carrier or
5 µM latrunculin B. The numbers of dissociated cells were
counted, and cell aggregation was calculated as percentages of the
total cell numbers. The values represent the means ± S.D.
(n = 3). B-E, following the latrunculin B
treatments, wild-type and mutant cells were deposited on coverslips,
fixed, stained, and imaged by confocal microscopy. Phalloidin staining
(B) and gp80 staining (C) of a wild-type cell
aggregate are shown. D, phalloidin staining of a
csaA cell aggregate. E, phalloidin
staining of a ponA cell aggregate.
Bars, 5 µm.
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When cell aggregates were stained with phalloidin to examine the actin
cytoskeleton of the wild-type and mutant strains, the overall patterns
of F-actin staining were indistinguishable between the strains and
resembled the data in Fig. 1B (data not shown). Since subtle
differences might be present, we treated the cells with latrunculin B
and examined the cytoskeleton remnants, reasoning that they might
represent stable actin complexes. In wild-type aggregates, small foci
of F-actin were observed, distributed more or less evenly along
cell-cell contacts (Fig. 8B). gp80 was also detected along
the contacts but with a more smooth and continuous distribution (Fig.
8C). However, in gp80-null aggregates, the latrunculin B
treatment resulted in a lower degree of F-actin staining at cell-cell
contacts, and a smaller number of strongly stained structures were
detected at random positions in the aggregates (Fig. 8D). In
ponticulin-null cell aggregates, residual F-actin was distributed along
the cell-cell contacts but in a more diffuse and continuous pattern
relative to wild-type (Fig. 8E). It is, therefore, possible
that gp80 and ponticulin are involved in remodeling cytoskeleton
structure at cell-cell contact regions.
gp80 Adhesion Complexes Are Elongated in Streams of Ponticulin-null
Cells--
Although ponticulin was apparently dispensable for
gp80-mediated cell-cell adhesion during development in suspension,
ponticulin may have effects on gp80 adhesion complexes during more
dynamic cell-cell interactions. Thus, we examined the distribution of gp80 in wild-type and ponticulin-null cells at the streaming stage of
development. In wild-type cell streams, gp80 was often enriched at
end-to-end contacts in C-shaped staining patterns (Fig.
9A). In ponticulin-null cells,
gp80 staining appeared to be more evenly distributed along and around
the cells (Fig. 9B). To quantify the length distribution of
the gp80 staining patterns, the tonal levels in the confocal images
were converted automatically into white, gray, or black (Fig. 9,
C and D). Measurement and statistical analysis of
the length distributions revealed a significant difference between the
wild-type and ponticulin-null cells. In wild-type streams, the average
complex length measured was 5.1 µm, and only 35% of the complexes
extended past 5 µm (Fig. 9E). In ponticulin-null streams,
the average complex length was 6.4 µm, and 53% of the complexes
extended past 5 µm (Fig. 9F). Thus, gp80 adhesion
complexes tended to be longer in the ponticulin-null cells.

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Fig. 9.
gp80 adhesion complexes are longer in streams
of ponA cells. Wild-type and
ponA cells were developed in parallel on
coverslips for 12 h, stained for gp80, and imaged by confocal
microscopy. Images were collected at the first focal plane above the
basal cell surface that showed both the strongest staining of the Golgi
apparatus and the largest cross-sectional area of individual cells. The
collections were maintained within the gray scale to minimize signal
saturation. Representative images are shown in for wild-type
(A) and ponA (B) cells.
Bars, 5 µm. To quantify the length of gp80 adhesion
complexes, the images were inverted and automatically posterized at
setting three in Adobe Photoshop to convert pixel intensities into
white, gray, or black. The inverted and posterized images of
A and B are shown in C and
D, respectively. All elongated detections containing a black
pixel were measured along their longest length, including black and
gray regions, until interruption by a white pixel. Ten cell streams
from two experiments were quantified for each strain. Histograms of
gp80 complex lengths are shown in E for wild-type cells and
F for ponA cells. The distributions
were shown to be significantly different using the Student's
t test (p < 0.001).
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DISCUSSION |
During Dictyostelium development, strong cell-cell
adhesion is mediated by a gp80 adhesion complex composed of a core of
oligomerized receptors integrated into opposing raftlike domains (31,
32). In this paper, we have demonstrated that specific cytoskeleton connections are established with the complex via ponticulin, a major
mediator of interactions between the actin cytoskeleton and the plasma
membrane in Dictyostelium (33). Our results frame a model
for complex assembly and provide insights into how the complex may
function during morphogenesis.
In contrast to transmembrane adhesion molecules that interact directly
with cytoskeleton adapter proteins, gp80 appears to interact indirectly
with ponticulin. Ponticulin is an atypical membrane protein containing
transmembrane domains and a phospholipid anchor (39). Its extracellular
loops are predicted to be short and close to the membrane, and its
cytoplasmic loops bind to the sides of actin filaments with high
affinity. The region of gp80 in proximity to the membrane is predicted
to form a linear stalk that extends perpendicularly away from the
membrane (40). If interactions occur between the short loops of
ponticulin and the linear stalk of gp80, they are probably weak, since
we were unable to co-immunoprecipitate the proteins after mild
detergent solubilization (Fig. 5). Although the possibility of direct
interactions could not be formally excluded, it is likely that these
two proteins interact indirectly through their association with
raftlike domains. Indeed, immunoprecipitation of plasma membrane
fragments has shown that gp80 and ponticulin are components of the same
raftlike membrane complex (32). The analyses of TIFF from null mutants
indicate that each protein can partition into such membranes in the
absence of the other. Sterols appear to play a role in linking the
proteins, since sterol sequestration depleted ponticulin from TIFF
without affecting gp80 (Fig. 6A).
We propose the following model for the assembly gp80 adhesion
complexes. Prior to cell-cell contact formation, individual membrane
rafts facilitate the assembly of gp80 cis-oligomers that can
be detected in 50-70-nm clusters on the cell surface (32, 41). Such
structures appear to be the limit of gp80 interactions prior to
cell-cell contact, since larger complexes were not detected by light
microscopy prior to cell aggregation (Fig. 1). By analyzing TIFF, we
have found that a basal level of actin is associated with raftlike
membranes through ponticulin, even without the expression of gp80 (Fig.
6B). However, gp80 oligomers are insensitive to latrunculin
B, and their assembly may not necessarily depend on the cytoskeleton
interaction (32).
In contrast to many adhesion complexes that require participation of
the actin cytoskeleton for their assembly (11), nascent gp80 adhesion
complexes appear to assemble with minimal cytoskeleton association. As
cells come into contact, the trans-homophilic interaction of
gp80 has the potential to cross-link the gp80 cis-oligomers into large adhesion complexes, and we have proposed that associated rafts are cross-linked and coalesced into enlarged raftlike domains as
a result (20, 32). Indeed, gp80 is able to assemble functional adhesion
complexes at cell-cell contacts of ponticulin-null cells, despite their
lack of cytoskeleton association. Consistent with this observation,
model membrane studies have demonstrated that gp80-gp80 interactions
alone can produce extensive expansion of intermembrane contacts (42).
Furthermore, large sterol-rich domains have been found at sites of
gp80-mediated cell-cell adhesion (20), as they have been observed at
bacterial attachment sites to macrophages (43) and mast cells (44).
Large domains enriched in raft lipids have also been reported for
cell-cell contacts among immune cells (45).
The formation of the nascent gp80 adhesion complexes may be critical
for the recruitment of ponticulin. In the formation of large raftlike
domains at cell-cell contacts, gp80 could relocalize the pool of
ponticulin normally partitioned into rafts found throughout the plasma
membrane. Indeed, gp80 is required for the accumulation of ponticulin
at cell-cell contacts (Fig. 7). Additionally, gp80 may promote the
recruitment of ponticulin into rafts. This effect is evident from the
markedly reduced level of ponticulin in TIFF isolated from gp80-null
cells (Fig. 6B). Since cross-linking and coalescence of
rafts at the nascent adhesion complexes would dramatically decrease the
ratio between the circumference and the interior area of the domains,
there might be fewer chances for ponticulin to transfer to the nonraft
part of the membrane, and its partitioning into the expanded raftlike
domains could increase. This effect may be particularly important for
the recruitment of ponticulin, since its association with TIFF is more
sensitive to sterol sequestration than gp80 and other TIFF components.
Ponticulin is required for the assembly of stable gp80 adhesion
complexes that co-fractionate with the actin-myosin cytoskeleton. Ponticulin is also known to assemble extensive networks of actin filaments in close apposition with the plasma membrane at the basal
cell surface (33). Thus, ponticulin may organize similar actin networks
at gp80 complexes. In fact, a large proportion of ponticulin is
apparently involved in TIFF-cytoskeleton interactions, and
ponticulin-based changes to cytoskeleton structure are evident at
gp80-mediated cell-cell contacts. Intriguingly, ponticulin displays
enhanced actin-binding activity with specific but unidentified lipids
(46). Certain lipids have been found to mediate raft-cytoskeleton interactions. For example, phosphatidylinositol 4,5-bisphosphate mediates interactions from intracellular rafts to the actin and tubulin
cytoskeletons in mammalian cells (47, 48). Additionally, the raft
protein comitin that mediates interactions between Golgi vesicles and
the actin cytoskeleton in Dictyostelium (49) has been
localized to gp80-mediated contacts (31). It is, therefore, possible
that other factors may be involved in the interactions between gp80
adhesion complexes and the cytoskeleton.
The effects on gp80 in ponticulin-null streams suggest that the
raftlike scaffold linking gp80 and ponticulin may mediate interactions
both to and from the cytoskeleton. We speculate that the aberrant
length and positioning of gp80 adhesion complexes in ponticulin-null
streams may reflect a role for ponticulin in coordinating gp80-mediated
cell-cell adhesion with cell motility. Actin-based cell motility occurs
through dynamic F-actin turnover that drives protrusions at the leading
edge and retrograde F-actin flow coupled to myosin-based contractility
that pulls the rear of the cell forward (50, 51). Since gp80-mediated
adhesion is insensitive to actin depolymerization with latrunculin B,
it may also be unaffected by natural F-actin turnover at the leading edge. It is conceivable that gp80-mediated cell-cell adhesion and
individual cell motility are coordinated through coupling to the myosin
II contractile system via ponticulin.
The gp80 adhesion complex is a paradigm for GPI-anchored cell adhesion
molecule complexes. The complex shares structural features with
adhesion complexes of transmembrane receptors, including receptor
oligomerization and cytoskeleton attachment, but is supported structurally and functionally by raftlike membrane domains (31, 32). We
have shown that the gp80 adhesion complex recruits the actin
cytoskeleton via ponticulin. The GPI-anchored receptors CD48 and CD59
also act through rafts to recruit the actin cytoskeleton in response
the antibody cross-linking or ligand binding in the immune system (52,
53). Additionally, the actin cytoskeleton is recruited to bacterial
attachment sites on HeLa cells through the raft component annexin II
(54). Annexin II also mediates raft-cytoskeleton interactions in
epithelial cells (55) and in muscle cells (56). To better understand
the interactions between adhesion complexes and the actin cytoskeleton,
it would be important to identify additional raft-cytoskeleton adapter proteins and determine their functions in various cellular processes associated with cell-cell adhesion.