Department of Cell Biology, University of Virginia School of Medicine, PO Box 800732, Charlottesville, VA 22908, USA
* Author for correspondence (e-mail: desimone{at}virginia.edu)
Accepted 27 February 2003
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Summary |
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Key words: Integrin, Cell migration, Cell adhesion, Xenopus, Fibronectin
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Introduction |
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Alterations in integrin activation state can trigger distinct cell adhesive
functions. For example, when the IIb cytoplasmic tail is replaced with
an integrin
5 tail or carries deletions in the GFFKR motif, the
receptor becomes competent to mediate the assembly of fibronectin (FN) fibrils
(Wu et al., 1995b
). The
cytoplasmic domains of
subunits can also initiate unique
post-ligand-binding signaling events
(Dedhar and Hannigan, 1996
;
Sastry and Horwitz, 1993
). The
4 tail can bind directly to the adapter protein paxillin, and this
property is important for
4ß1 to support strong migration
(Liu and Ginsberg, 2000
).
Nischarin, a novel protein that can affect Rho family GTPase function, is
reported to associate with the
5 tail
(Alahari et al., 2000
). Cells
expressing chimeric integrins with the
2 extracellular and
transmembrane domains fused to an
4 tail showed enhanced migration,
whereas strong collagen gel contraction was observed with the
2 and
5 tails (Chan et al.,
1992
). In myoblasts, enhanced paxillin and mitogen-activated
protein kinase (MAPK) activation is associated with integrin
5, whereas
focal adhesion kinase (FAK) and MAPK are suppressed on ectopic expression of
6A. In each case, the cytoplasmic tails of these subunits are believed
to act by regulating ß1 cytoplasmic tail functions
(Sastry et al., 1999
).
Differences in integrin signaling can also influence cell fate decisions.
Cytoplasmic domain-swapping experiments using quail skeletal muscle cells show
that the
5 tail can promote proliferation, whereas the
6 tail
affects differentiation (Sastry et al.,
1996
). Although studies such as these have led to important
general insights regarding the functional importance of integrin
cytoplasmic tails, specific results vary depending on the integrin and/or cell
type under investigation (Kassner and
Hemler, 1993
; O'Toole et al.,
1994
). To reveal the contribution of integrin
tail-specific signaling in various physiological and pathological processes,
therefore, it is important to conduct these studies in systems amenable to in
vivo analyses.
Studies of tail function in Xenopus gastrulae can provide
useful information about the molecular machinery that controls
position-specific changes in adhesive behaviors in development. The advantage
of the Xenopus system is that embryonic cells or tissue fragments can
be dissected and cultured under conditions that allow the progression of
normal in vivo behaviors and developmental fates. In early Xenopus
embryos, integrins act to assemble FN fibrils along the blastocoel roof (BCR),
mediate the adhesion and migration of mesendodermal cells
(Davidson et al., 2002
), and
are involved in regulating the polarity of cells engaged in cell intercalation
movements (Marsden and DeSimone,
2001
). At least three integrins,
5ß1,
3ß1
and an
V-containing integrin (Joos
et al., 1995
; Joos et al.,
1998
; Meng et al.,
1997
; Whittaker and DeSimone,
1993
), are expressed at these stages of development and may
potentiate the adhesive behaviors of cells on FN. Moreover, integrin adhesive
activity is regulated in both space and time by inductive interactions that
help to specify mesoderm and initiate gastrulation movements
(Ramos and DeSimone, 1996
;
Ramos et al., 1996
).
Our results establish that cytoplasmic tail sequences following the
membrane-proximal GFFK/RR motif are required to confer position-specific
cell-adhesive behaviors in embryos at the gastrula stage. Distinct
cytoplasmic tails differ in their ability to support FN fibrillogenesis on the
BCR and the migration of mesodermal cell and tissue explants. Our data also
suggest that integrin
cytoplasmic domains carry distinct, although
often overlapping, functional properties.
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Materials and Methods |
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Construction of 4 chimeric integrins
The Xenopus integrin 4 cDNA
(Whittaker and DeSimone, 1998
)
and the two cytoplasmic tail truncation mutants were prepared by Charles
Whittaker. The
4 chimeras were engineered using PCR-based methods. In
brief, the DNA fragment that encodes the
4 extracellular and
transmembrane domains, as well as the amino acids KVGFF of the cytoplasmic
tail was amplified from the Xenopus integrin
4 cDNA (basepairs
1 to 3113). The cytoplasmic domain segments of different integrin
subunits were amplified starting with sequence encoding K or R (
2
K760,
3 R977,
5 K1028,
6
R1057,
V K1007) of the conserved GFFK/RR
domain. The two DNA fragments were then digested with restriction enzymes,
ligated and subcloned into the pCS2+ vector.
In vitro transcription of capped RNA and micro-injection
Capped RNA was transcribed in vitro from linearized plasmid DNA using SP6
RNA polymerase (Promega, Madison, WI). Following the transcription reaction,
unincorporated nucleotides were removed using ProbeQuant G-50 Micro Columns
(Amersham Biosciences, Piscataway, NJ) and resuspended in distilled water at
desired concentrations. The amount of transcript injected varied according to
the specific construct, but it was typically in the range of 1.0-5.0
ng/embryo. Micro-injection was performed by targeting either the animal pole
or the future dorsal marginal zone regions of eggs or two-cell-stage
blastomeres, depending on the experiment.
Antibody generation
Two PCR-amplified fragments encoding the C- and N-termini of the
proteolytically cleaved extracellular domain of Xenopus 4 were
each subcloned into the pGEX-KG vector
(Guan and Dixon, 1991
) to
generate two glutathione-S-transferase (GST)-
4 fusion proteins;
4EX-S contained amino acids K557 to E606, and
4EX-LS contained amino acids G607 to E726. GST
fusion proteins were prepared as described in Guan and Dixon
(Guan and Dixon, 1991
) and
used to immunize rabbits for polyclonal antibody production. Immune-sera were
first precleared with a GST cross-linked glutathione-agarose bead column and
then affinity purified on a column containing a mixture of
4EX-S and
4EX-LS fusion proteins. GST fusion protein columns were prepared by
crosslinking GST to glutathione agarose beads using the method described by
Koff et al. (Koff et al.,
1992
). The affinity-purified polyclonal antibody (PcAb) directed
against the Xenopus
4 extracellular domain is named A4EX.
Biotinylation of Xenopus embryonic cells
Xenopus embryos were dissociated in Ca2+-free,
Mg2+-free MBS (CMF) in agarose-coated petri dishes.
EZ-linkTM Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6-biotinamido
Hexanoate) from Pierce (Rockford, IL) was used to biotinylate cell-surface
proteins according to the method described by Alfandari et al.
(Alfandari et al., 1995).
Immunoprecipitation and western blot
Xenopus embryos were extracted in embryo solublization buffer
(ESB) consisting of 100 mM NaCl, 50 mM Tris-HCl (pH=8.0), 1% NP-40 or Triton
X-100, 2 mM PMSF (phenylmethylsulphonylfluoride), 1 µg/ml aprotinin, 1
µg/ml leupeptin, 1 µg/ml, pepstatin A. For immunoprecipitation, embryo
lysates were first precleared with non-immune mouse or pre-immune rabbit serum
coupled to protein G or protein A agarose beads, followed by
immunoprecipitation with desired antibodies. Immunoprecipitated proteins were
then separated on 8% polyacrylamide gels and electrotransferred onto
nitrocellulose membranes for western blot and ECL detection. Horseradish
peroxidase (HRP)-labeled streptavidin was used to detect biotinylated proteins
by western blot.
Cell adhesion and migration assays
GST-FN fusion proteins were prepared and coated onto Falcon petri dishes at
a concentration of 0.23 µM according to Ramos et al.
(Ramos et al., 1996). The GST
fusion proteins used in these experiments included 9.11-GST, which contains
the synergy/RGD site of Xenopus FN, and V-GST, which contains only
the alternatively spliced V-region of FN
(Ramos and DeSimone, 1996
).
Xenopus animal cap cell adhesion assays and activin-induced cell
spreading assays were performed as described by Ramos et al.
(Ramos et al., 1996
).
Phase-contrast images were obtained using a Zeiss Axiovert microscope and an
MTI VE1000 CCD camera using the NIH Image software package.
For dorsal involuted marginal zone (DIMZ) cell migration assays, GFP mRNA
was co-injected with the 4 constructs targeting the future dorsal
marginal zone region. GFP-expressing cells from the DIMZ region of stage 11
embryos were dissected and dissociated in CMF-MBS. These cells were then
transferred onto coated substrates. Digital time-lapse images were recorded
with an Orca Camera (Hamamatsu, Bridgewater, NJ) using a Zeiss Axiophot
microscope equipped with GFP filter set, and the ISEE (Inovision, ISee
Imaging, Raleigh, NC) software package. Paths and parameters of cell migration
were tracked using the Nanotrack function within ISEE. Each experiment was
repeated with three different batches of embryos; a total of 40-65 cells were
tracked and analyzed.
Dorsal marginal zone explants and assays
Dorsal marginal zone (DMZ) explants were prepared as described previously
(Davidson et al., 2002). For
migration assays, DMZ explants expressing
4 constructs were first
allowed to adhere to FN or V-GST coated substrates for 1 hour before
time-lapse image recording. The culture chamber was then placed on a motorized
stage (Ludl, Hawthorne, NY) attached to a Zeiss Axiovert microscope.
Phase-contrast, time-lapse images were recorded using the MTI VE1000 CCD
camera and NIH Image at 1 minute intervals for 1 hour.
FN fibril assembly rescue assay
Embryos expressing 4 constructs targeted to the animal pole region
were injected into the blastocoel at blastula stage 9 with 400 ng of
monocloncal antibodies (mAbs) that block integrin
5ß1 (P8D4)
(Davidson et al., 2002
) or the
RGD site of Xenopus FN (4B12)
(Ramos et al., 1996
), as
previously described (Ramos et al.,
1996
). Embryos were cultured until stage 12, then animal caps were
dissected and washed in MBS, followed by fixation in 2% trichloroacetic acid
(TCA) at 4°C overnight.
For detection of 4 constructs, A4EX was used as the primary antibody
followed by FITC-labeled goat anti-rabbit immunoglobulin G (IgG) (Jackson
Labs, Bar Harbor, ME). For FN, mAb 4H2
(Ramos and DeSimone, 1996
) and
Rhodamine-labeled goat anti-mouse IgG (Jackson Labs) were used as primary and
secondary antibodies, respectively. Florescence was detected using a Zeiss
Axiophot microscope and recorded using a Hamamastu Orca Digital Camera and the
ISEE software. Each experiment was repeated with three different batches of
embryos, and more than ten animal caps for each
4 construct were
examined from every experiment.
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Results |
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A rabbit polyclonal antibody (PcAb) A4EX was generated against the
Xenopus integrin 4 extracellular domain. Similar to mammalian
4, Xenopus integrin
4 is expressed in two forms
a full-length form (140 kDa) and a proteolytically cleaved form consisting of
two products, the partial N-terminal extracellular domain (80 kDa) and the
remaining C-terminal portion that contains the cytoplasmic domain (60 kDa)
(Hemler et al., 1990
). As
shown in Fig. 1A,
4 is
not expressed in control embryos injected with H2O. A4EX recognizes
three bands in the embryos injected with
4wt transcript, representing
the full-length (140 kDa) and the cleaved form (80 kDa and 60 kDa) of the
4 protein. In the
4RR lane, the size of the full-length protein
and the C-terminal fragment containing the cytoplasmic domain is smaller than
that in the
4wt lane because of the tail truncation. D2AP is a PcAb
raised against the
4 cytoplasmic tail
(Whittaker and DeSimone, 1998
)
and, thus, only recognizes the 140 kDa and 60 kDa forms of
4wt
representing the full-length protein and the cleaved product that has the
cytoplasmic tail. As predicted, PcAb D2AP did not recognize
4RR.
|
To confirm that the 4 constructs were expressed on the surface of
embryonic cells, Xenopus embryos injected with RNA transcripts
encoding
4 constructs were cultured until stage 15 and dissociated in
CMF-MBS. Cells were surface labeled with biotin, solublized and subjected to
immunoprecipitation using the A4EX PcAb
(Fig. 1B). With the exception
of
4
2, each of the
4 chimeras and tail truncation mutants
was expressed on the embryonic cell surface at comparable levels (i.e. less
than a twofold difference based on quantification of pixel densities) and
primarily in the cleaved form. The ß1 subunit does not label as
efficiently as the
4 constructs and is therefore only detected on
longer exposures or at later stages when receptor levels at the surface are
increased (Whittaker and DeSimone,
1998
) (data not shown). Because we were unable to normalize the
surface expression level of
4
2 with other
4 constructs,
this chimera is not included in the functional studies described in this
paper. To confirm that each
4 construct formed heterodimeric receptors
with the ß1 subunit, mAb 8c8 (directed against Xenopus ß1
integrin) (Gawantka et al.,
1992
) was used to immunoprecipitate ß1 integrins from embryos
injected with
4 transcripts. All
4 chimeras and tail deletion
mutants were found to co-immunoprecipitate with the ß1 subunit
(Fig. 1C).
Cell adhesion assays were performed to test whether the 4ß1
constructs form functional receptors. Animal cap cells isolated from normal
gastrula-stage embryos attach to FN (Fig.
2A). These cells do not express integrin
4 and are unable
to attach to the V-region of FN (Fig.
2B). Animal cap cells expressing
4 constructs attach to a
V-region GST fusion protein substrate, similar to non-injected cells on intact
FN (Fig. 2C-I). The
4
cytoplasmic tail is not required for cell attachment; cells expressing either
4KV or
4RR were still able to attach to V-GST
(Fig. 2D,E). In all cases,
attached cells formed short filopodial protrusions on the substrate, but did
not spread. The interaction between the
4 extracellular domain and the
V-region is specific, because cells expressing
4 cannot adhere to GST
alone nor to bovine serum albumin (BSA)-coated surfaces (not shown).
|
These experiments confirm that 4 constructs are expressed on the
embryonic cell surface and act as functional heterodimeric receptors by
binding to the V-region of FN. The following studies investigate the
importance of a-subunit-specific tail sequences in supporting morphogenetic
cell adhesion behaviors in Xenopus. They include cell spreading in
response to mesoderm inducing signal, mesodermal cell and tissue migration,
and the assembly of FN matrix on the BCR.
The GFFK/RR motif is sufficient to support cell spreading in response
to activin induction
Cells dissected from the animal cap region can only attach to FN, whereas
involuted marginal zone cells can spread on FN
(Ramos et al., 1996). The
spreading behavior of marginal zone cells can be replicated in animal cap
cells following exposure to the mesoderm inducing factor activin
(Smith et al., 1990
). Previous
studies suggest that activin induction leads to changes in the functional
`activation states' of integrins, which affect adhesive behavior
(Ramos and DeSimone, 1996
).
Because integrin
cytoplasmic tails play important roles in the
regulation of integrin activation, we investigated whether specific a subunit
tail sequences are required for activin responsiveness. Animal cap cells from
embryos injected with
4 construct transcript were treated with 30
units/ml of activin and cultured on V-GST. Spread cells were scored as in
Ramos et al. (Ramos et al.,
1996
) on the basis of polygonal cell shape and the presence of
lamellipodial membrane protrusions. With the exception of
4KV, each of
the
4 constructs was able to support cell spreading
(Fig. 3C,E-I). Activin-treated
4KV cells failed to elongate their cell bodies
(Fig. 3D) but were able to form
some short ruffle-like protrusions, which were small and highly transient. The
4RR construct was able to support cell spreading in a manner similar to
each
4 chimera with a full-length cytoplasmic tail
(Fig. 3E). Thus, the conserved
GFFK/RR domain is the minimal sequence required to mediate cell spreading in
response to activin induction.
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Tracking single mesodermal cell migration mediated by 4
constructs
Individual mesodermal cells isolated from the dorsal involuted marginal
zone (DIMZ) region, unlike untreated animal cap cells, are able to migrate on
FN (Ramos et al., 1996;
Winklbauer, 1990
). To study
the migration behaviors of single mesoderm cells expressing different integrin
cytoplasmic domains,
4 transcripts were injected into the future dorsal
marginal zone region at the four-cell stage. GFP RNA transcripts were
co-injected as a marker to trace expression. At stage 10.5, DIMZ tissue with
GFP fluorescence was dissected, dissociated to single cells and plated on
artificial substrates. The migration of these cells was recorded over a 1 hour
period, then tracked and measured using the Nanotrack function within the ISEE
software package. `Spider' graphs were used to represent the migration
behaviors of these cells (Fig.
4). DIMZ cells migrate actively on 9.11-GST
(Fig. 4A), as do DIMZ cells
expressing
4 on V-GST (Fig.
4B). Many of these cells are able to move persistently and
translocate significant distances from their points of origin, whereas other
cells change their direction more frequently
(Fig. 4A,B). Cells expressing
4KV remain close to the point of origin during the 1 hour time-lapse
recording (Fig. 4C).
4RR
(Fig. 4D) supports slightly
increased migration compared with
4KV, but the tracks of these cells
remain significantly shorter than
4 chimeras containing a full-length
cytoplasmic tail (Fig. 4B,E-H).
Among all of the chimeras,
4
5 has slightly shorter and less
persistent migration tracks (Fig.
4F).
|
The travel distance (the length of a cell migration track) and radial
displacement (the straight-line distance from the start point to the end point
of the migration) within 1 hour were quantified to compare the migration
behaviors of cells expressing different 4 constructs
(Fig. 5A,B). Statistical
analyses confirm and extend the data represented by the `spider' graphs
(Table 2). Thus, the sequences
following the conserved GFFK/RR motif are important for cell motility.
Although the interactions between integrin
5 and the synergy/RGD site
of FN is required for normal DIMZ cell adhesion and migration on FN
(Davidson et al., 2002
;
Ramos and DeSimone, 1996
;
Ramos et al., 1996
), the
5 cytoplasmic domain alone did not support the highest migration rates
in these assays.
|
|
A full-length cytoplasmic domain is required for intact
dorsal marginal zone tissue explant migration on V-GST
At the onset of gastrulation, dorsal marginal zone cells involute at the
site of the dorsal blastopore lip and migrate towards the animal pole along
the FN matrix deposited on the BCR
(Winklbauer and Nagel, 1991).
This directional migration is important for gastrulation movements and the
formation of the three-layered basic body plan. When isolated dorsal marginal
zone (DMZ) tissue explants are placed on FN-coated substrates, the explants
are able to reproduce the in vivo behaviors of the DMZ tissue by spreading and
migrating out from the dorsal lip as a coherent sheet
(Davidson et al., 2002
). The
DMZ explants are schematically represented in
Fig. 6A. These explants were
used to investigate the ability of
4 constructs to support the
migration of intact mesodermal tissue.
|
DMZ explants injected with GFP RNA transcripts spread and migrate on FN but
not the V-region GST fusion protein (Fig.
6B, GFP). DMZ explants expressing 4 chimeras containing a
full-length cytoplasmic domain were each able to migrate out on V-GST, similar
to explants on FN (Fig. 6C;
4
5 is shown as a representative example in
Fig. 6B). The leading edge
cells from the dorsal lip margin of the explants send out frequent
lamellipodial protrusions followed by advancement of their cell bodies. This
eventually leads to the spreading and migration of the whole mesodermal tissue
sheet. In contrast to explants derived from embryos injected with
4
chimera transcripts, DMZ explants expressing
4KV were unable to migrate
on V-GST. Despite the ability of
4RR to support cell spreading in
response to activin induction and some cell motility in single DIMZ cells
(Fig. 3E,
Fig. 5D), this construct also
failed to mediate migration of intact DMZ explants on V-GST. The leading edges
of both
4KV and
4RR explants were able to send out some membrane
protrusions; however, these protrusions were not capable of forming stable
anchors with the substrate. The distance of leading edge advancement in 1 hour
was measured as the migration rate (Fig.
6C). The
4KV and
4RR explants failed to migrate,
whereas
4
5 explants migrated the slowest among each of the
4 chimera explants observed, which is consistent with our previous
observation of single DIMZ cell migration behaviors
(Fig. 5). Thus, the
cytoplasmic domain sequences beyond the GFFK/RR motif play a general positive
role in supporting mesodermal tissue explant migration. The adhesive and
migratory behaviors of
4-expressing explants and dissociated cells on
V-region substrates (e.g. Figs
2,
3 and
6) are similar to those on
intact FN in the presence of antibodies that block endogenous
5ß1
binding to the CCBD of FN (data not shown).
Only 5,
6 and
3 cytoplasmic domains are able to
mediate FN fibril assembly on the blastocoel roof (BCR)
During gastrulation, cells lining the inner surface of the BCR assemble FN
into a fibrillar network (Winklbauer and
Stoltz, 1995). This process is dependent on integrin
5ß1 in vivo. Antibodies that block either
5ß1 or its
ligand, the RGD/synergy domain of FN, can abolish FN fibrillogenesis, leading
to defects in later development (Marsden
and DeSimone, 2001
; Ramos and
DeSimone, 1996
). Integrin
4ß1 is competent to assemble
FN fibrils in vitro when exogenously activated with Mn2+ or
activating antibody (Sechler et al.,
2000
). Because ectopically expressed
4ß1 is able to
bind to FN through the V-region, we investigated whether
4 constructs
could assemble FN into a fibrillar matrix in vivo under conditions that block
endogenous
5ß1-dependent assembly.
Early cleavage-stage embryos injected in the animal pole with transcripts
encoding the 4 constructs were cultured to blastula stages and then
injected with mAb P8D4 (Fig.
7A), which blocks integrin
5ß1 function and FN
fibrillogenesis (Davidson et al.,
2002
). By the end of gastrulation (stage 12), a dense FN fibrillar
network is assembled normally on the BCR of control embryos
(Fig. 7B). Injection of P8D4
into the blastocoel completely abolished the formation of FN fibrils
(Fig. 7C). The
4wt and
two cytoplasmic tail truncation mutants failed to rescue fibril assembly
(Fig. 7D,E,F). However,
4 with the
5 or
6 cytoplasmic domains was able to
assemble significant numbers of fibrils
(Fig. 7H,I). A few short
fibrils were also frequently observed on the BCR injected with the
4
3 transcript (Fig.
7G). No fibrils were observed on animal caps expressing
4
v (Fig. 7J).
|
To further confirm that the observed FN fibril assembly by 4
chimeras was independent of interactions of endogenous integrins with the RGD
site of FN, we repeated the above experiments with mAb 4B12, which blocks the
activity of the RGD motif in the 10th type III repeat of Xenopus FN
(Marsden and DeSimone, 2001
;
Ramos and DeSimone, 1996
).
Similar results were obtained with this antibody, thus we conclude that the
4 chimera-dependent rescues of fibril assembly occur in the absence of
any contribution of endogenous
5ß1 binding to FN (data not shown).
To exclude the possibility that the observed differences in fibril assembly by
various
4 constructs were due to differences in cell-surface expression
levels,
4 transcripts were injected over a range of concentrations.
Differences in cell-surface expression were confirmed by biotinylation and
immunoprecipitation with the A4EX PcAb. When the amount of transcript injected
was varied within a fourfold range, the same results were obtained (data not
shown). Thus, we can conclude that the
5,
6 and
3
cytoplasmic tails enable an RGD-independent fibrillogenesis by the integrin
4 extracellular domain, whereas the
4wt,
4av and
4
cytoplasmic tail truncation mutants are unable to mediate fibril assembly.
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Discussion |
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|
Integrin cytoplasmic domains and mesodermal cell
migration
Integrin 4 chimeras with a full-length cytoplasmic domain are each
able to support the migration of mesoderm cells and tissue explants. This
suggests that a subunit tails play a general positive role in promoting cell
migration. Quantitative analysis of mesoderm cell and tissue explant migration
reveals that
4
5 has a slightly reduced migration ability
compared with
4 chimeras containing the
6,
4 and av
cytoplasmic domains. Cell migration is a multistep dynamic process involving
cell attachment and subsequent detachment from the substrate
(Lauffenburger and Horwitz,
1996
). Migration speed is directly related to receptor and ligand
concentrations, as well as the affinity of the receptor for its ligand
(Palecek et al., 1997
).
Maximal migration speed is achieved at an intermediate force:adhesiveness
ratio (DiMilla et al., 1993
;
Palecek et al., 1997
). In our
experiments, the substrate concentration and the amount of receptor expressed
on the cell surface were normalized. Thus, it is possible that the
5
tail confers the
4 extracellular domain with an activation state not
optimized for rapid cell migration. High-affinity ligand binding may delay
cell detachment from the substrate, hence resulting in a reduction in the
migration speed. The
5 cytoplasmic domain is reported to confer
integrin receptors with `high-affinity' binding in a variety of contexts
(O'Toole et al., 1994
;
Wu et al., 1995b
). Moreover,
an
2 chimera with an
5 tail is reported to support strong
collagen gel contraction, but not enhanced migration
(Chan et al., 1992
).
Cytoplasmic domains have different abilities to support FN
fibril assembly
In this study, we show that 4 chimeras with
5,
6 or
3 cytoplasmic tails, but not
4 or
v tails, can mediate
RGD-independent FN fibril assembly in vivo during Xenopus
gastrulation. Fibrillogenesis is important to many biological processes
including development, wound healing and tumorigenesis; thus, mechanisms of
matrix assembly are of considerable interest. Integrin activation and
cytoskeletal interaction have been shown to be essential for FN matrix
assembly (Wu et al., 1995b
).
Integrin
4 is reported to exist in multiple activation states
(Masumoto and Hemler, 1993
).
In the case of FN fibrillogenesis, it cannot initiate matrix assembly unless
exogenous stimuli such as Mn2+ or `activating' antibodies are
present (Sechler et al., 2001
;
Wu et al., 1995a
). In our
experiments, it is highly likely that the
5,
6 and
3
tails are able to confer the
4 extracellular domain with the
appropriate conformation needed to promote FN matrix assembly on the BCR,
whereas the
4 and
v tails can not. In support of this
hypothesis, O'Toole et al. (O'Toole et
al., 1994
) have shown that the
5,
6A,
6B and
2 tails are all able to confer an energy-dependent high-affinity state
to the
IIb extracellular domain in CHO cells, whereas
IIb,
v, aL and aM tails lack such activities. Thus,
cytoplasmic
domain-specific inside-out signaling is probably essential for FN matrix
assembly on the BCR during Xenopus gastrulation. This may help to
explain why the matrix is preferentially deposited on the BCR and not the
blastocoel floor (although cells in both regions express
5ß1).
Cytoskeletal tension is also crucial for FN fibrillogenesis. Integrin
tails can directly interact with some cytoplasmic scaffolding
proteins. For example, the
4 tail associates with paxillin
(Liu et al., 1999
), and the
5 tail has been show to bind Nischarin, a novel protein that can affect
Rho GTPase function (Alahari et al.,
2000
). These
tail-specific interactions probably result in
differential composition and/or dynamics of adhesive contacts organized by
these integrins. Ultimately, this may affect the organization of the
cytoskeleton. It has been proposed that the
5 tail preferentially
regulates prolonged force, which can help to `pull open' the coiled FN dimer
and expose cryptic sites that promote FN self-assembly
(Chan et al., 1992
;
Schwarzbauer and Sechler,
1999
). Thus, it is possible that, aside from defining integrin
activation states,
cytoplasmic tails can also differentially modulate
cytoskeletal interactions that are crucial for the preferential assembly of FN
matrix.
Other possible roles of integrin cytoplasmic domains
Aside from their signaling functions, integrin a subunit cytoplasmic
domains may also play a role in controlling integrin trafficking at the cell
surface. When the 2 cytoplasmic tail is joined to the
4
extracellular and transmembrane domains, most of the protein synthesized is
retained intracellularly, even when high concentrations of
4
2
mRNA are injected. Biochemical analyses suggest that the
4
2
chimeric protein is translated in proportion to the amount of RNA injected
(Fig. 1C) but is not
efficiently mobilized to the cell surface
(Fig. 1B and data not shown).
The
4 chimeras with other
cytoplasmic tails are expressed on
the cell surface approximately as efficiently as the two cytoplasmic tail
truncation mutants. Thus, the sequences after the GFFKR motif within the
2 cytoplasmic domain probably account for the low cell-surface
expression of the
4
2 chimera. During Xenopus
development,
2 mRNA is detected at late gastrula/neurula stages
(Whittaker and DeSimone, 1993
)
but the
2 protein is not expressed at the cell surface until much later
in development (Meng et al.,
1997
). Our findings suggest the existence of a trafficking
checkpoint in embryonic cells, which may limit the transit of some integrins
to the cell surface. Such checkpoint machinery may recognize information
within specific
cytoplasmic tail sequences.
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Conclusions |
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Acknowledgments |
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References |
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