1 Department of Microbiology, Umeå University, 901 87 Umeå,
Sweden
2 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC,
Box 582, 751 23 Uppsala, Sweden
3 Max Planck Institute for Biochemistry, Department of Molecular Medicine, Am
Klopferspitz 18A, 82152 Martinsried, Germany
* Author for correspondence (e-mail: maria.fallman{at}molbiol.umu.se )
Accepted 18 April 2002
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Summary |
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Key words: Invasin, ß1-integrin, Yersinia pseudotuberculosis, Focal complexes, Bacterial internalization
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Introduction |
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In addition to binding ECM proteins such as fibronectin and laminin, the
ß1A subunit, dimerized with 3,
4,
5,
6 or
v, also binds to the bacterial adhesin, invasin, which is expressed on
enteropathogenic Yersinia species
(Isberg and Leong, 1990
;
Leong et al., 1990
;
Rankin et al., 1992
).
Interaction of invasin with integrins results in internalization of the
bacteria, whereas binding to matrix-protein-coated bacteria or beads usually
does not (Tran Van Nhieu and Isberg,
1991
). This difference is thought to be caused by the
high-affinity binding of integrins to invasin and maybe also result from
qualitative differences in signal transduction. Invasin, which bind with about
100-fold higher affinity than fibronectin to integrins, can efficiently
compete out fibronectin from integrin binding
(Tran Van Nhieu and Isberg,
1991
). In addition, invasin expressed by Y.
pseudotuberculosis contains a central dimerization region enabling
multimerization of the integrin receptor that greatly contributes to efficient
internalization of bacteria (Dersch and
Isberg, 1999
). The crystal structures of the integrin-binding
regions of invasin and fibronectin showed that these two ligands recognize
similar residues on the integrin, although invasin lacks an
alanine-glycine-asparagine (RGD) sequence and the overall contour was
different (Hamburger et al.,
1999
). The higher affinity seen for invasin is probably due to the
involvement of more hydrogen bonds in the binding to the integrin and also to
a less flexible interdomain region on invasin
(Leahy et al., 1996
;
Hamburger et al., 1999
).
ß1-integrin-mediated phagocytosis of invasin-expressing bacteria is an
actin-dependent process involving local membrane protrusions. It also requires
tyrosine kinase activity, and earlier studies have implicated the tyrosine
kinases Src and FAK as well as the small G-protein Rac as important players in
this signaling (Alrutz and Isberg,
1998; McGee et al.,
2001
). However, pathogenic Yersinia strains can actively
block the uptake process by injecting antiphagocytic factors into host cells
(Rosqvist et al., 1988a
;
Persson et al., 1997
). The
antiphagocytic effect has been attributed to two Yersinia outer
proteins, YopE and YopH, which are delivered into host cells through a type
III secretion mechanism (Cornelis et al.,
1998
). YopE is a GTPase-activating protein acting on Rho family
proteins (Black and Bliska,
2000
; Von Pawel-Rammingen et
al., 2000
). YopH is a protein tyrosine phosphatase that recognizes
focal complex structures and dephosphorylates p130Cas, paxillin and FAK, which
all are proteins implicated in ß1-integrin signaling and focal complex
dynamics. The effect of the dephosphorylation by YopH is that the focal
contacts are disrupted and bacterial uptake by cells is blocked
(Black and Bliska, 1997
;
Persson et al., 1997
). The
YopH protein contains a sequence that mediates its localization to
integrin-containing focal complex structures
(Persson et al., 1999
).
Interestingly, mutations that abolish YopH colocalization with these
structures strongly reduce YopH's capacity to block phagocytosis
(Persson et al., 1999
),
suggesting that integrin-associated focal complex structures are important for
ß1-integrin-mediated uptake of bacteria.
The aim of this study was to identify features of the ß1-integrin that
are important for invasin-promoted phagocytosis. For this purpose we took
advantage of the fibroblast-like cell line GD25, which was derived from
ß1-integrin-deficient embryonic stem cells
(Fässler et al., 1995;
Wennerberg et al., 1996
). The
GD25 cells and stable transfectants of these, expressing wild-type and mutated
variants of the ß1-integrin, were used to investigate the mechanism
behind invasin-promoted bacterial internalization. We found that invasin, in
contrast to fibronectin, can bind to inactive ß1B-integrin but without
triggering uptake of bacteria. The first NPXY motif and the double threonine
site in the ß1A cytoplasmic chain were found to be of importance for
internalization of bacteria, but there was no requirement for tyrosine
phosphorylation of the ß1-integrin cytoplasmic NPXY motifs. There were
also a correlation between integrin-mediated internalization of
invasin-expressing bacteria and invasin-induced spreading.
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Materials and Methods |
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Cell culture
The GD25 cell line lacks the ß1-integrins owing to disruption of the
ß1 gene (Fässler et
al., 1995); all the other cell lines were derived from GD25 by
stable transfection with cDNA encoding wild-type ß1-integrin or mutated
ß1 subunit. The cell lines GD25ß1A, GD25ß1B, GD25ß1AY795F,
GD25ß1AY783F, GD25ß1AYY783/795FF and GD25ß1ATT788-9AA have been
described previously (Wennerberg et al.,
1996
; Sakai et al.,
1998
; Wennerberg et al.,
1998
; Armulik et al.,
2000
). The GD25ß1Y783A, GD25ß1AT788A, GD25ß1AT789A
and GD25ß1AD130A lines where generated as previously described for the
GD25ß1ATT788-9AA cell line
(Wennerberg et al., 1998
). The
GD25 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing
10% heat inactive fetal calf serum (FCS), L-glutamine (2 mM), penicillin (100
U/ml) and streptomycin (100 µg/ml). The transfected cells were grown in the
same medium supplemented with puromycin (20 µg/ml).
Fluorescence activated cell sorter (FACS) analysis
Cells (1x106) were trypsinized and washed twice with fresh
medium. Subsequently, the cells were suspended in primary antibody
(HMß1-1, 1:100) in FACS-PBS (phosphate buffered saline containing 10% FCS
and 0.001% NaN3) and incubated on ice for 20 minutes. After
washing, the cells were incubated with secondary antibody (FITC-conjugated
goat anti-Armenian hamster antibody, 1:60) for 20 minutes in FACS-PBS, washed
cells were resuspended in FACS-PBS and analyzed (10,000 cells per sample)
using a FACScan® (BD Biosciences).
Bacterial uptake
The multiple Yop mutant strain (MYM) lacking YadA, YopM, E, K, H and YpkA
of Yersinia pseudotuberculosis
(Håkansson et al., 1996)
was used to investigate bacterial uptake into the cells. An over night culture
of MYM, grown in Luria broth at 26°C, was diluted 1:120 in cell culture
medium and incubated for 1 hour at 26°C. This culture was used to infect
cells grown for 18 hours on cover slips, at a multiplicity of infection of
150:1. The infection was carried out for 30 minutes at 37°C in an
atmosphere of 5% CO2. The cells were then fixed in 2%
paraformaldehyde and double immunofluorescence labeling was applied to
distinguish between extracellular and total cell-associated bacteria
(Heesemann and Laufs, 1985
;
Rosqvist et al., 1988a
). The
extracellularly localized bacteria were labeled by incubating the samples with
rabbit anti-Yersinia serum (1:500). The cells were subsequently
permeabilized in 0.5% Triton X-100, and extracellularly localized bacteria
were stained by incubating with FITC-conjugated anti-rabbit antibodies
(1:100). All cell-associated bacteria were then stained with
anti-Yersinia serum (1:500) and LRSC-conjugated anti-rabbit serum
(1:100). The cells were finally mounted using the Ultimate Mounting Media
(UMM) (Fällman et al.,
1995
), and the specimens were analyzed in a fluorescence
microscope (Zeiss axioskop 50), where 50 cells per slide were analyzed in at
least five separate experiments.
Cell attachment assay
Cell attachment was quantified as previously described
(Wennerberg et al., 1996).
Briefly, 96-well plates were coated with vitronectin (10 µg/ml) or various
concentrations of GST or GST-inv
1 in PBS (overnight at 4°C), and
thereafter blocked with 1% heat-treated bovine serum albumine (BSA) for 2
hours at 37°C. Cells were suspended in serum-free DMEM, plated to coated
wells (1x105 cells/well) and allowed to attach for 1 hour at
37°C. Unattached cells were washed away, and the remaining cells were
fixed in 96% ethanol and stained in crystal violet (0.1%) for 30 minutes.
Excess stain was washed away with water and attached cells were dissolved in
Triton X-100 in PBS (0.2%). The absorbance was read in a microtiter plate
reader at 600 mm. All samples were analyzed in triplicates. The amount of
cells of each cell line that were attached to wells coated with vitronectin
(10 µg/ml) was set as 100% binding.
Immunoprecipitation and western blotting
Serum-starved cells were trypsinized and treated with soybean trypsin
inhibitor type II (Sigma-Aldrich, Stockholm, Sweden). Cells were washed once
with serum-free DMEM and plated on dishes (6x106 cells)
coated with GST-invasin (10 µg/ml) or BSA. The cells were allowed to attach
at 37°C for 60 minutes and subsequently lysed in RIPA (20 mM Tris-HCl pH
7.4, 150 mM NaCl, 5 mM EDTA, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS, 200
µM sodium orthovanadate, 2 mM PMSF, 2 mM NEM, 1 µg/ml pepstatin A) for
15 minutes on ice. Thereafter the lysates were clarified by centrifugation at
14,000 g for 15 minutes. The spun lysates were precleared with
ProteinA-sepharose (Amersham Biosciences) for 1 hour at 4°C. The beads
were removed by centrifugation, and the supernatant was divided in two and
incubated for 3 hours at 4°C with protein A-sepharose beads precoupled to
either anti-p130Cas or anti-paxillin antibodies. Lysates used for
precipitation of paxillin were later used to immunoprecipitate FAK. The beads
were washed twice with RIPA buffer and subjected to SDS-PAGE followed by wet
transfer to a nitrocellulose membrane (Scheicher and Schnell). Tyrosine
phosphorylated proteins were detected by anti-phosphotyrosine antibodies
conjugated with horseradish peroxidase (RC20) and then enhanced
chemiluminescence (Amersham Biosciences). To confirm that an equal amount of
proteins was immunoprecipitated in all samples, the antibodies were stripped
from the membrane by incubation in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, 100 µM
ß-mercaptoethanol for 30 minutes at 50°C, whereafter membranes were
restained with relevant antibodies.
Immunofluorescence
Cover slips were coated with GST-inv1 (10 µg/ml) overnight at
4°C and blocked with 1% heat-treated BSA in PBS at 37°C for 1 hour.
Cells were detached and incubated in serum-free DMEM containing cycloheximide
(25 µg/ml; Sigma-Aldrich, Stockholm, Sweden) on BSA-blocked plates for 40
minutes at room temperature, then the GRGDS peptide (0.1 mg/ml) was added and
the cells were incubated for an additional 20 minutes. After this,
5x104 cells were allowed to attach on invasin-coated cover
slips at 37°C for 3 hours. Unattached cells were washed away and the
remaining cells were fixed in 2% paraformaldehyde for 10 minutes.
Alternatively, 2.5x104 cells were seeded on cover slips and
cultured at 37°C for 20 hours in serum-containing medium. Subsequently the
wells were washed and the cells were fixed as above. The fixed cells were
permeabilized with 0.5% Triton X-100 and further processed for
immunofluorescence labeling. To visualize ß1-integrins, cells were
incubated with primary antibodies against ß1-integrins (HMß1-1) and
then with the FITC-conjugated goat anti-Armenian hamster secondary antibodies.
In double labeling experiments, the cells were incubated as above followed by
incubation with antibodies against phosphotyrosine (PY20) or vinculin and with
the LRSC-conjugated secondary donkey anti-mouse antibodies. All incubations
were performed at room temperature. Samples were mounted onto microscope
slides using ProLong Antifade (Molecular Probes, Leiden, The Netherlands). The
pictures were captured using a microscope (Zeiss axioskop 50) and a CCD camera
(ORCA, Hamamatsu) and processed using Adobe software (Adobe).
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Results |
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To further evaluate the ß1-integrininvasin interaction, cells
were plated for 1 hour onto dishes coated with invasin or vitronectin. With
this setup, the ligand is immobilized on a surface and the integrins on the
cells are forced to find its ligands, in contrast to the bacterial uptake
assay where the bacteria are involved in finding receptors on the cells
(Andersson et al., 1999). The
recombinant invasin protein used was a truncated derivative corresponding to
the 500 C-terminal amino acids of invasin, containing both the dimerization
and integrin recognition parts (Dersch and
Isberg, 1999
). Vitronectin, which is a ligand for
vß3-integrin but not for ß1-integrins, was used to set the
100% attachment for each cell line. GD25 and GD25ß1A cells attached to
vitronectin to a similar extent, but, as in the uptake assay, only the
GD25ß1A cells bound to invasin (Fig.
1C). Cells expressing a ß1A subunit unable to bind to ligand
because of the mutated extracellular domain (GD25ß1AD130A)
(Takada et al., 1992
) failed
to attach to invasin. However, GD25ß1B-expressing cells readily bound to
invasin-coated surfaces, although somewhat less efficiently than the
ß1A-expressing cells (Fig.
1C). Hence, invasin can bind to ß1-integrins exhibiting an
inactive conformation for binding to fibronectin, but the binding itself is
insufficient to promote integrin-mediated internalization.
Ligation and clustering of ß1-integrins is associated with tyrosine
phosphorylation of several focal adhesion proteins
(Vuori, 1998). The ß1B
isoform does not mediate such phosphorylations and neither does it promote
formation of focal adhesion structures when exposed to ECM proteins
(Balzac et al., 1993
;
Balzac et al., 1994
;
Armulik et al., 2000
). This is
believed to be because of the lack of ligand binding, and therefore it was of
interest to investigate invasin-induced ß1B-integrin signaling. To
elucidate this, GD25 cells expressing the ß1A or the ß1B subunit
were plated on invasin-coated dishes, and the focal contact components FAK,
p130Cas and paxillin were immunoprecipitated from cell lysates. Western
blotting using anti-phosphotyrosine antibodies showed that all three proteins
were tyrosine phosphorylated in an invasin-dependent manner in cells
expressing ß1A (Fig. 2).
By contrast, in ß1B-expressing cells no induced tyrosine phosphorylation
of FAK or paxillin and only a slight increase in p130Cas phosphorylation was
seen after adhesion to invasin. Hence, the occupancy of the ß1B-integrins
is not sufficient for induction of tyrosine phosphorylation of focal contact
components.
|
Phosphorylation of the ß1-integrin NPXY or TT motifs are not
important for bacterial uptake
The inability of GD25 cells expressing the ß1B subunit to internalize
Yersinia indicated that the cytoplasmic region specific to ß1A
was required for bacterial uptake. There are several potential phosphorylation
sites in this region, among these two tyrosines (amino acid 783 and 795 in
murine ß1A), which are within the NPXY motifs, and two threonines (amino
acids 788 and 789 in murine ß1A) located between the two NPXY motifs.
These sites have been implicated in downstream signaling activities, such as
FAK activation (Wennerberg et al.,
1998; Wennerberg et al.,
2000
) and were therefore of particular interest. To elucidate the
importance of these motifs for bacterial internalization, uptake studies were
performed using GD25 cells expressing ß1A mutants with the tyrosines
exchanged to phenylalanines (GD25ß1AY783F, GD25ß1AY795F and
GD25ß1AYY783/795FF; Fig.
3) or to alanine (GD25ß1AY783A), or with the threonines
exchanged to alanines (GD25ß1AT788A, GD25ß1AT789A and
GD25ß1ATT788-9AA). The ß1AY783A- or ß1ATT788-9AA-expressing
cells showed a marked reduction in bacterial internalization, whereas cells
expressing the tyrosine to phenylalanine mutants or the single threonine
mutants were as effective as wild-type ß1A
(Fig. 4A). Hence, these results
indicate that phosphorylation of these motifs is dispensable for
internalization. GD25ß1AY783F, GD25ß1AY795F and GD25T789A cells
bound to as many bacteria as cells expressing wild-type ß1A, whereas
those expressing ß1AY783A, ß1ATT788-9AA, ß1AYY783/795FF and
ß1AT788A bound to slightly less bacteria
(Fig. 4B). However, the uptake
efficiencies by the two latter were similar or higher compared to cells
expressing wild-type ß1A, indicating that the amount of bound bacteria is
not correlated with internalization efficiency. Moreover, there was no
striking difference in integrin-expression
(Table 1) or in adhesion to
invasin-coated surfaces between the different ß1A-integrin mutants
(Fig. 4C).
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|
Cells affected in bacterial uptake also have altered spreading
Earlier work has suggested that peripheral focal-complex-like structures
play a role in uptake of Yersinia
(Persson et al., 1997;
Persson et al., 1999
).
Therefore it was of interest to investigate whether the different
ß1-integrin mutants were affected in their ability to form focal contact
structures. For this analysis, cells expressing different mutants were seeded
on cover slips and incubated overnight in similar conditions to those used in
uptake studies. The cells were then fixed and the integrins were visualized by
indirect immunofluorescence using ß1-integrin-specific antibodies. All
cells, including GD25, exhibited spread morphology, which was expected since
they all express integrin
Vß3, which will bind to serum-derived
vitronectin. ß1-integrins were detected in all cells (apart from GD25),
and they localized to focal-contact-like structures except in cells expressing
ß1B or ß1AY783A where the ß1-integrins were distributed along
the cell periphery (Fig.
5).
|
To evaluate invasin-induced focal contact formation, the cells were allowed
to adhere to invasin for 3 hours under serum-free conditions. After this, the
cells were fixed and double stained with ß1-integrin-specific antibodies
together with antibodies against the focal contact marker vinculin or with
antibodies against phosphotyrosine. To exclude binding of Vß3 to
secreted matrix proteins, the cells were pretreated with cycloheximide; in
addition, the GRGDS peptide was included at a concentration known to block
Vß3 binding without severely affecting ß1A-mediated
attachment to fibronectin (Wennerberg et
al., 1998
). With this pretreatment, the GD25 cells did not attach
to invasin-coated dishes (data not shown). The ß1A-expressing cells
spread with a uniform morphology, in some cases with a pointed end, and they
formed vinculin-, phosphotyrosine- and ß1-integrin-containing focal
contacts at the edges as well as central focal adhesion-like structures spread
throughout the cell (Fig.
6A,B). The cells expressing the single and double tyrosine to
phenylalanine ß1A mutants spread and formed focal contacts similar to
cells expressing wild-type ß1A (Fig.
6E-G). The ß1B-expressing cells did adhere to the invasin
surface, but were clearly affected in spreading. No focal contact structures
containing vinculin could be observed at cell edges, only a diffuse staining
surrounding the nucleus could be seen (Fig.
6Ca-c). Noteworthy, despite the lack of detectable focal adhesion
markers, are the very weak ß1-integrin staining and clear punctuate
structures of phosphotyrosine staining observed mainly at the very edges of
the cells (Fig. 6Cd). The
GD25ß1AY783A cells, which like GD25ß1B cells exhibited a reduced
ability to internalize Yersinia, were also affected in spreading on
invasin. These cells spread in a `neuron-like' pattern with long protrusions
extending from the center of the cells
(Fig. 6D). The
ß1-integrins as well as vinculin were distributed all over the cells with
some accumulation at the tip of the extensions
(Fig. 6D). Here, a somewhat
different pattern was also seen for phosphotyrosine proteins, which could be
seen in punctuate structures, although they were not as distinct as in
GD25ß1B cells (Fig. 6Dd). The double threonine mutant of ß1A that also was affected in bacterial
uptake was markedly delayed in spreading compared with wild-type ß1A,
although focal-complex-like structures could be seen at the cell edges
(Fig. 6J). The single threonine
to alanine mutants of ß1A resembled wild-type ß1A in spreading and
focal contact localization of integrin, vinculin and phosphotyrosine
(Fig. 6H,I).
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Discussion |
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The ability of ß1B-integrins as well as the ß1ATT788-9AA to bind
to invasin-expressing bacteria and attach to invasin-coated surfaces was
somewhat surprising since these variants have been found to be defective in
binding to physiological ligands (e.g. fibronectin and laminin)
(Retta et al., 1998;
Wennerberg et al., 1998
;
Armulik et al., 2000
). There
was, however, a discrepancy between the binding of bacteria and adhesion to
invasin-coated surfaces, where the ß1B-expressing cells showed lower
binding compared with the ß1A-expressing cells in the latter case. Thus,
the binding of invasin to ß1B is obviously weaker than that to ß1A,
but still sufficient for binding bacteria. It is likely that the bacteria
present a denser and defined surface of correctly exposed ligands compared
with that on the coated dish where the binding epitope is likely to be
distributed more randomly. In addition, the shear force during the washing
procedure is higher in the cell attachment assay than in the bacterial binding
assay.
ß1B-expressing cells binding to invasin had an impaired signal
transduction as tested by immunoprecipitation experiments. Furthermore, they
showed a defective spreading on invasin. It is obvious that this integrin
isoform does not participate in establishing typical focal adhesion sites on
the invasin substrate, in analogy with previous studies of fibronectin and
laminin (Balzac et al., 1993;
Armulik et al., 2000
). However,
our finding of the involvement of the so far non-identified phosphotyrosine
protein complex(es) in spreading GD25ß1B cells implies that some kind of
signaling is associated with this truncated receptor. The signaling can be
derived from either the very short ß1B cytoplasmic tail or from the
associated
-chain. Interestingly, p130Cas was weakly tyrosine
phosphorylated in response to adhesion to invasin, which suggests that p130Cas
could be activated upon ß1B binding to invasin. Work to identify the
phosphotyrosine protein(s) is currently ongoing, and this will hopefully
contribute to a better understanding of the molecular events involved in
formation of integrin adhesions, dissecting early microspike-associated
signaling from that associated with later events (lamellipodia and final
adhesion).
The ability of ß1B-integrins to bind to invasin without being able to
internalize invasin-expressing bacteria clearly indicates that bacterial
internalization is an active process and that the cytoplasmic tail of the
ß1A subunit mediates the necessary signaling. However, when analyzing the
importance of different amino acid motifs in the ß1A-integrin cytoplasmic
tail, only mutations causing more severe alterations in the structure resulted
in reduced internalization capacity. The finding that the Y783F mutation is
tolerated, whereas the Y783A mutation abrogates internalization of bacteria,
implies that the NPIY motif is important for uptake but that there is no need
for tyrosine phosphorylation of the NPXY motifs for bacterial internalization
to occur. This result is consistent with the fact that the corresponding motif
in the major phagocytic receptor, integrin Mß2 (CD11b/CD18,
Mac-1), that is present on professional phagocytes contains phenylalanine
instead of tyrosine at this position
(Kishimoto et al., 1987
;
Law et al., 1987
). Moreover, a
tyrosine to glutamic acid mutation in the first NPXY motif of
ß1-integrins has been shown to result in an integrin that is unable to
internalize bacteria (Tran Van Nhieu et
al., 1996
). Since glutamic acid may mimic a phosphorylated
tyrosine (Maciejewski et al.,
1995
), this finding suggests that phosphorylation of this residue
is unfavorable for internalization. However, the more drastic mutation of the
tyrosine in the first NPXY motif to alanine probably disturbs the predicted
tight turn conformation at this motif
(Haas and Plow, 1997
), and
this could explain the inability for this mutant to mediate internalization of
bacteria. Similarly, different effects on integrin localization to focal
contact structures when exchanging the tyrosine in the integrin NPIY motif to
phenylalanine or to alanine had been seen for chicken ß1-integrins
overexpressed in a background of wildtype ß1-integrins in NIH3T3 cells
(Reszka et al., 1992
). Under
these conditions the phenylalanine variant localized to focal contacts upon
adhesion to fibronectin, whereas the corresponding alanine variant exhibited
reduced localization to these sites
(Hayashi et al., 1990
;
Reszka et al., 1992
). The
defective internalization of bound bacteria by the ß1ATT788-9AA mutant
integrin could be caused by the removal of phosphorylation sites or by
conformational disruption. Since mutations of the individual threonines did
not have any major effects, the first alternative appears unlikely. These
threonines are flanked by hydrophobic amino acids, and the change of both
threonines to alanines will remove the intervening polar groups and markedly
change the nature of the region between the two NPXY motifs. The importance of
keeping these regions structurally intact probably reflects that they are part
of interaction domains important for downstream signaling. Both the NPXY and
the double threonines have been implicated as interaction domains for
cytoskeletal and signaling proteins
(Burridge and Chrzanowska-Wodnicka,
1996
; Liu et al.,
2000
). The Y783A mutation of ß1A disrupts the binding of the
cytoskeletal proteins talin and filamin to the integrin
(Pfaff et al., 1998
;
Kääpä et al.,
1999
), and the TT788-9AA mutation impairs binding of the integrin
interactive protein ICAP-1 (Stroeken et
al., 2000
; Degani et al.,
2002
). However, since the ß1ATT788-9AA, like
ß1B-integrins, also has an altered extracellular conformation
(Wennerberg et al., 1998
), it
can not be excluded that part of the observed effect on bacterial
internalization capacity is caused by a lower affinity for invasin.
A common feature for the mutations causing reduced capacity to internalize
Yersinia was that they also affected cell spreading on immobilized
invasin. This correlation is not surprising since both processes involve
integrin-mediated adhesion and membrane extensions. The Yersinia
virulence factor YopH, which mediates phagocytic inhibition of target cells,
specifically disrupts peripheral focal contact structures, and YopH that are
deficient in focal adhesion recognition exhibit a reduced inhibitory effect
(Persson et al., 1997;
Persson et al., 1999
).
Peripheral focal contact structures have therefore been implicated as
important for uptake of Yersinia. In accordance, all ß1-integrin
variants that could mediate uptake of Yersinia also localized to
peripheral focal contact structures, albeit that these structures were not
always morphologically similar to wild-type focal contacts, and two out of
three mutations that affected uptake also caused a dislocation of the integrin
from peripheral focal contacts. This suggests that the capacity to form
peripheral focal complex structures might be important, but not sufficient,
for integrin-dependent bacterial internalization. Tran Van Nhieu et al. have
reported that several integrin mutants that exhibited reduced focal contact
association were more efficient in promoting bacterial uptake
(Tran Van Nhieu et al., 1996
).
This might appear contradictory to our data, but since that study was made
with a background of endogenous wild-type ß1-integrins it is difficult to
draw firm conclusions on the effect of the mutations. However, in spite of the
different systems used, it is noteworthy that in both cases several of the
integrin mutants exhibiting reduced focal contact localization were still
observed in peripheral complex-like structures. Furthermore, FAK null cells,
which have more stable and higher numbers of focal contacts compared with
cells expressing FAK (Ilic et al.,
1995
), exhibit an impaired capacity to internalize bacteria
(Alrutz and Isberg, 1998
) in
addition to being defective in cell migration. Since FAK is implicated in the
turnover of focal contacts, these data indicate that it is the dynamics of
focal complexes rather than the presence of such structures per se that is
important in the bacterial uptake process. This appears likely, considering
that bacterial internalization is a dynamic process involving initial adhesion
of bacteria to the cell surface followed by membrane extensions and closure of
a phagosome. Hence, if the ability to form and reshape focal contact-like
structures is critical for bacterial internalization, integrin variants that
are less potent in forming rigid and stable focal adhesions may be more
efficient, whereas variants that cannot form these structures at all, in this
case ß1B and ß1AY783A, are defective in phagocytosis. In the case of
the double threonine to alanine mutant the receptor was defective in uptake
and spreading, even though it could localize to peripheral focal-contact-like
structures. It is possible that the nature of these adhesion complexes differ
from these of uptake-competent integrins and that there are certain protein
interactions that are required at some step of the internalization process and
which fail because of the missing threonines.
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Acknowledgments |
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References |
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