1 Craniofacial Developmental Biology and Regeneration Branch, National Institute
of Dental and Craniofacial Research, National Institutes of Health, Bethesda,
MD 20892-4370, USA
2 Department of Tissue Biology, Jerome H. Holland Laboratory, American Red
Cross, Rockville, MD 20855, USA
* Author for correspondence (e-mail: kenneth.yamada{at}nih.gov )
Accepted 19 February 2002
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Summary |
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Key words: Adhesion, Integrin, Kinectin, Kinesin-binding proteins, Endoplasmic reticulum, Fibronectin
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Introduction |
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To analyze cellular responses to integrin clustering, an experimental
approach was developed using beads coated with integrin ligands to mimic
cell-to-ECM adhesion formation (Grinnell
and Geiger, 1986; Plopper and
Ingber, 1993
). For example, fibronectin-coated beads can rapidly
induce the accumulation of vinculin, paxillin,
-actinin, talin and
F-actin (Grinnell and Geiger,
1986
; Mueller et al.,
1989
; Plopper and Ingber,
1993
; Lewis and Schwartz,
1995
; Miyamoto et al.,
1995b
). This procedure also permits the rapid isolation of intact
integrin-based adhesion complexes (IAC) after integrin crosslinking and allows
searches for cytoskeletal and other components that associate with IAC
(Plopper and Ingber,
1993
).
Previous studies using beads coated with integrin ligands or integrin
antibodies have shown that both integrin occupancy and clustering into
aggregates play important roles in IAC formation, and that these two signals
can synergize; a `hierarchy' of molecules requiring either one or both of
these signals to accumulate was identified
(Miyamoto et al., 1995a;
Miyamoto et al., 1995b
).
Numerous cytoskeletal proteins have been localized to the IAC by morphological
and biochemical approaches, but integrin clustering can also induce the
aggregation of growth factor receptors, stimulate the phosphorylation of FGF,
EGF and PDGF receptors, and activate downstream signal transduction
(Plopper et al., 1995
;
Schwartz et al., 1995
;
Miyamoto et al., 1996
;
Sundberg and Rubin, 1996
). In
addition, recent studies have shown that mRNA and ribosomes also localize to
IAC when integrins are clustered with FN-coated beads
(Chicurel et al., 1998
).
Furthermore, cell adhesion induces increased translation of pre-existing mRNAs
(Benecke et al., 1978
;
Farmer et al., 1978
). Taken
together, these studies indicate that IAC are important for redistribution of
cytoplasmic components and a variety of integrin functions.
In the present study, we have used beads coated with a specific integrin ligand to isolate intact IAC in order to identify new proteins that associate with these complex cellular structures. We searched for novel component(s) that might be particularly markedly sequestered from the cytoplasm into IAC compared with other components to potentially establish the concept of differential recruitment. Unexpectedly, we found that kinectin, an integral membrane protein prominent in endoplasmic reticulum (ER) that binds to kinesin and is involved in vesicle transport, becomes strikingly localized to intracellular sites at regions where cells are in contact with FN ligand. Over 50% of total cellular kinectin becomes associated with fibronectin-integrin adhesion sites, an enrichment many times higher than the classical components vinculin and paxillin. Furthermore, two ER-resident proteins, RAP and calreticulin, accumulate at these sites. Our results demonstrate that IAC formation can trigger integrin-mediated differential recruitment of cytoplasmic components, and suggest control of the localization of a portion of the ER, thereby potentially influencing sites of ER-based protein synthesis and secretion.
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Materials and Methods |
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Polystyrene microbeads (mean diameter 10 µm) were obtained from
Polysciences, Inc. Tosyl-activated magnetic microbeads (mean diameter 4.5
µm) were purchased from Dynal. Human plasma fibronectin (FN) and
vitronectin (VN) were purified as previously described
(Miekka et al., 1982;
Yatohgo et al., 1988
).
Collagen I (Vitrogen 100) was purchased from Collagen Corp. Concanavalin A
(ConA) and poly-DL-lysine were obtained from Sigma. FN fragments containing
type III repeats 5-10 (III5-10) and type III repeats 5-10 with the
RGD sequence mutated to KGE (III5-10KGE) were described previously
(Danen et al., 1995
) and were
generously provided by Shinichi Aota (CDBRB, NIDCR, NIH).
Plasmid DNAs for pRK-VSV-tensin and pRK-VSV were kindly provided by Kazue
Matsumoto (CDBRB, NIDCR, NIH). The pRK-VSV plasmid was prepared by inserting
two continuous VSV sequences with a Kozak consensus sequence in a
cytomegalovirus promoter-based expression system. The puromycin resistance
plasmid pHA262pur was generously provided by Hein te Riele (Netherlands Cancer
Institute, Amsterdam, The Netherlands)
(Lacalle et al., 1989).
Preparation of cDNAs encoding kinectin and its fragments
Plasmid constructs designed to express five portions of kinectin and
full-length kinectin are depicted in Fig.
7A. The eukaryotic expression vector pRK-VSV containing a VSV tag
was used to place the kinectin cDNAs under the transcriptional control of the
cytomegalovirus promoter.
|
The cDNAs encoding various portions of kinectin shown in
Fig. 7A were assembled from
restriction fragments of PCR-generated kinectin cDNAs, which were generated
using as template the full-length chick kinectin cDNA plasmid pGinKNT
(Yu et al., 1995) kindly
provided by Michael Sheetz (Columbia University). The fragment I construct
(designated PCR fragment 1; Table
1) encoding residues 1-326 of kinectin was generated by PCR using
primers 1 and 2 (Table 2). PCR
fragment 1 (1007 bp) was cleaved by EcoRI and SalI and
ligated into EcoRI-SalI linearized vector pRK-VSV.
Similarly, constructs for fragments II, III, IV and V
(Table 1) encoding various
portions (residues 327-629, 630-935, 935-1365 and 327-1365) of kinectin were
prepared as described for fragment I.
|
|
For construction of full-length kinectin, pGinKNT
(Yu et al., 1995) plasmid DNA
was digested with BglI and BglII to liberate a 2759 bp
kinectin fragment encoding kinectin nucleotides 1015 to 3774. Kinectin
contains both BglI and BglII sites (1015 and 3774 in the
kinectin sequence). PCR-generated fragment 1 was subjected to EcoRI
and BglI digestion to obtain an EcoRI-BglI fragment
encoding kinectin nucleotides 58-1015. PCR-generated fragment 4 was digested
with BglII and SalI restriction enzymes to generate a
BglII-SalI fragment encoding kinectin nucleotides 3774 to
4176. Full-length 4126bp kinectin cDNA was prepared by ligation of
EcoRI-BglI fragment 1, the 2759bp
BglI-BglII kinectin fragment, and
BglII-SalI fragment 4 together into a linearized
EcoRI-SalI pRK-VSV vector. Plasmid DNAs of all PCR-generated
fragments were subjected to complete DNA sequencing analysis to confirm the
integrity of each construct.
Cell culture and transfection
Primary human foreskin fibroblasts (HFF) and 293 cells were maintained in
Dulbecco's modified Eagle (DME) medium (Gibco/BRL Life Technologies)
supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin
and 100 µg/ml streptomycin. The cells were a gift from Susan Yamada (NIDCR,
NIH) and were used at cell passages 9-22. The NIH3T3 cell line was obtained
from American Type Culture Collection. These cells were maintained in DME
medium supplemented with 10% bovine calf serum, 100 units/ml penicillin and
100 µg/ml streptomycin. Transient transfections of kinectin and its
fragments into NIH3T3 cells were performed using Lipofectamine Plus (Gibco/BRL
Life Technologies) according to the protocol provided by the manufacturer. In
other experiments, kinectin plasmid DNAs were introduced into HFF by
electroporation. Briefly, pRK-VSV containing either no insert or full-length
kinectin, kinectin fragments or tensin (30 µg each) was transfected into
1.5x106 cells by electroporation. Cells were then cultured in
serum-containing medium for 24 hours with 5 mM sodium butyrate to enhance
expression of transfected genes. In some experiments, plasmid DNAs encoding
kinectin or its fragments were transfected into HFF together with 6 µg of
the puromycin selection vector pHA262pur. Cells were subcultured at a 1:3
dilution for 24 hours after transfection and were selected for 2 days in 1
µg/ml puromycin-containing medium.
Isolation of integrin-based adhesion complexes (IAC)
IAC were isolated as described previously
(Plopper and Ingber, 1993)
with some minor modifications. Tosyl-activated magnetic beads (4.5 µm;
Dynal) were coated with either anti-integrin antibodies, FN, FN fragment
III5-10KGE, or ConA at 5 µg per 107 beads
in 0.1 M phosphate buffer, pH 7.4, according to the manufacturer's protocol.
Cells were removed from flasks by trypsin-EDTA and allowed to recover in DME
medium containing 10% fetal bovine serum. Cells were then washed twice with
DME medium containing 1% BSA and 20 mM Hepes, pH 7.6 and resuspended at
1x106 cells/ml. Cells were mixed with FN-, ConA-,
III5-10-, III5-10KGE-, mAb13-, or ES66-coated beads (10
beads/cell) and rotated gently for 20 minutes using a rotator at 37°C. The
complex of beads and bound cells was collected by a magnetic particle
concentrator (Dynal) and was washed with 1 ml cold CSK buffer [50 mM NaCl, 300
mM sucrose, 3 mM MgCl2, protease inhibitor mixture (Boehringer
Mannheim), and 1 mM PMSF in 10 mM PIPES, pH 6.8] without detergent. The pellet
containing cell-bead complexes was extracted with 0.5 ml of cold CSK buffer
containing 0.5% Triton X-100 and sonicated for 10 seconds with a 50 watt
Ultrasonic Processor (Aldrich, model GE 50, set at amplitude 20). The
beads-protein complexes were washed five times with 1 ml cold CSK buffer
containing detergent. In experiments to test for nonspecific binding, beads
were incubated with cell homogenates. Cells were homogenized in CSK buffer
containing detergent and clarified by centrifugation in a microfuge at 19,000
g for 15 minutes at 4°C. The supernatants were incubated
with FN- or ConA-coated beads for 15 minutes at 4°C; the beads were then
washed and processed as described above.
Proteins were extracted from beads and subjected to SDS-PAGE and immunoblot analyses. The ratio of bead-bound to non-bound (soluble) protein was determined by quantitative densitometry and correction for the proportion of sample used for immunoblotting. For example, if gels were loaded with all of the material extracted from beads and 1/10 of the sonicated supernatant containing non-bound material, we calculated the ratio of bead-bound protein as R=B/10S, where R is the ratio, B is the absorbance of immunoblotted bound material, and S is the absorbance of immunoblotted supernatant fraction (1/10 volume). Alternatively, we calculated P=[B/(B+10S)]x100, where P is the percentage of a protein that was bead-bound.
Immunoprecipitation and immunoblot analyses
Confluent 293 cells (15 cm diameter dish) were lysed with 1 ml of RIPA
lysis buffer [50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(Hepes; pH 7.5), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM
EGTA, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 100 mM NaF, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), protease
inhibitor mixture (Boehringer Mannheim) and 1 mM phenylmethylsulfonyl
fluoride]. The cell extracts were clarified by centrifugation at 20,000
g for 15 minutes at 4°C. The supernatants were absorbed
with protein G-Sepharose for 1 hour at 4°C and centrifuged at 5000
g for 5 minutes at 4°C. Antibodies (4 µg/ml) and a
fresh aliquot of protein G-Sepharose (30 µl/1 ml of lysate) were added to
the supernatants and incubated for 1 hour or overnight at 4°C. The
immunoprecipitated proteins were washed five times with RIPA buffer at
4°C. Protein complexes bound to beads were solubilized in reducing
SDS-PAGE sample buffer, boiled for 5 minutes, separated by SDS-PAGE, and
transferred to nitrocellulose membranes (Novex) for 1.5 hours at 30 V.
Unoccupied protein binding sites were blocked by incubating the membranes with blocking buffer [5% nonfat dried milk in TBS-T (150 mM NaCl, 50 mM Tris-HCl, 0.1% Tween-20, pH 7.4)]. Antibodies were diluted in blocking buffer and incubated with membranes for 1 hour at room temperature. The membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated goat antimouse or -rabbit IgG (Bio-Rad). Bound antibodies were visualized using a SuperSignal kit (Pierce) and Hyperfilm (Amersham).
Partial amino acid sequence analysis
Partially immuno-affinity purified 160 kDa polypeptide was separated
by SDS-PAGE and transferred to nitrocellulose. The polypeptide band migrating
at
160 kDa was visualized by staining with Ponceau S (Sigma) and excised
from the membrane. The nitrocellulose strip containing the 160 kDa polypeptide
was digested with endoproteinase Lys-C (Boehringer Mannheim) in 0.1 M
NH4HCO3 as described
(Aebersold et al., 1987
). The
liberated digest was fractionated on a Vydac C18 column using a
microbore HPLC (Model 130; Applied Biosystems). Peptides from individual peaks
were then subjected to Edman degradation using a protein sequencer (Model
477A; Applied Biosystems).
Indirect immunofluorescence microscopy and bead-binding assay
Polystyrene beads (mean diameter 10 µm, or 4.5 µm for comparisons)
were coated with 50 µg/ml FN, 100 µg/ml ConA, or 100 µg/ml polylysine
in 0.1 M carbonate buffer, pH 9.5, at 4°C overnight. The ligand-coated
beads were blocked with 1% BSA in Dulbecco's PBS (D-PBS) for 1 hour at room
temperature. Glass coverslips (12 mm; Carolina Biological Supply Company) were
coated overnight with 20 µg/ml type I collagen (Vitrogen 100, Collagen
Corp.) in D-PBS and then blocked with 1% heat-denatured BSA for 1 hour at room
temperature. To inhibit FN synthesis so as to retain a diffuse distribution of
FN receptors, HFF cells were incubated with DME medium containing 25 µg/ml
of cycloheximide and 10% FN-depleted fetal bovine serum for 2 hours at
37°C. Cells were detached with trypsin-EDTA and allowed to recover in DME
medium containing cycloheximide and FN-depleted serum for 45 minutes. Cells
(2x104) were plated on collagen-coated coverslips for 30
minutes at 37°C. Ligand-coated beads were then added to the cells at a
ratio of 10 beads per cell and incubated for 20 minutes at 37°C.
Subsequently, the cells were fixed with 4% paraformaldehyde with 5% sucrose in
D-PBS for 20 minutes and permeabilized with 0.4% Triton X-100 in PBS for 5
minutes at room temperature.
For kinectin staining, HFF were plated on FN-coated coverslips (10
µg/ml) for time periods ranging from 20 minutes to 2 hours,
fixed-permeabilized for 3 minutes with the above fixative containing 0.5%
Triton X-100, and post-fixed for an additional 20 minutes with the fixative
alone. The coverslips were incubated with an appropriate dilution of different
primary antibodies in D-PBS for 1 hour at room temperature. The coverslips
were then washed with D-PBS three times and incubated with Cy3-conjugated
secondary IgG in D-PBS. Double staining with two primary mouse antibodies was
achieved as follows: The fixed samples were blocked with 20% donkey serum in
D-PBS for 30 minutes, washed, and stained with mouse anti-kinectin antibody
for another 30 minutes. After three 5-minute washes with D-PBS containing
0.05% Tween 20 (D-PBST), the samples were incubated for 30 minutes with goat
anti-mouse IgG (25 µg/ml in D-PBST, 0.5% donkey serum) and washed as above.
To prevent possible redistribution of bound antibodies, the samples were fixed
briefly again (10 minutes), washed, and blocked again with 20% donkey serum in
D-PBS for 15 minutes. The coverslips were then stained with the appropriate
dilution of the second primary mouse antibody (anti--actinin) for 30
minutes. After three washes with D-PBST, the samples were incubated for 30
minutes with a mixture of labeled secondary antibodies consisting of
FITC-conjugated donkey anti-goat and Cy3-conjugated donkey antimouse IgGs to
visualize kinectin and
-actinin, respectively. Specificity was
confirmed by appropriate controls.
After three washes with D-PBST, each coverslip was mounted on a glass slide
with mounting medium (Biomeda) containing 1 mg/ml 1,4 phenylenediamine to
minimize photobleaching. Immunofluorescent samples were examined using a Zeiss
Axiophot fluorescence microscope. Accumulation of a particular protein at a
bead was assayed as described previously
(Miyamoto et al., 1995b) and
scored as positive if it localized as aggregates or thick zones around at
least half of the bead.
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Results |
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Partial amino acid sequence analysis of 160 kDa protein
Internal proteolytic peptides were generated from the 160 kDa protein by
Lys-C digestion. These peptides were purified by HPLC and subjected to
microsequencing. The amino acid sequence of these peptides was used to search
protein databases. As shown in Fig.
2, the sequence of these peptides matched exactly with the human
CG1 or kinectin sequence (Toyoshima et
al., 1992; Futterer et al.,
1995
; Print et al.,
1996
).
|
Kinectin accumulation with FN-coated beads
Immunoblot analysis using the monoclonal KR160 antibody against kinectin
confirmed that the 160 kDa protein associated with FN was kinectin
(Fig. 3A). In order to test the
specificity of kinectin association with IAC induced by integrin clustering,
we used beads coated with an FN fragment containing the cell-binding domain
(III5-10) or the adhesion-blocking/mimicking ß1
integrin monoclonal antibody mAb13 to induce formation of IAC. As shown in
Fig. 3, kinectin accumulated
with wildtype fragment III5-10RGD
(Fig. 3A) or mAb13-coated beads
(Fig. 3B) but not with a
mutated version of III5-10 that had its RGD site substituted by KGE
(III5-10KGE, Fig.
3A), nor with class-matched control monoclonal antibody
ES66-coated beads (Fig. 3B). We
next examined whether kinectin specifically accumulates with IAC or merely
binds directly to FN-coated beads. As shown in
Fig. 3C), kinectin did not bind
to FN- or ConA-coated beads that were mixed with cell homogenates
(Fig. 3C, Lysate). In contrast,
we again detected kinectin accumulation with IAC after integrin clustering
induced by the same FN-coated beads interacting with the exterior of living
cells (Fig. 3C, Cells). Taken
together, these results indicate that kinectin is specifically recruited to
IAC after integrins are clustered.
|
The proportion of total cellular kinectin bound to beads was determined by
densitometry of western immunoblots (e.g.
Fig. 4). The ratio of
bead-bound to soluble kinectin was 1.3±0.31 (s.e.m.), indicating that
57% of total cellular kinectin became associated with bead-bound complexes. In
this bead-binding system, cytoskeletal molecules such as actin, vinculin,
paxillin, talin, tensin and -actinin also accumulated with beads coated
with FN or FN fragment III5-10
(Fig. 4, Beads). None of these
proteins was clustered and isolated with FN mutant fragment
III5-10KGE-coated beads (Fig.
4) or with control ConA-coated beads (data not shown).
Interestingly, the bead-binding ratios for the well-known IAC constituents
vinculin and paxillin were only 1.6% and 0.2%, respectively, of the total
cellular content of these proteins.
|
Since kinectin is a kinesin-binding protein and an ER integral membrane protein, we examined whether other proteins involved in vesicular transport could accumulate with FN-coated beads. As shown in Fig. 4, in comparison to kinectin, relatively little dynein accumulated after binding of FN-coated beads; mutant FN fragment III5-10KGE-coated beads showed neither kinectin nor dynein accumulation (Fig. 4). Interestingly, neither FN-coated beads nor FN fragment III5-10-coated beads (nor FN fragment III5-10KGE-coated beads) induced the accumulation of either kinesin or tubulin (Fig. 4). These results indicated that kinectin can be clustered specifically upon integrin aggregation by fibronectin in the absence of substantial accumulation of kinesin and tubulin.
Cellular distribution of kinectin
Immunofluorescence analysis of human fibroblasts with anti-kinectin
antibodies at early times after adhesion to tissue culture substrates revealed
a linear staining pattern in addition to the expected endoplasmic reticulum
staining. This staining was less intense than within the ER, but it was highly
reproducible and particularly prominent at early times of cell spreading
(Fig. 5). This kinectin
localization was organized as fibrils (Fig.
5A,B) that were parallel to actin stress fibers
(Fig. 5B,b,b',c,c'). Kinectin
staining was absent from -actinin-positive focal adhesions
(Fig. 5A,c,c'). After 1 hour of
spreading, only occasional cells demonstrated this fibrillar pattern of
staining, indicating that this kinectin accumulation was transient.
|
In order to test further for the accumulation of kinectin after integrin
crosslinking, we used immunofluorescence staining of human foreskin
fibroblasts after binding of FN-coated beads in a system for visualizing rapid
experimental induction of adhesive complexes. As shown in
Fig. 6A, kinectin showed marked
accumulation in a zone around the FN-coated beads. In the experiment shown in
Fig. 6, semi-quantitative
analysis showed that 53% of the FN-coated beads induced clear kinectin complex
formation, while polylysine-coated beads were only 3% positive (significant at
the P<0.0001 level by unpaired t-test). Similar results
were observed when smaller-sized beads (4.5 µm) coated with FN were used
(data not shown). Under the same conditions, activated ß1
integrin staining also showed similar aggregation around FN beads
(Fig. 6I). As expected,
cell-substrate adhesion proteins such as paxillin and tensin also accumulated
around the FN-coated beads (Fig.
6G; and data not shown). In addition, we observed similar results
when cells were clustered with beads coated with the
adhesion-blocking/mimicking monoclonal antibody mAb13 to the
ß1 integrin (data not shown). Similar observations were also
seen in WI-38 lung fibroblasts after clustering with FN-coated beads (data not
shown). In contrast, polylysine-coated beads did not induce any such
transmembrane accumulation (Fig.
6B,H,J) nor did ConA-coated beads (data not shown). The
possibility that kinectin might bind directly to accumulations of F-actin was
tested using a standard actin polymerization assay; no binding could be
detected, even though -actinin in the same samples bound efficiently to
the F-actin (data not shown).
|
Since kinectin is an endoplasmic reticulum-associated protein, we next examined whether other ER-resident proteins could be accumulated after cells were clustered with FN-coated beads. Calreticulin and RAP were both clustered around the FN-coated beads (Fig. 6C and E, respectively). In the experiment shown in Fig. 6, clear calreticulin accumulation was observed around 39% of the FN-coated beads and around only 3.5% of the polylysine-coated beads (P<0.0001). Similarly, RAP exhibited 51% positive accumulation around FN-coated beads, but only 4.7% around the polylysine-coated beads (P<0.0001). In contrast, neither fibronectin- nor polylysine-coated beads induced clustering of tubulin (Fig. 6K,L). In addition, treating HFF with microtubule-modifying drugs such as nocodazole had no effect on the recruitment of kinectin to FN-coated beads (data not shown). Taken together, these results show that kinectin and two other ER-associated proteins are recruited to the sites of integrin clustering.
Both N-terminal and C-terminal portions of kinectin are clustered
with FN-coated beads
To localize the region(s) of kinectin involved in clustering after integrin
crosslinking, NIH3T3 cells were transfected with various fragments of
kinectin, and the cells were incubated with FN-coated beads. Proteins bound to
FN-coated beads were separated by SDS-PAGE, transferred to nitrocellulose
membranes, and probed with VSV antibody. As shown in
Fig. 7B, both N-terminal and
C-terminal portions of kinectin (residues 1-326 and 935-1365, respectively)
were also clustered with FN-coated beads. Other fragments encoding residues
327-629 and 630-935 were not detected in the beads-protein complexes, whereas
a large fragment (327-1365) containing the C-terminal region was bound
(Fig. 7B). In addition, as
expected, VSV-tensin was clustered around the FN-coated beads (not shown). The
protein expression levels of VSV-kinectin and its fragments in NIH3T3 cells
are shown in Fig. 7C. As
expected in controls expressing VSV alone, we did not detect any VSV-reactive
protein bands of sizes 30-200 kDa clustered around FN-coated beads or in
the extract of cells transfected with VSV alone
(Fig. 7B,C). These data
indicate that the N-terminal hydrophobic and C-terminal
-helical
coil-coiled regions of kinectin contain binding site(s) involved in the
localization of kinectin at IAC.
Next, we examined whether the accumulation of N-terminal and C-terminal portions of kinectin at IAC can be detected by immunofluorescence staining of cells after integrin clustering using FN-coated beads. Human foreskin fibroblasts were transfected with various fragments of kinectin and then clustered with FN-coated polystyrene beads. As shown in Fig. 7D, fragments from the middle half of kinectin (containing residues 327-629 and 630-935) were not significantly accumulated at the FN-coated beads (Fig. 7Dc,d). In contrast, the hydrophobic N-terminal region (1-327) and both fragments containing the C-terminal domain (935-1365 and 327-1365) accumulated with FN-coated beads (Fig. 7Db,e). These results confirm that both N-terminal and C-terminal portions of kinectin are individually capable of associating with IAC.
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Discussion |
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It is noteworthy that the molecule for which kinectin is the receptor, kinesin, is not co-accumulated during the striking clustering of kinectin. Moreover, tubulin is not co-accumulated, and the process is not inhibited by disruption of microtubules. Thus, the integrin-mediated clustering of kinectin appears to be independent of the classical microtubule-kinesin-kinectin system.
Although the redistribution of kinectin to IAC in bead-induced integrin
clustering assays is specific and massive, thereby establishing the principle
that integrins have the ability to control the global distribution of a
specific cytoplasmic protein, the normal biological role of this phenomenon is
not yet clear. In a popular but also arguably artificial type of assay system
the attachment of cells to a flat tissue culture substrate a
portion of the kinectin molecules show a transient, novel redistribution to
oriented fibrillar structures overlapping actin stress fibers. Thus, both
adhesion to beads and to a planar surface induce temporary kinectin
redistribution. Kinectin is not found in focal adhesions on these planar
surfaces, yet it is found in experimental IAC. However, bead-induced IAC are
not direct equivalents of focal adhesions: they transiently accumulate high
levels of many signaling molecules, such as tyrosine kinase receptors, which
are difficult to demonstrate in normal focal adhesions. We speculate that
these adhesion complexes induced by beads represent a maximal cellular
adhesive response to integrins and are structures combining the properties of
several types of integrin-based adhesion [i.e. focal complexes, focal
adhesions, fibrillar adhesions and/or others (see also
Geiger et al., 2001)].
The co-accumulation of ER-resident proteins calreticulin and RAP with
kinectin at IAC suggest that integrin clustering might play a role in helping
to direct proteins newly synthesized in the ER to the site where signals are
initiated by integrin aggregation. This result extends the observation that
ribosomes and mRNA are targeted to IACs that form when cells bind to FN-coated
beads (Chicurel et al., 1998)
to include localization of kinectin and a fraction of the endoplasmic
reticulum itself. The redistribution of mRNA and ribosomes, kinectin,
calreticulin and RAP to the IAC might contribute to the rapid increase in
protein synthesis in response to cell adhesion, which occurs before changes in
transcription are detectable (Benecke et
al., 1978
; Chicurel et al.,
1998
). Taken together, our results suggest the possibility that
directing ER, and consequently newly ER-synthesized proteins, to the vicinity
of IAC may constitute a novel function of integrin signaling. Calreticulin has
been shown to associate with the cytoplasmic domains of integrin
-subunits although, in these studies, it was originally thought to be
free in the cytoplasm rather than still within the ER
(Rojiani et al., 1991
;
Leung-Hagesteijn et al., 1994
;
Coppolino et al., 1995
). Other
ER-resident proteins involved in chaperone function or vesicular transport may
also participate in this novel kinectin-related integrin accumulation
function. RAP (39 kDa receptor-associated protein) is another ER-resident
protein that binds to the low-density lipoprotein receptor-related protein
(LRP) (Bu et al., 1995
).
Binding of RAP to LRP inhibits all ligands from binding to LRP in vitro.
Recent studies have demonstrated that RAP interacts with LRP in vivo,
functions as a molecular chaperone for LRP, and regulates its ligand binding
activity along the secretory pathway (Bu et
al., 1995
; Willnow et al.,
1996
; Willnow,
1998
).
Kinectin is an integral membrane protein of the ER, and its best-known
function is to serve as a receptor for the molecular motor kinesin
(Toyoshima et al., 1992;
Kumar et al., 1995
). This
latter motor protein is involved in microtubule-based vesicle transport
towards microtubule plus-ends, thereby directing vesicle movement towards the
cell periphery; in contrast, the dynein motor protein is involved in vesicle
movement towards the cell center (Vallee
and Shpetner, 1990
; Walker and
Sheetz, 1993
). Antibody to kinectin has been shown not only to
inhibit kinesin-membrane binding and plus-end-directed vesicle movement, but
also to diminish the binding of dynein to membranes and reduce
minus-end-directed vesicle movement in vitro to a lesser extent
(Kumar et al., 1995
). Kinectin
antibody inhibits the movement of the glycoprotein of vesicular stomatitis
virus (VSV G protein) from the ER to the Golgi and to the plasma membrane.
Kinectin fragments derived from the
-helical coil-coiled region bind to
kinesin and dynein and inhibit vesicle transport in vitro
(Kumar et al., 1997
).
Therefore, one important function of kinectin may be as a component in vesicle
transport (Sheetz, 1996
),
serving as a receptor for both kinesin and dynein and helping these motor
proteins direct the cycle of vesicle movement in cells. The exact mechanisms
by which cells control vesicle transport, directionality and particularly
secretion at specific membrane sites are unclear. Recent studies have shown
that small G-proteins, such as Rac1, RhoD, RhoA and RhoG, bind to kinectin and
can modulate its motile activities (Hotta
et al., 1996
; Vignal et al.,
2001
). Others have also shown that endocytic vesicles contain a
variety of cytoskeletal proteins including kinesin, dynein, actin and tubulin
(Pol et al., 1997
). Kinectin
can also regulate the activity of RhoG, indicating that it has functions
beyond serving as a kinesin receptor
(Vignal et al., 2001
).
Although the mechanisms of kinectin accumulation at IAC are still unknown in the absence of any link to the classical kinesin-microtubule system, the rapid accumulation of over half of the total cellular content of this particular protein appears to be the most dramatic extent of redistribution of a cytoplasmic protein to IAC yet reported. It establishes the novel concept that integrin clustering can exercise global control over the cytoplasmic localization of at least one protein. We suggest that determining the relative enrichment of the many other constituents of IAC may provide further insight into the hierarchy of assembly and function of these complex structures.
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