From the Banting and Best Department of Medical
Research and § Department of Biochemistry, University of
Toronto, Toronto, Ontario M5G 1L6, Canada,
Integrative
Proteomics Inc., Toronto, Ontario M5G 1L5, Canada, and the ** Department
of Biology, Concordia University,
Montreal, Quebec H3G 1M8, Canada
Received for publication, November 3, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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We have isolated and characterized a
Triton-insoluble floating fraction (TIFF) from
Dictyostelium. Ten major proteins were consistently
detected in TIFF, and six species were identified by mass spectrometry
as actin, porin, comitin, regulatory myosin light chain, a novel member
of the CD36 family, and the phospholipid-anchored cell adhesion
molecule gp80. TIFF was enriched with many acylated proteins. Also, the
sterol/phospholipid ratio of TIFF was 10-fold higher than that of the
bulk plasma membrane. Immunoelectron microscopy showed that TIFF has
vesicular morphology and confirmed the association of gp80 and comitin
with TIFF membranes. Several TIFF properties were similar to those of
Dictyostelium contact regions, which were isolated as a
cytoskeleton-associated membrane fraction. Mass spectrometry
demonstrated that TIFF and contact regions shared the same major
proteins. During development, gp80 colocalized with F-actin, porin, and
comitin at cell-cell contacts. These proteins were also recruited to
gp80 caps induced by antibody cross-linking. Filipin staining revealed
high sterol levels in both gp80-enriched cell-cell contacts and gp80
caps. Moreover, sterol sequestration by filipin and digitonin inhibited
gp80-mediated cell-cell adhesion. This study reveals that
Dictyostelium TIFF has structural properties previously
attributed to vertebrate TIFF and establishes a role for
Dictyostelium TIFF in cell-cell adhesion during development.
Cell membranes are thought to exist primarily in a fluid, liquid
crystalline phase. However, certain membranes display elevated acyl
chain order and exist in a liquid-ordered phase. These membranes exhibit Triton X-100 insolubility and can be separated from other insoluble cellular material by floatation into density gradients after
isopycnic centrifugation (1-3). We will refer to these membranes as a
Triton X-100-insoluble floating fraction
(TIFF).1 TIFF has distinctive
structural properties and is involved in a variety of cellular
functions. TIFF is typically isolated as membrane vesicles (4-6).
These membranes are enriched in lipids, such as cholesterol and those
with saturated acyl chains, which are expected to pack closely within
the liquid-ordered environment (7). In addition, many cell
membrane-associated structural and signaling proteins have been found
in TIFF (8). TIFF proteins are often anchored to the membranes through
a lipid moiety (7).
TIFF was originally characterized in vertebrate cells (4, 5). However,
TIFF analyses have been extended to yeast and Drosophila (6,
9, 10), and a CHAPS-insoluble floating fraction (CHIFF) has been
reported in Dictyostelium discoideum (11). The physiological
importance of TIFF has been shown by perturbation of TIFF protein
activities by cholesterol sequestration and through the co-localization
of TIFF components at sites of cellular activity. Processes that
involve TIFF include cellular trafficking (7), T-cell signaling
(12-16), integrin signaling (17, 18), and bacterial interactions with
macrophages (19) and mast cells (20).
Dictyostelium is a favorable model organism for the study of
plasma membrane structure and function. Dictyostelium is
amenable to both biochemical and molecular genetic analyses of its
cellular and developmental processes. During Dictyostelium
development, unicellular amoeboid cells aggregate through chemotaxis
toward cAMP and embark on a multicellular developmental program (21). Three components of the cAMP signaling pathway (the cAMP receptor cAR1,
adenylate cyclase, and the cell surface phosphodiesterase) are found in
CHIFF, suggesting that they are components of specialized microdomains
on the plasma membrane (11).
Multicellularity in Dictyostelium is maintained by several
cell adhesion molecules, including DdCAD-1/gp24 (22-24), gp150/LagC (25-27), and gp80 (28, 29). gp80 is expressed during the aggregation stage of development (30-32) and mediates the so-called contact sites
A by a Ca2+/Mg2+-independent homophilic binding
mechanism (33-35). gp80 is both necessary for strong adhesion during
development (36-38) and sufficient for the aggregation of otherwise
nonadhesive vegetative cells (39, 40).
It is apparent that gp80-gp80 interactions mediate cell-cell adhesion
among Dictyostelium cells. However, structural details of
gp80 adhesion complexes are largely unknown. Intriguingly, gp80 is
enriched in Triton X-100-insoluble, cytoskeleton-associated contact
regions (41). This subcellular fraction contains stacked membranes with
dimensions of intact cell-cell contacts and can only be isolated after
the cell aggregation stage of development. Considering that gp80 is
phospholipid-anchored (42, 43), we hypothesized that these Triton
X-100-insoluble contact regions may be a form of TIFF and that gp80
adhesion complexes may be organized as distinct membrane domains within
the plasma membrane.
In the present study, we have isolated Dictyostelium TIFF,
characterized its morphology, identified its protein constituents, and
analyzed its lipid composition. We demonstrate that TIFF membranes share many common physical and biochemical properties with the Triton
X-100-insoluble contact regions. Furthermore, a role for TIFF
components during gp80-mediated adhesion is established through colocalization, co-capping, and adhesion perturbation studies.
Cell Growth and Development--
Dictyostelium cells,
including the wild-type axenic strain AX2 and the gp80-null strain GT10
(36), were cultured either in association with Klebsiella
aerogenes or axenically in HL5 liquid medium (44). For
development, cells at the late exponential growth phase were collected,
washed, and resuspended at 1.5 × 107 cells/ml in
MCM buffer (2 mM MgCl2, 0.2 mM
CaCl2, 20 mM MES, pH 6.8) (45) and then shaken at
180 rpm. To stimulate gp80 expression, cells were pulsed with cAMP at a
final concentration of 2 × 10 Construction of GFP-Comitin Vector and Cell
Transformation--
A GFP-comitin construct was produced by
end-filling an EcoRI fragment containing the comitin
cDNA (46), followed by subcloning into the end-filled
HindIII site of the Dictyostelium expression vector pBS18-74E (obtained from Dr. R. Firtel, University of California at San Diego, La Jolla, CA). Expression of comitin was under the control of the actin 15 promoter. The BamHI/BglII
fragment of pBS-GFPII (obtained from Dr. H. MacWilliams, Ludwig
Maximillian University, Munich, Germany), containing the entire coding
region of GFP, was then subcloned into the BglII site of
comitin in the resulting plasmid. Hence, GFP was inserted in-frame
between Arg15 and Ser16 of comitin. Plasmid DNA
was introduced into AX2 cells by the calcium phosphate co-precipitation
method (47). Stable transformants were selected and maintained in HL5
medium containing 20 µg/ml of G418.
Isolation of TIFF--
TIFF was isolated from cell aggregates
that were collected at 12 h of development and gently resuspended
at 5 × 107 cells/ml in cold buffer 1 (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 centrifuged at 14,500 × g, and the pellet was washed with buffer 1, centrifuged at
14,500 × g, washed with buffer 2 (1 mM
EGTA, 5 mM Tris-HCl, pH 7.6), and centrifuged at
14,500 × g. The resulting pellet was resuspended in
buffer 2 and mixed at a 1:2 ratio with 60% (w/w) sucrose, placed at
the bottom of a centrifuge tube, and then overlaid with an 11-ml
continuous gradient of 10-40% (w/w) sucrose. The gradients were
centrifuged at 120,000 × g for 15-17 h at 2 °C
using a Beckman SW40 rotor. The TIFF material banded at ~34% sucrose
was collected. In subsequent experiments, TIFF was isolated in
discontinuous gradients from the interface between 28 and 38% sucrose.
TIFF collected from the sucrose gradients was washed with 20 mM sodium phosphate buffer, pH 7.6, and then pelleted by
centrifugation at 39,000 × g for 20 min at
2 °C.
Isolation of Plasma Membranes and Triton X-100-insoluble Contact
Regions--
Plasma membranes were isolated using the aqueous
two-phase polymer system (48). Triton X-100-insoluble contact regions
were isolated according to Ingalls et al. (41). Cells were
treated with 0.2% Triton X-100 at 4 °C and then centrifuged at
4000 × g. The pellet was washed and resuspended in
buffer 2 and then layered on the top of a discontinuous gradient of 5.5 ml of 50% and 5.5 ml of 60% (w/w) sucrose in 20 mM
phosphate buffer, pH 6.8, for centrifugation at 120,000 × g for 3 h at 2 °C. The material banded at the
interface was collected, washed, and dialyzed in a cytoskeleton
depolymerizing solution containing 0.1 mM EDTA, 0.2 mM sodium phosphate buffer, pH 7.6. The dialyzed material was pelleted, resuspended in 1.5 ml of buffer 2, and layered on top of
an 11-ml continuous gradient of 26-51% (w/w) sucrose. The gradient
was centrifuged at 120,000 × g for 3 h at
2 °C. Membranes banded at 32-34% sucrose were collected.
Gel Electrophoresis--
Protein concentrations were determined
using the bicinchoninic acid protein assay kit (Pierce). For SDS-PAGE,
proteins were solubilized and reduced by boiling for 5 min in 3% (w/v)
SDS, 3 M urea and 5% (v/v) MALDI-TOF Mass Spectrometry--
For protein identification by
mass spectrometry, silver-stained gel bands were excised, macerated,
reduced, alkylated, and then digested with trypsin. The tryptic
peptides were extracted using the protocol of Shevchenko et
al. (50). The peptides were purified using Sephasil C18 resin,
applied to MALDI plates, and mixed with a matrix solution of
Immunogold Electron Microscopy--
Cells expressing GFP-comitin
were collected at 10 h of development for TIFF isolation. TIFF
membranes were incubated overnight with anti-gp80 mAb 80L5C4 (29) or
anti-GFP rabbit antibody (Molecular Probes, Inc., Eugene, OR) in TBS
(20 mM Tris, pH 7.6, 137 mM NaCl) plus 0.1%
(w/v) bovine serum albumin, with rotation at 4 °C. After washing,
samples were incubated with either goat anti-mouse or goat anti-rabbit
antibodies conjugated to 10-nm gold (Sigma) at 1:10 dilution in TBS
plus 0.1% (w/v) bovine serum albumin. Samples were rotated overnight
at 4 °C. After four washes, pellets were fixed overnight at 4 °C
with 1% (v/v) glutaraldehyde in 0.1 M sodium phosphate
buffer, pH 7.2. After several washes, the pellets were incubated in 1%
(w/v) OsO4 in phosphate buffer for 30 min at room
temperature. Samples were dehydrated with an ethanol series followed by
propylene oxide and then embedded in Spurr's standard resin. Ultrathin
sections were cut with a diamond knife and then stained with uranyl
acetate and lead citrate prior to examination under a Hitachi 8600 analytical transmission electron microscope.
In Situ Triton X-100 Extraction--
To perform Triton X-100
extraction of living cells, cell aggregates on coverslips were chilled
to 4 °C by replacing the MCM buffer with cold buffer 1 and placing
the coverslips on an ice water bath for 15 min, and Triton X-100 was
added to a final concentration of 0.2% (v/v). After 5 min, the Triton
X-100 solution was aspirated, and the coverslips were fixed with cold
10% (v/v) formaldehyde for 10 min on ice, followed by 3.7% (v/v)
formaldehyde at 22 °C for 10 min. Cells were stained and prepared
for confocal microscopy.
Metabolic Labeling with Palmitic Acid--
Cells were developed
at 2 × 107 cells/ml for 2 h in 17 mM
phosphate buffer, pH 6.1, containing streptomycin (0.5 mg/ml). Cells were collected and resuspended in phosphate buffer containing 0.1 mCi/ml of [9,10-3H]palmitic acid (PerkinElmer Life
Sciences) and developed for another 6 h with cAMP pulsing. Cell
aggregates were collected, and proteins in different subcellular
fractions were analyzed by SDS-PAGE. Protein blots were coated with
EN3HANCE spray (PerkinElmer Life Sciences) and exposed to
Biomax MR film (Eastman Kodak Co.) at Lipid Analyses--
Samples were analyzed by gas-liquid
chromatography as described previously (51). Briefly, sample lipids
were extracted with chloroform/methanol (2:1, v/v) and digested with
phospholipase C (Clostridium welchii). The mixture was
extracted with chloroform/methanol (2:1, v/v) containing 100 µg of
tridecanoylglycerol as an internal standard. The samples were then
incubated in SYLON BFT plus 1 part dry pyridine for 30 min at 20 °C.
All extracted lipids are converted into neutral species by this
procedure, and they were quantified after separation on a nonpolar
capillary column.
Cell Staining--
Cells were developed for 12 h on
coverslips and then fixed, stained, and mounted as previously described
(52, 53). Samples were first incubated with primary antibodies against
gp80 or porin, followed by Alexa 568/488-conjugated secondary
antibodies (Molecular Probes) at 1:300 dilution. Filamentous actin
(F-actin) was stained with fluorescein-phalloidin (Molecular Probes) at
1:10 dilution. Laser-scanning confocal microscopy was performed using a
Zeiss Axiovert 135 inverted microscope equipped with a × 63 Neofluor objective and an LSM 410 confocal attachment. Detection was
maintained within the range of the gray scale to prevent signal saturation.
For filipin staining, cells were grown axenically, developed on
coverslips, and then fixed in 3.7% (v/v) formaldehyde in MCM buffer
for 15 min at room temperature. Cells were incubated with 0.025% (w/v)
filipin in MCM buffer for 15 min at room temperature, washed, and then
mounted for epifluorescence microscopy.
Capping of Cell Surface Proteins--
Cells were developed in
suspension for 6-8 h with cAMP pulsing. Cell aggregates were dispersed
and deposited on coverslips in MCM buffer (3 × 105
cells/coverslip). After 10 min, anti-gp80 mAb or polyclonal antibodies were added to cells for 15 min at room temperature. The coverslips were
washed gently with two changes of 10 ml of MCM buffer for 3 min. Alexa
568-conjugated secondary antibodies were added at 1:50 dilution for 15 min, followed by two washes. The cells were fixed at 40 min after the
initial addition of the primary antibody. To identify co-capped
molecules, fixed cells were stained with appropriate antibodies.
Cell Cohesion and Cell Dissociation Assays--
Cell cohesion
was assayed as described previously (54). Alternatively, the effects of
various reagents on cell cohesion were assayed by measuring aggregate
dissociation under high shear force. Cells were cultured on bacteria
plates and then collected for development in liquid culture at 2 × 107 cells/ml. At different time points, cell aliquots
were taken and diluted in 17 mM phosphate buffer, pH 6.1, plus the reagent under test. Stock solutions of filipin and digitonin
were prepared fresh in Me2SO prior to each experiment.
Equivalent amounts of Me2SO were added to control samples.
Cell aggregates were incubated in various reagents for 10 min at room
temperature with gentle shaking. Shear force was then applied by
continuous vortexing for 30 s using a Vortex Genie 2 at setting 8. Cell dissociation was quantified by counting cells with a
hemacytometer. Singlets, doublets, and triplets were scored as
dissociated cells, and the percentage of cell dissociation was
calculated relative to the number of cells obtained at 0 h, which
was ~2 × 106 cells/ml.
Isolation and Characterization of TIFF--
TIFF was isolated from
Dictyostelium cells after 10-12 h of development in liquid
medium. After fractionation of detergent-insoluble material in a
continuous sucrose density gradient (Fig.
1A), TIFF formed a sharp band
at ~34% sucrose (Fig. 1A, lane 9),
whereas the cytoskeleton pelleted (Fig. 1A, lane
13). The highly resolved banding pattern suggested that TIFF
had a relatively homogeneous composition. This isolation protocol
routinely yielded 0.8-0.9 mg of TIFF protein from 1010
cells.
Silver staining of TIFF proteins revealed a highly reproducible profile
(Fig. 1B). The 10 most strongly stained bands were designated with "t" followed by their apparent molecular mass in
kDa. Protein identification was attempted using MALDI-TOF mass spectrometry and data base search engines. Protein bands designated t103, t88, t43, t28, t23, and t20 were identified to be the
Dictyostelium expressed sequence tag (EST) C84888, gp80,
actin, porin, comitin, and regulatory myosin light chain (RMLC),
respectively (Fig. 1C). These proteins produced the
strongest mass spectra of the 10 analyzed. As an example, the mass
spectrum of t103 is shown (Fig. 1D). Their identifications
displayed similar ranks and scores, based on two search engines. They
each had similar sequence coverage with their matches and displayed the
expected electrophoretic mobility.
Since the identification of t103 was limited to one search engine, a
postsource decay analysis was performed (Fig. 1, D and E). Every postsource decay product of the 2498-Da peptide
corresponded to the predicted sequence of the EST, thus confirming the
match (Fig. 1E). To annotate the EST, the position-specific
iterated basic local alignment search tool was used to search for
similar proteins. The search results showed that the C termini of
members of the CD36 family had the highest scores, and t103 was
therefore designated DdCD36. The identities of gp80, actin, porin, and
comitin were confirmed by Western blot analysis (Fig.
1F).
Since proteins anchored to the plasma membrane via a lipid moiety are
known to be associated with TIFF (7), we labeled cells metabolically
with [3H]palmitic acid and determined whether labeled
proteins were enriched in the TIFF fraction. Labeled cells were
fractionated into three parts: the Triton X-100-soluble fraction, the
Triton X-100-insoluble pelleted fraction after floatation
centrifugation, and TIFF. Equal amounts of protein from each fraction
were separated by SDS-PAGE and compared after fluorography (Fig.
2). TIFF was enriched with many
palmitoylated proteins. Several intensely labeled species displayed gel
mobilities corresponding to gp80, t46, actin, RMLC, and t18, suggesting
that these proteins or some co-migrating species were heavily
palmitoylated.
Enrichment of Sterols in TIFF--
Since membrane insolubility in
cold Triton X-100 is a characteristic of liquid-ordered membrane
structure (1-3), we expected Dictyostelium TIFF to be
enriched in lipids conforming to this structure. Total lipids in plasma
membranes and TIFF were analyzed and compared (Table
I). TIFF contained a 15-fold higher
sterol level and a 1.5-fold higher phospholipid level than the plasma membrane. The elevated lipid/protein ratios in TIFF probably accounted for its low density (1.13-1.14 g/ml). The sterol/phospholipid ratio in
TIFF was ~10-fold higher than that of plasma membranes. The sterol
species in TIFF were identified to be stigmasterol (78%), campesterol
(14%), and sitosterol (8%) based on column retention times. The
stigmasterol species was probably Morphological Characterization of Dictyostelium TIFF--
To
assess the effects of Triton X-100 extraction on the plasma membrane of
cells, the subcellular distribution of gp80 was examined, since it was
the most prominent TIFF protein and a good plasma membrane marker.
Cells developed on coverslips were extracted with Triton X-100 and then
fixed prior to staining with anti-gp80 mAb. Confocal microscopy
revealed a lattice-like pattern of gp80 staining along the cell
periphery, suggesting the formation of gp80-enriched vesicular
structures on the plasma membrane (Fig. 3A).
The morphology of TIFF was examined by electron microscopy. Membrane
vesicles with diameters ranging from 0.1 to 1.0 µm were observed
(Fig. 3, B-D). These membranes often stacked upon one another to form multilayered structures similar to those observed for
the Triton X-100-insoluble contact regions (41). The samples appeared
relatively homogeneous and were devoid of other membranous organelles.
Since antibodies against comitin showed cross-reactivity with several
protein bands, transformants expressing GFP-comitin were made.
GFP-comitin could be detected specifically using an anti-GFP antibody,
and immunoblot analysis showed that the fusion protein was enriched in
TIFF (data not shown). Immunogold labeling of gp80 and GFP-comitin
showed the association of both proteins with the TIFF membranes (Fig.
3, B and C). gp80 displayed a patchy distribution, which was observed over a 10-fold range of antibody concentrations, while GFP-comitin was distributed more evenly along the membranes.
Co-capping of F-actin, Comitin, and Porin with gp80--
Since
gp80 is the predominant protein in TIFF and Triton-insoluble gp80
represented 50-55% of total cellular gp80, it was important to
determine whether gp80 existed together with other TIFF components as
complexes in the plasma membrane of intact cells. If this was the case,
clustering of gp80 on living cells by antibody cross-linking should
lead to the co-capping of other TIFF proteins. Cells were induced to
form gp80 caps, and double immunofluorescence labeling revealed the
co-capping of both porin and F-actin with gp80 (Fig.
4, A, B,
E, and F). GFP-comitin was also found to
co-localize with gp80 caps (Fig. 4, C and D).
Therefore, porin, F-actin, and comitin were membrane-associated
components that could be induced to form large complexes with gp80 via
antibody cross-linking. In addition, cross-linking of gp80 induced cell rounding, indicative of cytoskeleton reorganization.
Subcellular Localization of Sterols by Filipin
Staining--
Results presented earlier revealed high levels of
sterols in the gp80-enriched TIFF membranes (see Fig. 1 and Table I).
The relationship between gp80 and sterols was examined further by capping gp80 on living cells, which were then fixed and stained with
filipin. Filipin staining revealed a high concentration of sterols
associated with gp80 caps (Fig. 5,
A and B). In addition to gp80 caps,
co-localization of sterols with gp80 at cell-cell contacts was observed
(Fig. 5, C and D). To avoid perturbations by
antibody exposure, cells were also stained with filipin without prior
incubation with anti-gp80 mAb. Epifluorescence microscopy again
revealed high sterol levels at cell-cell contacts between aggregating
cells (Fig. 5E). These results indicate that sterols are
enriched in cell-cell contact regions as well as TIFF.
Co-localization of TIFF Proteins at Cell-Cell Contacts--
The
subcellular distribution of TIFF proteins in aggregating cells was
examined by confocal microscopy. Co-localization of gp80 and F-actin
was evident at cell-cell contact regions (Fig. 6, A and B). Double
staining for gp80 and porin also revealed their co-localization at
cell-cell contact regions (Fig. 6, C and D). In
addition, porin displayed punctate cytoplasmic staining, consistent
with its mitochondria association (56). Finally, GFP-comitin was also
co-localized with gp80 at cell-cell contact regions (Fig. 6,
E and F). In addition to contact regions,
GFP-comitin displayed punctate cytoplasmic staining.
Comparison of Triton-insoluble Contact Regions with TIFF--
The
Triton X-100-insoluble contact regions were also enriched in gp80 (41),
suggesting that this subcellular membrane fraction might be related to
TIFF. To compare the properties and composition of these two membrane
fractions, contact regions were isolated from cell aggregates at 10-12
h of development (Fig. 7). In contrast to
TIFF, the contact regions co-fractionated initially with the cytoskeleton and displayed a relatively high density after equilibrium centrifugation. Following actin depolymerization, the cytoskeleton-free contact regions shifted to a lower density and formed a highly resolved
band between 32 and 34% sucrose after equilibrium centrifugation through a continuous sucrose gradient. This density was similar to that
of TIFF. The silver-stained protein profile of the purified contact
regions was also similar to that of TIFF (Fig. 7). Protein bands were
excised from the gel, digested with trypsin, and analyzed by MALDI-TOF
mass spectrometry. The 10 proteins with the strongest mass spectra were
designated with "cr" followed by their apparent molecular mass in
kDa. The contact region proteins cr22, cr28, cr42, cr88, and cr110 were
identified as comitin, porin, actin, gp80, and DdCD36, respectively
(Fig. 7). Proteins lacking positive identifications were also shared
between the TIFF and contact region fractions. Lipid analysis showed
that the isolated contact regions were also highly enriched in sterols
(data not shown). Thus, TIFF and Triton X-100-insoluble contact regions
share many common properties, although the latter are associated
with the cytoskeleton.
Sterol Sequestration Perturbs Both TIFF Recovery and Cell-Cell
Adhesion--
In vertebrates, cholesterol is required for the
structural integrity of TIFF (57). Although Dictyostelium
cells do not synthesize cholesterol, the above results show that TIFF
and intact cell-cell contacts are enriched in sterols. Both filipin and
subcritical micelle concentrations of digitonin are known to sequester
sterols within Dictyostelium plasma membranes (58).
Therefore, we tested whether these reagents had an effect on the
structural integrity of TIFF. When cell aggregates were treated with
either 0.01% digitonin or 0.004% filipin prior to Triton X-100
extraction, TIFF recovery was reproducibly reduced to less than 10% of
their respective controls (Fig.
8A).
The effects of sterol sequestration on gp80-mediated cell-cell adhesion
were also examined. The EDTA-sensitive cell adhesion sites mediated by
DdCAD-1 are fully developed during the first few hours of development
(23, 59), and they can be easily disrupted by mechanical force. The
gp80-dependent EDTA-stable adhesion sites appear later and
confer stronger adhesion among cells (36-38). At first, cell-cell
adhesion was monitored using the established cell reassociation assay
(54). At low concentrations when cell viability was not affected,
neither digitonin nor filipin had a significant effect on cell
reassociation (data not shown). Since similar concentrations have
perturbed cellular distributions and functions of vertebrate TIFF
proteins (60, 61), it was possible that a more stringent assay was
needed to detect perturbation of the strong adhesion mediated by gp80.
Therefore, a cell dissociation assay was developed to monitor the
resistance of cell aggregates to high shear forces. At 0 and 3 h
of development, cells were subjected to high shear forces, and
microscopic examination revealed complete cell dissociation at both
time points (Fig. 8B). The small drop in the cell
dissociation curve at 3 h was probably due to cell loss via
attachment to the container surface with development. After 6 h,
aggregates that resisted shear forces were observed, and the number of
dissociated cells was diminished. Cell-cell adhesiveness was further
strengthened by 9 h. The resistance of cell dissociation coincided
temporally with the accumulation of high levels of gp80, suggesting
that this strong cell-cell adhesion was mediated by gp80. Cell
aggregates of gp80-null cells, formed through the EDTA-sensitive
adhesion molecules, were subjected to high shear forces and microscopic
examination revealed complete cell dissociation at all developmental
time points (Fig. 8B).
Next, we determined whether the gp80-dependent resistance
to shear forces was sensitive to treatment with low concentrations of
filipin and digitonin. Incubation of cells with either 0.005% filipin
or 0.001% digitonin for 10 min led to high levels of cell dissociation
at all time points (Fig. 8C). Sterol sequestration is a
common activity of these agents that otherwise have dissimilar cellular
effects (62). To control for other effects, such as the formation of
transmembrane pores, cells were incubated with 0.001% Triton X-100.
This treatment had no effect on the resistance of cell aggregates to
shear forces. Both filipin and digitonin treatments yielded results
similar to those obtained with the gp80-null cells, consistent with the
idea that the adhesive function of gp80 is sensitive to sterol sequestration.
We have isolated and characterized TIFF from
Dictyostelium cells. In many ways, Dictyostelium
TIFF is similar to other detergent-insoluble floating fractions. Its
protein components have counterparts in vertebrate TIFFs, such as CD36,
actin, porin, and RMLC (8). Dictyostelium TIFF is highly
enriched in gp80 (an analogue of GPI-anchored proteins), palmitoylated
proteins, and sterols, as found in vertebrate TIFF (1, 7). Furthermore,
Dictyostelium TIFF has the common vesicular TIFF morphology
(4-6).
High levels of cholesterol and ergosterol have been found in TIFF (4,
6, 9). Although Dictyostelium cells synthesize primarily
stigmasterol, campesterol, and sitosterol, the sterol content of
Dictyostelium TIFF is 15-fold higher than that of plasma membranes. Another notable feature of Dictyostelium TIFF is
its relatively high sterol/phospholipid molar ratio in comparison with
vertebrate TIFF (4) and Drosophila TIFF (9). The lipids of
Dictyostelium CHIFF are also predominantly sterols (11). These high sterol levels may be due to the high level (75-90%) of
unsaturated fatty acid side chains in Dictyostelium (55). The unsaturated acyl chains would not conform to the liquid-ordered membrane structure expected for TIFF and CHIFF, but they probably allow
membrane fluidity at the normal Dictyostelium growth
temperature of 22 °C. These distinct lipid compositions of the bulk
plasma membrane versus the detergent-insoluble floating
fractions may have pronounced effects on the structure and function of
the sterol-rich domains in Dictyostelium membranes.
Although TIFF and CHIFF have a similar lipid composition, these
fractions contain different proteins. The cAMP receptor cAR1 is the
predominant protein in CHIFF, but it was absent from TIFF, and the
opposite is true for the cell adhesion molecule gp80 (11). gp80 and
cAR1 also display distinct subcellular distributions during cell
aggregation. gp80 becomes concentrated in cell-cell contact regions
(52), where it co-localizes with other TIFF proteins and sterols,
whereas cAR1 is evenly distributed over the plasma membrane (63).
Therefore, the Dictyostelium plasma membrane may be
organized as a mosaic of domains with specialized functions. Similarly,
different types of detergent-insoluble domains have been shown to
segregate within the plasma membranes of vertebrate cells (64, 65).
This compartmentalization of specialized membrane components could
enable cells to distinguish and efficiently respond to various
external stimuli.
Several lines of evidence suggest that TIFF components form membrane
domains that are involved in gp80-mediated adhesion. First, gp80
co-localizes with TIFF proteins and sterols in large membrane domains
involved in cell-cell contact formation. Second, co-capping experiments
show that TIFF components are directly or indirectly linked to gp80.
Third, TIFF shares many properties with the Triton-insoluble contact
regions (41). Finally, sterol sequestration reduces TIFF recovery and
perturbs gp80-dependent intercellular cohesiveness.
How do TIFF properties influence the strong cell-cell binding activity
mediated by gp80? Typically, adhesion is strengthened when adhesion
molecules cluster to form large complexes that are associated with the
cytoskeleton. For transmembrane cell adhesion molecules, their
cytoplasmic domains can interact with adapter proteins that link to the
cytoskeleton (66-68). In contrast, GPI-anchored adhesion molecules
lack direct access to the cytoskeleton. However, TIFF membranes could
produce a lipid scaffold for the assembly of both extracellular and
cytoplasmic proteins. Specifically, a membrane domain consisting of
liquid-ordered lipids may stabilize adhesion complexes by both
clustering GPI-anchored adhesion molecules and facilitating their
interactions with the cytoskeleton. Indeed, we have observed that
gp80-mediated contacts encompass membrane domains, which are enriched
in sterols and associated with the cytoskeleton. Moreover, we found
that the Triton-insoluble contact regions were basically
cytoskeleton-associated forms of TIFF. Therefore, we speculate that
sterol sequestration perturbed gp80-mediated adhesion by disrupting
liquid-ordered membrane scaffolds within gp80 adhesion complexes.
We have mapped TIFF components to large membrane domains involved in
cell-cell contact formation. However, TIFF can also be derived from
plasma membrane microdomains termed rafts (7, 62). Thus, it is possible
that gp80 adhesion complexes may form from precursory rafts. In fact,
immune electron microscopy studies have revealed gp80 clusters
on single cells (69). Most clusters had diameters between 30 and 70 nm,
values remarkably close to sizes ascribed to vertebrate rafts (70, 71).
Thus, it is likely that gp80 exists within raft-like precursors on the
cell surface prior to cell-cell contact. Such rafts may be primed for
efficient adhesion complex construction. Preassembly of components
within rafts would facilitate rapid complex formation, and avid
trans interactions between gp80 clusters could expand the
microdomains, eventually leading to the formation of a large cell
adhesion complex.
A variety of molecular mechanisms likely underlie cell-cell adhesion.
Cell-cell adhesion mediated by lipid-anchored adhesion molecules, such
as gp80, is inherently distinct due to the lipid moiety. Many
vertebrate cell adhesion molecules are GPI-anchored, including
NCAM-120, contactin/F3/F11, axonin-1/TAX-1/TAG-1 (72), T-cadherin (73),
neurin-1 (74), LFA-3 (75), and members of the IgLON family (76, 77). In
fact, several GPI-anchored cell adhesion molecules have been found in
TIFF (78-80). We have demonstrated a role for TIFF in gp80-mediated
adhesion that may involve lipid scaffolds and precursory rafts. It
would be of interest to determine whether TIFF is involved in adhesion
mediated by other lipid-anchored adhesion molecules. Moreover, adhesion
complexes constructed from TIFF may display unique regulation and
signaling. To explore such signaling and regulatory events, TIFF
component interactions that connect gp80 to the cytoskeleton are
currently under investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8
M every 7 min. Cells were also developed in MCM buffer on coverslips.
-mercaptoethanol and then
separated in a slab gel (49). For immunoblot analysis, bound antibodies
were detected using horseradish peroxidase-conjugated secondary
antibodies and the enhanced chemiluminescence kit (Amersham Pharmacia
Biotech). Immunoblots were quantified using the Bio-Rad Fluor-S Max
multiimager system.
-cyano-4-hydroxy-trans-cinnamic acid (Sigma) at 20 mg/ml
in 50% (v/v) acetone and 50% (v/v) isopropyl alcohol. The
samples were dried and then subjected to mass spectrometry. Peptide
masses were determined using a PerSeptive Biosystems Voyager Elite
MALDI-TOF mass spectrometer (PerSeptive Biosystems, Inc.) in the linear
mode, with 92% grid voltage, 0.15% guide wire voltage, laser
intensities between 1600 and 2100, and delayed extraction of 200 ns.
Trypsin autolysis products and matrix molecules were used for
calibration. The remaining masses were submitted to the ProFound search
engine and the Protein Prospector search engine (both available on the
World Wide Web) for matches. Search parameters were held
constant, including tolerance for peptide mass error of ±1 Da,
tolerance for protein mass error of ±10 kDa from apparent molecular
masses determined by SDS-PAGE, and a maximum of one missed cut
per peptide.
70 °C for 3 weeks.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TIFF isolation and characterization of TIFF
proteins by mass spectrometry. A, a schematic drawing
of the isolation protocol is shown at the top. TIFF was
isolated from total Triton X-100-insoluble material by floatation into
a continuous sucrose density gradient. Equal volumes of the gradient
fractions were analyzed by SDS-PAGE and subjected to silver staining.
TIFF floated to ~34% (w/w) sucrose (lane 9,
arrowhead), whereas the material associated with the
cytoskeleton was pelleted (lane 13).
B, silver staining profile of TIFF proteins separated on a
12% polyacrylamide gel. The 10 most prominent bands are listed at the
right. C, summary of protein identification data
based on mass spectrometry and data base searches. All matches were the
top ranking Dictyostelium proteins from each search engine.
The ProFound scores represent probabilities with a maximum score of
1.0. The Protein Prospector utilizes MOWSE scoring that does not have a
defined upper limit. Sequence coverage refers to the maximum percentage
of an identified protein sequence that can be matched to peaks in the
mass spectra. The known mass refers to each candidate identified.
D, MALDI-TOF mass spectrum of t103. Peaks that matched
(error of <1 Da) the masses of peptides encoded by the EST C84888
sequence are marked with an asterisk. Peaks marked with a
T are trypsin autolysis products. E, postsource
decay analysis of the 2498-Da peak marked with two
asterisks in D. Decay products are marked with
the fragment ions expected from the decay of the corresponding peptide
encoded by EST C84888 (error of <1 Da). F, confirmation of
protein identity by immunostaining. Immunoblots of TIFF proteins were
stained with antibodies against gp80, actin, porin, and comitin.
View larger version (99K):
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Fig. 2.
Fluorograms of subcellular fractions of cells
labeled with palmitic acid. [3H]Palmitic acid was
added to cells at 2 h of development, and cell aggregates were
collected at 8 h and treated with 0.2% Triton X-100 at 4 °C
and then separated into three fractions: the Triton X-100-soluble
fraction (TSF), the Triton X-100-insoluble pelleted fraction
(TIPF) collected from the bottom of the floatation gradient,
and TIFF. Equal amounts of protein from these fractions were separated
by SDS-PAGE, blotted, and prepared for fluorography. The apparent
molecular masses in kDa of the major labeled proteins are indicated at
the right.
22-stigmasten-3
-ol
because it accounts for 88% of the sterols in Dictyostelium
cells (55). Both TIFF and plasma membranes had similar sterol
compositions.
Lipid compositions of Dictyostelium plasma membranes and TIFF
View larger version (60K):
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Fig. 3.
Immunolocalization of TIFF components and
TIFF morphology. A, a confocal image showing the
subcellular distribution of gp80 after in situ Triton X-100
extraction of 10-h cells. Living cells were treated with the detergent,
fixed, and then stained with anti-gp80 mAb. Most cells showed punctate
staining along the cell periphery. Bar, 5 µm. The
inset shows the boxed area at higher
magnification, where gp80 displayed a lattice-like staining pattern.
Bar, 0.5 µm. B-D, electron micrographs of
isolated TIFF material. TIFF samples were labeled with primary
antibodies followed by 10-nm colloidal gold-conjugated secondary
antibodies. B, immunogold labeling of gp80 on TIFF
membranes. C, TIFF was isolated from cells expressing
GFP-comitin, and comitin was labeled with antibodies against the GFP
tag. D, to control for antibody trapping, TIFF was probed
with secondary antibodies alone. Bars in
B--D, 0.25 µm.
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Fig. 4.
Co-capping of TIFF components with gp80.
Cells were collected at 10 h of development and incubated with mAb
against gp80, followed by a goat anti-mouse secondary antibody.
Subsequently, cells were fixed and permeabilized and then stained with
antibodies against porin and actin (GFP-comitin was used to visualize
comitin). Double-labeled coverslips were examined by confocal
microscopy. The membrane domains encompassed by the gp80 caps
(A, C, and E) were enriched with
F-actin (B), GFP-comitin (D), and porin
(F), respectively. Bars, 5 µm.
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Fig. 5.
Membrane distribution of sterols visualized
by filipin staining. A and B, cells were
developed on coverslips for 10 h and then incubated with anti-gp80
mAb to induce cap formation. After 40 min of incubation at room
temperature, cells were fixed with formaldehyde, and sterols were
stained with 0.025% filipin. Confocal images revealed sterol
enrichment (A) in membrane domains associated with gp80 caps
(B). In addition to gp80 caps, intense filipin staining
(C) coincided with gp80 staining (D) in cell-cell
contact regions (arrowheads). E, fixed cells that
were incubated with filipin alone also revealed high levels of sterol
at cell-cell contacts (arrowheads). Bars, 5 µm.
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Fig. 6.
Co-localization of TIFF proteins with gp80 in
cell-cell contact regions. Aggregating cells were double-stained
for several combinations of TIFF protein components. In each
vertical pair of panels, a region of
an aggregation stream, involving contacts between several cells, is
shown. Cells were double-stained for gp80 (A) and F-actin
(B) or for gp80 (C) and porin (D). In
addition, transformants expressing GFP-comitin (F) were
stained for gp80 (E). Bars, 5 µm.
View larger version (52K):
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Fig. 7.
Characterization of Triton X-100-insoluble
contact regions. A schematic drawing of the subcellular
fractionation steps is shown at the top. Silver-stained gel
profiles of proteins at consecutive steps of the isolation protocol are
shown below. Equal protein amounts were loaded per lane,
separated by SDS-PAGE, and then subjected to silver staining.
Lane 1, whole cell aggregates; lane
2, Triton-insoluble pellet; lane 3,
material banded at the 45%/60% sucrose interface; lane
4, postdialysis pellet; lane 5,
Triton-insoluble contact membranes banded at 32-34% sucrose. To the
right of the gel is a list of the 10 contact region proteins
(designated with "cr" followed by the apparent molecular mass in
kDa) with the strongest mass spectra. In the next
column, protein identification based on data base searches
is shown. In the far right column,
matching masses (error of <1.5 Da) between the contact region protein
and the corresponding TIFF protein (shown in parentheses) are shown.
Proteins were compared by first selecting the 10 strongest peaks in the
contact region protein and scoring matching peaks in the corresponding
TIFF protein. Then the opposite operation was performed. Thus, a total
of 20 peaks were compared between each pair of proteins, and the
comparisons are presented as fractions with the maximum of 20 possible
matches as the denominator.
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Fig. 8.
Effects of sterol sequestration on TIFF and
gp80-mediated cell-cell adhesion. A, the effect of
sterol sequestration on TIFF recovery was examined by incubating cell
aggregates with either 0.01% (w/v) digitonin or 0.004% (w/v) filipin
for 30 min on ice prior to TIFF isolation. The amounts of protein
recovered in TIFF were normalized to their respective control, and the
data represent the mean ± S.D. (n = 3).
B, intercellular cohesiveness assessed by cell dissociation
under high shear forces (see "Experimental Procedures"). Wild-type
AX2 cells ( ) and gp80-null GT10 cells (
) were
developed in liquid culture and collected at different time points for
the assay. The data represent the mean ± S.D. (n = 4). The inset shows the time course of gp80 protein
expression in wild-type cells (arrowhead). C, the
effect of sterol sequestration on cell-cell adhesion was
evaluated during development. Wild-type cells (AX2) were collected from
different developmental time points and incubated for 10 min with
0.001% (w/v) digitonin (
), 0.0005% (w/v) filipin
(
), or 0.001% (v/v) Triton X-100 (
). Cells were
then subjected to the cell dissociation assay. The data represent the
mean ± S.D. (n = 4). Controls for the
Me2SO carrier were indistinguishable from the wild-type
cells shown in B.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Opas and R. Reithmeier for advice and discussion, Dr. G. Gerisch for the gift of anti-porin mAb, and Tak Yee Lam for expert assistance. We also thank Steven Doyle and Battista Calveiri of the Electron Microscopy Laboratory of the Faculty of Medicine, University of Toronto for assistance with electron and confocal microscopy.
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FOOTNOTES |
---|
* This work was supported by Canadian Institutes of Health Research Operating Grant MT-6140.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada.
To whom correspondence should be addressed: Charles H. Best
Inst., University of Toronto, 112 College St., Toronto, Ontario M5G
1L6, Canada. Tel.: 416-978-8766; Fax: 416-978-8528; E-mail: chi.hung.siu@ utoronto.ca.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M010016200
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ABBREVIATIONS |
---|
The abbreviations used are: TIFF, Triton-insoluble floating fraction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHIFF, CHAPS-insoluble floating fraction; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; GFP, green fluorescent protein; RMLC, regulatory myosin light chain; GPI, glycosylphosphatidylinositol; PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag; mAb, monoclonal antibody; MES, 4-morpholine ethanesulfonic acid.
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