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Vol. 14, Issue 7, 2890-2899, July 2003
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*Université de Genève, Centre
Médical Universitaire, Département de Morphologie, CH-1211
Geneva, Switzerland; Laboratoire de Biochimie
et Biophysique des Systèmes Intégrés, Unité Mixte
Recherche 314, Centre National de la Recherche Scientifique, CEA, Grenoble,
France; and Institut de Biologie et de Chimie
des Protéines, Unité Mixte Recherche 5086 Centre National de la
Recherche Scientifique, Université Lyon I, 69367 Lyon, France
Submitted November 12, 2002;
Revised March 25, 2003;
Accepted March 25, 2003
Monitoring Editor: Mark Ginsberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Due to this high degree of conservation in sequence and
predicted topology (Chluba-de Tapia et
al., 1997; Schimmoller
et al., 1998), genes encoding TM9 proteins can be
unambiguously identified in eukaryotic genomes. The family includes many
members in organisms ranging from yeast to plants and human. Although
conservation throughout evolution strongly suggests an important biological
role, their function(s) remains largely unknown
(Chluba-de Tapia et al.,
1997).
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), and it was then proposed that TM9 proteins might represent
a family of adhesion receptors. Their variable extracellular domains could
confer distinct receptor specificities, whereas the conserved
membrane-associated domain would establish the link with the intracellular
machinery. However, this interpretation remains highly hypothetical and a
regulatory role in cellular adhesion was not excluded
(Cornillon et al.,
2000). To gain further insight into the function of proteins belonging to the TM9 family, knockouts of two genes encoding distinct members of the family were obtained and characterized in D. discoideum. Together, our results indicate that TM9 proteins play synergistic roles and strongly support a model where they function as regulators of cellular adhesion by regulating the protein composition of the cell surface.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Cells were grown at
20°C in HL5 medium (Cornillon et
al., 1998) and subcultured twice a week to maintain a density
of <106 cells/ml. D. discoideum cell transfection was
performed as described previously
(Cornillon et al.,
2000). For experiments at 25°C, cells were incubated overnight
at this temperature in HL5 medium before the experiment.
For developmental analysis, cells were plated on 0.45-µm membrane
filters laid onto two layers of Whatman paper grade 1 soaked with phosphate
buffer (5 mM Na2HPO4, 5 mM KH2PO4,
1 mM CaCl2, 2 mM MgCl2, pH 6.5) (Whatman, Maidstone,
United Kingdom) (Sussman,
1987). For each mutant used herein, three independent mutant
strains were tested and identical phenotypes were observed. Thereafter, only
one clone was chosen for extensive characterization.
Rabbit polyclonal antiserum against a Phg1a peptide (YC1) was described
previously (Cornillon et al.,
2000). An anti-peptide rabbit polyclonal antibody against Phg1b
was directed to a sequence in the lumenal domain of the Phg1b protein
(GEYYYAEMIYDDLP).
Nucleotide Sequences and Plasmids
PHG1b Sequence and Expression Plasmid. The full-length
PHG1b open reading frame is represented in cDNA clone SSE715 from the
D. discoideum cDNA project in Japan
(Morio et al., 1998)
(GenBank accession no. C92378
[GenBank]
). This clone was obtained and fully sequenced
(accession no. AJ507828
[GenBank]
). PHG1b was subcloned into a derivative of
pDXA-3C (Manstein et al.,
1995; Cornillon et
al., 2000) to obtain the pSC3B vector that upon transfection
into D. discoideum cells drives overexpression (actin 15 promoter) of
Phg1b protein.
PHG1c Sequence. The full-length PHG1c open reading
frame is represented in cDNA clone dda25m17 (GenBank accession no. BJ329614
[GenBank]
)
from the D. discoideum cDNA project in Japan
(Morio et al., 1998).
This clone was obtained and fully sequenced (accession no. AJ507829
[GenBank]
).
PHG1b Knockout Plasmid. The pSC4 plasmid was produced by
introducing into pDXA-3C (Manstein et
al., 1995), two fragments of the PHG1b gene:
XbaI/ClaI DNA fragment and a polymerase chain reaction DNA
fragment amplified with the two following oligonucleotides,
5'-GGTACAGTTGGTTTCTAC-3' and
5'-ACCCTATTTGTAGCACCA-3'.
A/B Fusion Protein. The pMB25 plasmid harbors a fusion gene encoding a chimera made of the hydrophilic extracellular domain of Phg1a (M1 to V276) and the nine transmembrane domains of Phg1b (H221–D587).
B/A Fusion Protein. The pMB24 plasmid harbors a fusion gene encoding a chimera made of the hydrophilic extracellular domain of Phg1b (M1 to I220) and the nine transmembrane domains of Phg1a (H277 to N642). pMB24 and pMB25 were linearized with pvUI and used to transfect PHG1a knockout cells to obtain the G418 resistant cells expressing the corresponding fusion protein.
Phg1a-GST Fusion Protein. The pMB30 plasmid harbors a hybrid gene encoding an N-terminal fusion of glutathione transferase to the full-length Phg1a protein and allows its expression in D. discoideum cells. pMB30 was linearized with pvUI and used to transfect PHG1a knockout cells to obtain the G418-resistant cells expressing the fusion protein.
Multiple alignment and phylogenetic analysis were performed at the Pôle Bio-Informatique Lyonnais Web site with ClustalW. Homology searches and sequences submission were performed at the European Molecular Biology Laboratory Web site.
Phagocytosis and Fluid Phase Uptake Assays
Rhodamine-labeled bacteria were produced as described previously
(Cornillon et al.,
2000). Fluoresbrite YG 1-µm carboxylate microspheres,
fluoresbrite YG 1-µm plain microspheres, and fluospheres 1-µm
amine-modified microspheres were purchased from Polysciences (Warrington,
PA).
Measurements of phagocytic substrate internalizations were carried out as
described previously (Cornillon et
al., 2000). Phagocytosis assays were done at 20°C and
25°C in either HL5 medium or phosphate buffer (SB; 2 mM
Na2HPO4, 14.7 mM KH2PO4, pH 6.5).
When the phagocytosis assay using latex beads in HL5 medium was carried out at
25°C with cells grown at 20°C, a 50% defect in phagocytosis by
PHG1b knockout cells relative to wild-type cells was observed (our
unpublished data). Because the thermosensitive growth phenotype of
PHG1b knockout cells became significant after 48 h, a 16-h
preincubation at 25°C was chosen to assess the effect of temperature on
phagocytosis in PHG1b knockout cells. Longer incubations at 25°C,
however, did not enhance the defect in phagocytosis (our unpublished data).
Fluid-phase uptake was measured using fluorescein isothiocyanate-dextran
(Molecular Probes, Eugene, OR) as described previously
(Cornillon et al.,
2000).
Adhesion of Cells to Substrate
Cell adhesion to glass surfaces was probed using the radial flow detachment
assay, performed at 20°C and 25°C in either HL5 or phosphate buffer,
according to Decave et al.
(2002). The flow was applied
during 7 min. Adhesion was quantified by measuring the value of the shear
stress detaching 50% of the cells.
Affinity Purification of Phg1a-GST Fusion Protein
D. discoideum cells (5 x 106) expressing the
Phg1a-GST fusion protein were lysed with either 0.5% Triton X-100 or 0.5%
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) in 20 mM
phosphate buffer, pH 7.4, containing 150 mM of NaCl. Under these conditions,
Phg1b as well as Phg1a are found in the cellular lysate and absent from the
insoluble pellet (our unpublished data). Lysates were cleared by
centrifugation, 15 min at 10,000 x g at 4°C, and applied to
either glutathione S-transferase GST or FLAG affinity resin (Amersham
Biosciences AB, Uppsala, Sweden, and Sigma-Aldrich, St. Louis, MO,
respectively) for 1 h at 4°C. Both resins were washed five times with the
corresponding lysate buffer and bound proteins were eluted with sample buffer
(0.103 g/ml sucrose, 5 x 10–2 M Tris, pH
6.8, 5 x 10–3 M EDTA, 0.5 mg/ml bromophenol
blue, 2% SDS). D. discoideum cell equivalent (2 x
106) was loaded on SDS-PAGE for Western blot analysis.
Protein Electrophoresis and Immunodetection
To determine the presence of Phg1a and Phg1b proteins, cells were washed
once in HL5 medium, resuspended at 2 x 105 cells/10 µl in
sample buffer (0.103 g/ml sucrose, 5 x 10–2
M Tris, pH 6.8, 5 x 10–3 M EDTA, 0.5 mg/ml
bromophenol blue, 2% SDS), and each sample was run on a 9% acrylamide gel
under nonreducing conditions. The proteins were then transferred to a
nitrocellulose BA 85 membrane (Schleicher & Schuell, Dassel, Germany). The
membrane was incubated with a primary rabbit antibody to Phg1a or Phg1b and
then a horseradish peroxidase-coupled donkey antiserum to rabbit Ig (Amersham
Biosciences AB), washed, and revealed by enhanced chemiluminescence.
Cell Surface Protein Biotinylation
Cells were grown in HL5 medium at a concentration of 5 x
105 cells/ml. After cooling down to 4°C, cells were collected
and washed once with ice-cold SB, pH 7.8, by centrifugation. Cells were then
resuspended in ice-cold SB containing 2 mg/ml NHS-Sulfobiotin (Pierce
Chemical, Rockford, IL). After 30-min incubation on ice, the cells were washed
twice with ice-cold SB containing 100 mM glycine, pH 7.2, and resuspended in
nonreducing sample buffer. Lysate of 2.5 x 105 cells was
loaded onto a 9% acrylamide gel and electrophoresed under nonreducing
conditions. The lane was cut out of the gel, incubated in reducing sample
buffer for 20 min, and loaded onto a second 9% reducing acrylamide gel.
Biotinylated proteins were detected with ImmunoPure Avidin horseradish
peroxidase conjugate (Pierce Chemical).
Fluorescence Microscopy
Cells were grown on glass coverslips in HL5 medium for 3 d at 20°C and
were processed for p80 immunofluorescence analysis as described previously
(Ravanel et al.,
2001). Briefly, cells were allowed to internalize a fluid-phase
marker (fluoresbrite YG 50-nm microspheres; Polysciences) in HL5 for 90 min
and then fixed in 4% paraformaldehyde, permeabilized with saponin, and
incubated successively with H161 monoclonal antibody specific for the p80
endosomal protein and the corresponding fluorescent secondary antibody. Cells
were visualized with a confocal microscope (LSM510; Carl Zeiss, Jena,
Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Phg1b and Phg1c are 24.5 and 21.2% homologous to the Phg1a protein,
respectively (16.3 and 12.9% in the extracellular domain, respectively; 33 and
31.3% in the membrane domain, respectively). A previous report classified the
TM9 proteins into two subgroups, referred to herein as I and II
(Sugasawa et al.,
2001). Subgroup I is characterized by a shorter (
220 amino
acids) hydrophilic N-terminal domain and a characteristic motif at position 50
(VGPYxNxQETY), whereas subgroup II contains a conserved sequence
[FY(V/L)PG(VL)AP] immediately after the signal peptide and a longer
N-terminal domain (280 amino acids). Phg1a belongs to subgroup II
(FYLPGMIP at amino acid position 25) and Phg1b to subgroup I (VGPYSNPTETY at
amino acid position 42) (Figure
1B). The Phg1c sequence does not contain either of these two
distinctive motifs and therefore cannot be classified unambiguously into
either subgroup. Phylogenetic analysis shows that Phg1a and b proteins are
distantly related members of the TM9 family in D. discoideum,
differing to the same extent as the most divergent members of the human family
TM9SF1 and TM9SF2 (Figure 1B). For comparison, TM9SF1 and TM9SF2 are 27.2% homologous (15% in the
extracellular domain, 35.8% in the membrane domain). Because Phg1a and b are
divergent members of the TM9 family and belong to two distinct subclasses,
analysis of their function(s) may provide a general view of the function of
TM9 proteins. Functional analysis was therefore focused on these two
proteins.
TM9 Proteins Play Synergistic Roles in Growth and Development
To investigate the respective roles of the Phg1a and Phg1b proteins, the
PHG1b gene was inactivated by homologous recombination in wild-type
cells to obtain the PHG1b knockout or in PHG1a knockout
cells to obtain the PHG1a/PHG1b double knockout. Expression of Phg1a
and Phg1b proteins in the resulting knockout strains was assessed by Western
blot (Figure 2). In wild-type
and PHG1a knockout cells, the polyclonal antiserum directed to the
Phg1b protein recognized one major band at a molecular weight of 57 kDa
(Figure 2, lanes 1 and 2). This
band was absent in PHG1b and PHG1a/PHG1b knockout cells
(Figure 2, lanes 3 and 4) and
clearly more intense in cells overexpressing Phg1b
(Figure 2, lane 5), confirming
that it corresponds to the Phg1b protein. Several other bands represented
nonspecific cross-reactivity of the antiserum with unrelated proteins. The
Phg1a antiserum detected one band at 65 kDa in wild-type cells and in
PHG1b knockout cells (Figure
2, lanes 1 and 3) that was absent in PHG1a and
PHG1a/PHG1b knockout cells (Figure
2, lanes 2, 4, and 5).
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Although both PHG1a and PHG1b knockout cells grew almost as well as wild-type cells in HL5 medium at 20°C, growth of PHG1a/PHG1b double knockout cells was severely reduced (Figure 3). Furthermore, a thermosensitive growth phenotype was observed for PHG1b knockout cells: growth was normal at 20°C (usual growth temperature for D. discoideum) but severely inhibited at 25°C (Figure 3). A temperature shift to 25°C also further impaired the growth of PHG1a/PHG1b double knockout cells (Figure 3). Both PHG1a and PHG1b knockout cells also showed defects in development upon starvation (Figure 4). At 20°C, development of PHG1a knockout cells was arrested at the finger stage, whereas PHG1b knockout cells formed normal fruiting bodies. The double knockout strain exhibited a more severe phenotype, development of these cells stopped at the early culmination stage. At 25°C the development of PHG1a knockout cells was similar to that observed at 20°C (arrest at finger stage), whereas the development of PHG1b knockout cells was stopped at the early culmination stage (Figure 4).
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Development of PHG1a/PHG1b double knockout cells was blocked at an even earlier stage: the tipped mound stage (Figure 4).
In summary the combination of PHG1a and PHG1b mutations led to more than additive phenotypes on D. discoideum growth. This synergistic effect strongly suggests that the Phg1 proteins are involved in the same cellular function(s).
Different TM9 Knockout Cells Exhibit Similar Phagocytosis and
Adhesion Defects
A previous study established that PHG1a knockout cells are
specifically defective for adhesion to and phagocytosis of hydrophilic
particles (latex beads coated with HL5 medium) but show no defect in
phagocytosis of hydrophobic particles (plain latex beads in phosphate buffer)
(Cornillon et al.,
2000) (Figure 5A).
Herein, this selectivity was further documented by showing that in phosphate
buffer phagocytosis of other hydrophilic beads (carboxylate or amino group
substituted beads) was also inhibited in PHG1a knockout cells
(Figure 5B, columns 3 and 4).
Phagocytosis rates of Klebsiella pneumoniae and Escherichia
coli bacteria were also decreased in PHG1a knockout cells
(Figure 5B, columns 2 and 5).
At least three different types of phagocytosis receptors are present in D.
discoideum: a lectin, a receptor for hydrophobic surfaces, and a receptor
for hydrophilic surfaces (Vogel et
al., 1980). Because PHG1a knockout cells are
specifically defective in adhesion to hydrophilic substrates, Phg1a protein
might represent the hydrophilic receptor or be specifically involved in its
regulation. Herein, we also report that PHG1a knockout cells
displayed a slight temperature-sensitive phenotype that increased the
phagocytosis defect at 25°C compared with 20°C
(Figure 5C).
At 20°C, PHG1b knockout cells phagocytosed all of the substrates tested as efficiently as wild-type cells (Figure 5C). On the contrary, at 25°C, PHG1b knockout cells displayed a strong phagocytosis defect (Figure 5C). Remarkably, PHG1a and PHG1b knockout cells showed a similar specificity in their phagocytosis defect at 25°C (Figure 5C). Interestingly, even at 20°C PHG1a/PHG1b double knockout cells showed a strong phagocytosis defect even more severe than PHG1a knockout cells alone (Figure 5C). This is again suggestive of a functional synergy between Phg1a and Phg1b proteins.
To confirm that the phagocytosis defect observed was due to a defect in adhesion, the ability of knockout cells to adhere to a substrate was measured and is presented in Table 1: PHG1a knockout cells showed a strong decrease in adhesion to a hydrophilic surface (glass coated with HL5 components), but not to a more hydrophobic surface (glass in phosphate buffer). At 20°C, PHG1b cells adhered normally to both substrates but at 25°C they exhibited a significant defect in adhesion to hydrophilic surfaces. PHG1a/PHG1b double knockout cells did not adhere to a hydrophilic substrate and showed a small decrease in adhesion to a more hydrophobic surface. Together, these results demonstrate that Phg1a and Phg1b proteins both play a role in adhesion to and phagocytosis of hydrophilic particles.
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As observed previously, PHG1a knockout cells did not show any
defect in fluid phase internalization
(Cornillon et al.,
2000). We did not observe any fluid phase uptake defect in
PHG1b or PHG1a/PHG1b double knockout cells (our unpublished
data). Because fluid phase uptake in Dictyostelium discoideum cells
is mostly accomplished by macropinocytosis, the basic machinery for
controlling rearrangement of the actin cytoskeleton is still functional in the
PHG1 knockout cells. Consequently, in these cells the phagocytosis
defect is limited to the first step in phagocytosis, i.e., adhesion to the
particles.
Together, these results are compatible with a receptor function of Phg1a and Phg1b for phagocytosis of hydrophilic particles.
TM9 Proteins Are Not Functionally Redundant
To test further the functional relationship between Phg1a and Phg1b
proteins, we performed complementation analysis. Expression of Phg1a protein
in PHG1a knockout cells restores phagocytosis
(Cornillon et al.,
2000) and adhesion (Table
1). In contrast, overexpression of Phg1b protein in PHG1a
knockout cells did not complement the defect in phagocytosis
(Figure 6B) or in adhesion
(Table 1). These results
clearly demonstrate that the Phg1a and Phg1b proteins do not share redundant
functions.
TM9 proteins differ most significantly in their N-terminal extracellular domain. To investigate the nature of the functional difference between Phg1a and Phg1b, we generated two hybrid proteins in which the extracellular domains were swapped (Figure 6A). Whereas expression of the B/A fusion largely complemented the phagocytosis defect of PHG1a knockout cells (Figure 6B), overexpression of the A/B fusion had no detectable effect (our unpublished data). Surprisingly, these results suggest that the main functional difference between the Phg1a and Phg1b proteins resides in the conserved hydrophobic domain, whereas the more divergent N-terminal domain is functionally similar.
No Detectable Association between the TM9 Proteins
The results described so far demonstrate that the functions of the Phg1a
and Phg1b proteins are similar but not redundant. A possible interpretation is
that the two proteins interact physically to form a Phg1a-Phg1b complex or are
part of a multisubunit complex essential for cellular adhesion and
phagocytosis. To test this possibility, a fusion protein encoding the
full-length Phg1a fused to GST and tagged with FLAG epitope
(Figure 7A) was expressed in
PHG1a knockout cells. This hybrid protein fully complemented the
adhesion and phagocytosis defect of PHG1a knockout cells (our
unpublished data). Two different cell solubilization conditions were used
(Triton-X 100 or CHAPS), and coimmunoprecipitation was then performed using
either a GST or a FLAG affinity resin. Western blot analysis revealed that
Phg1a was efficiently immunoprecipitated in both cases. Although the amount of
material loaded (2 x 106 cells) corresponded to 10 times more
cells than in Figure 2, Phg1b
was not detected in these samples even after prolonged exposure
(Figure 7B). Although it is
virtually impossible to rule out a weak or detergent-sensitive interaction,
this result argues against the existence of a stable protein complex between
Phg1a and Phg1b.
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Composition of the Cell Surface Is Altered in TM9 Knockout Cells
The defective adhesion properties of PHG1 knockout cells prompted
us to examine the protein cell surface composition. The cell surface was
biotinylated and total protein lysates were then resolved by two-dimensional
nonreduced/reduced gels. This technique allows visualization of transmembrane
proteins and resolution of those containing disulfide bridges because they
migrate differently in nonreducing and reducing conditions.
Approximately 20 proteins were detected outside the diagonal in the wild-type cells (Figure 8), whereas only a weak background was observed only on the diagonal for nonbiotinylated wild-type cells (our unpublished data). None of these proteins corresponded to Phg1a or Phg1b as both migrated on the diagonal (our unpublished data). Interestingly, the same analysis performed in PHG1a, PHG1b, and PHG1a/PHG1b knockout cells revealed a number of differences (Figure 8). In particular, in conditions where cellular adhesion is defective, at least three proteins are clearly down-regulated in all PHG1 knockout cells compared with wild-type cells (Figure 8, arrowheads). In addition, a number of other changes were observed, for example up-regulated proteins in PHG1b and PHG1a/PHG1b knockout cells (Figure 8).
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These results demonstrate that deletion of PHG1 genes changes dramatically the cell surface composition and this might be the cause of the loss of cellular adhesion in the knockout cells.
p80 Endosomal Protein Is Shifted to the Cell Surface in PHG1a/PHG1b
Knockout Cells
The p80 membrane protein was characterized previously in D.
discoideum as an integral membrane protein, mostly localized to the
endocytic compartments (Ravanel et
al., 2001). Minor amounts of p80 can also be found at the
cell surface, but it is absent from the contractile vacuole
(Ravanel et al.,
2001). Subcellular localization of p80 was determined to detect
any modifications in the intracellular transport. Although in wild-type cells
p80 was mostly localized in endosomes
(Figure 9), in
PHG1a/PHG1b knockout cells p80 was mainly present at the cell surface
and often absent from intracellular compartments
(Figure 9). To quantify this,
the number of cells with no visible intracellular p80 was counted for
wild-type cells and PHG1a/PHG1b knockout cells. In two independent
experiments only very few wild-type cells seemed devoid of intracellular p80
(1.3 and 0%, n = 304 and 100, respectively). On the contrary, almost half of
the PHG1a/PHG1b knockout cells exhibited pure p80 surface labeling
(42.4 and 48%, n = 309 and 100, respectively). Interestingly in
PHG1a/PHG1b knockout cells exhibiting no internal p80, endosomes were
still visible as evidenced by the presence of an endocytosed fluid-phase
marker (Figure 9). Together,
these experiments indicate that p80 is relocalized from endosomal membranes to
the cell surface in PHG1a/PHG1b knockout cells. In single
PHG1a or PHG1b knockouts, no major p80 relocalization was
observed (our unpublished data), again suggesting a synergistic role of the
Phg1a and Phg1b proteins. To conclude alteration in the intracellular
transport, in particular in the endocytic pathway, might account for the
modified cell surface composition in PHG1 knockout cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The presence of TM9 proteins in
evolutionary distant organisms (four members in human, three in yeast, and
several in plants) raises several interesting questions regarding their
function. For instance, do these proteins carry out a common evolutionary
conserved function in these organisms? In the same organism do different
members of the family play redundant roles? Does the variable extracellular
domain confer distinct properties to each member of the family? The D.
discoideum genome contains at least three distinct genes encoding TM9
proteins: Phg1a, Phg1b, and Phg1c. The function of two divergent TM9 members,
Phg1a and Phg1b, was investigated by analyzing the phenotype of the
corresponding knockout cells.
Synergy in phagocytosis, adhesion, growth, and development suggests
strongly that Phg1a and Phg1b proteins function in the same cellular process.
Compared with the non-selective phagocytosis defect of LvsA mutant
cells (Cornillon et al.,
2002), PHG1a and PHG1b knockout cells display a
selective phagocytosis defect. This is compatible with the hypothesis that
Phg1a and Phg1b proteins represent receptors necessary for adhesion to
hydrophilic particles, whereas the LvsA protein is only involved at later
stages in the phagocytic process (Cornillon
et al., 2002). However, the fact that overexpression of
PHG1b failed to complement PHG1a knockout cells argues
against a role for Phg1a and Phg1b proteins as two phagocytosis receptors
sharing similar specificity. Similarly, PHG1a knockout
complementation by the B/A fusion protein suggests that the variable
extracellular domain of TM9 proteins does not confer a specific role in
cellular adhesion to each member of the family. Biochemical experiments also
excluded the possibility that Phg1a and Phg1b proteins might together form one
oligomeric phagocytosis receptor.
We are thus brought to propose that TM9 proteins function as regulators of
cellular adhesion rather than as adhesion molecules. Inactivation of two
converging regulatory pathways would indeed be expected to result in similar
and synergistic effects but no complementation. Interestingly, regulator role
for a TM9 protein (SMBP) has recently been suggested in rat: SMBP binds to a
3-adrenergic agonist and is involved in regulation of eosinophil
chemotaxis and relaxation of depolarized rat colon
(Sugasawa et al.,
2001). This suggests that TM9 proteins could act as receptors for
as yet unidentified ligands and regulate a number of important cellular
functions, such as adhesion in D. discoideum or chemotaxis in
eosinophil. This model would also account for the presence of TM9 proteins in
nonphagocytic species such as Saccharomyces cerevisiae and plants.
Presumably in these organisms, TM9 proteins regulate other cellular
functions.
At least two mechanisms by which regulators modulate cellular adhesion can
be proposed: first, modulation of the adhesive properties of adhesion
molecules; second, modulation of their cell surface expression. In support of
the second mechanism, we observed an alteration of the protein composition at
the cell surface in PHG1 knockout cells. Any down-regulated cell
surface protein in PHG1 knockout cells might represent a potential
receptor for hydrophilic substrates and at least three of them were
down-regulated. Hence, TM9 proteins might control cellular adhesion in D.
discoideum by determining the cell surface expression of as yet
unidentified receptor(s). The selective phagocytosis defect of PHG1
knockout cells might then reflect the specificity of the regulated
receptor(s). Additionally, the large number of up- or down-regulated cell
surface proteins in PHG1 knockout cells points to a more general
function of TM9 proteins, i.e., regulation of the cell surface composition.
Interestingly, at least three TM9 proteins have been found in the endosomal or
phagosomal pathways: the human p76 was predominantly localized in endosomes
(Schimmoller et al.,
1998); the yeast Emp70 cleavage product p24 has been identified in
endosome-enriched membranes (Singer-Kruger
et al., 1993); the D. discoideum Phg1a was
reported to be present in phagosomes
(Cornillon et al.,
2000). It is therefore tempting to postulate that TM9 proteins
regulate the cell surface composition by modulating intracellular transport of
proteins in the endocytic pathway. This hypothesis is further supported by the
observation that the intracellular localization of an endosomal marker protein
such as p80 is affected in PHG1 knockout cells.
In conclusion, both TM9 proteins Phg1a and Phg1b are required for cellular adhesion. Our results strongly suggest a regulatory role of TM9 proteins rather than a receptor role in adhesion and phagocytosis. Additional studies, such as analysis of Phg1c function in D. discoideum or TM9 proteins in mammalian system are necessary to gain further insight into the regulatory function of the TM9 proteins and to establish their putative function in intracellular transport.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Corresponding author. E-mail address:
mohammed.benghezal{at}medecine.unige.ch.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Cornillon, S., Dubois, A., Bruckert, F., Lefkir, Y., Marchetti, A.,
Benghezal, M., De Lozanne, A., Letourneur, F., and Cosson, P.
(2002). Two members of the beige/CHS (BEACH) family are involved
at different stages in the organization of the endocytic pathway in
Dictyostelium. J. Cell Sci.
115,
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Phg1a-specific antibody (top)
or anti-Phg1b-specific antibody (bottom). Arrows indicate bands corresponding
to the Phg1a and Phg1b proteins. Lane 1 shows proteins from wild-type cells;
lane 2, PHG1a knockout cells; lane 3, PHG1b knockout cells;
lane 4, PHG1a/PHG1b double knockout cells; and lane 5, PHG1a
knockout cells overexpressing PHG1b. The low molecular weight Phg1a
species visible in PHG1a/PHG1b knockout cells corresponds to an
unstable truncation product of Phg1a produced in PHG1a knockout cells
(Cornillon et al.,
2000
