* Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School,
Boston, Massachusetts 02114; Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4;
and § Department of Biology, University of Utah, Salt Lake City, Utah 84112
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Abstract |
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We describe here the molecular and functional characterization of the Caenorhabditis elegans
unc-97 gene, whose gene product constitutes a novel
component of muscular adherens junctions. UNC-97
and homologues from several other species define the
PINCH family, a family of LIM proteins whose modular composition of five LIM domains implicates them as
potential adapter molecules. unc-97 expression is restricted to tissue types that attach to the hypodermis,
specifically body wall muscles, vulval muscles, and
mechanosensory neurons. In body wall muscles, the
UNC-97 protein colocalizes with the -integrin PAT-3
to the focal adhesion-like attachment sites of muscles.
Partial and complete loss-of-function studies demonstrate that UNC-97 affects the structural integrity of the
integrin containing muscle adherens junctions and contributes to the mechanosensory functions of touch neurons. The expression of a Drosophila homologue of
unc-97 in two integrin containing cell types, muscles,
and muscle-attached epidermal cells, suggests that unc-97 function in adherens junction assembly and stability has been conserved across phylogeny. In addition to its
localization to adherens junctions UNC-97 can also be
detected in the nucleus, suggesting multiple functions
for this LIM domain protein.
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Introduction |
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THE nematode Caenorhabditis elegans has proven to
be a useful system to study the development of muscle (Moerman and Fire, 1997; Waterston, 1988
).
Myoblasts arise after the end of gastrulation (at ~290 min
of embryonic development) and are defined by the accumulation of structural components such as myosin, vinculin, and integrin (Epstein et al., 1993
; Coutu-Hresko et al.,
1994
). Myoblasts then migrate to their final positions, polarize, and flatten. In mid-embryogenesis (450 min), the
muscle components organize into sarcomeres and attachment structures (Coutu-Hresko et al., 1994
; see Fig. 1).
These attachment structures, also termed dense bodies,
are adherens junctions that provide a physical linkage between muscles and the hypodermis and are of critical importance to translate the mechanical movement of myofibrillar components to motion of the whole animal. These
adherens junctions contain integrin heterodimers as central structural units that anchor cytoskeletal components
to the extracellular matrix (Gettner et al., 1995
). They also
contain cytoskeletal adapter proteins such as vinculin,
-actinin, and talin (see Fig. 1) (Francis and Waterston, 1985
; Barstead and Waterston, 1991
; Moulder et al., 1996
);
thus, dense bodies are reminiscent of the structural composition of focal adhesions in tissue culture cells (Clark
and Brugge, 1995
). The regulatory steps that coordinate
the assembly of adherens junction components into functional attachment structures that are capable of enduring
and transmitting mechanical stress are largely undefined.
We describe here a new LIM domain protein, UNC-97,
that is required for assembly and stability of focal adhesion-like muscle attachment structures in C. elegans.
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LIM domains are defined by the presence of two zinc
coordinating, Cys/His-containing motifs and represent protein-protein interaction domains (Schmeichel and Beckerle, 1994; Feuerstein et al., 1994
; Dawid et al., 1998
).
Some LIM proteins (LIM-only) are comprised almost exclusively of LIM domains whereas other LIM proteins
(LIM-plus) display LIM domains linked to other functional domains including homeodomains, kinase domains
or other protein-protein interaction domains (Dawid et al.,
1998
). LIM domain proteins display considerable specificity in their sites of subcellular localization. LIM homeodomain proteins and the LIM-only proteins LMO1, -2, and -3 are transcriptional regulatory proteins that function
in the nucleus (Dawid et al., 1998
). On the other hand, the
LIM-only proteins zyxin and paxillin localize to actin
stress fibers and focal adhesions, CRP family members localize to actin filaments and to the Z lines of myofibers in
vertebrates (Turner et al., 1990
; Crawford and Beckerle,
1991; Arber et al., 1997
; Louis et al., 1997
) and the Drosophila CRP family members, MLP60A and MLP84B, distribute along muscle fibers and to sites of muscle attachment (Stronach et al., 1996
). Several of these cytoskeletal LIM proteins have been shown to display a remarkable
dual subcellular localization; apart from its cytoskeletal localization, CRP3/MLP protein has also been reported to
localize to the nucleus (Arber and Caroni, 1996
), where it
may participate in MyoD dependent transcriptional regulation (Kong et al., 1997
). Moreover, although the LIM
protein zyxin appears to be restricted to focal adhesions at
steady-state levels, recent experiments have shown that it shuttles between focal adhesions and the nucleus (Nix and
Beckerle, 1997
).
The functional role of LIM proteins has mostly been addressed for those LIM proteins involved in transcriptional
control. Mutations in LIM homeobox genes revealed their
participation in lineage determination and neural differentiation in vertebrates and invertebrates (Dawid et al., 1998).
Disruption of the LMO transcriptional regulator LMO1
causes haematopoietic lineage defects (Warren et al., 1994
).
In spite of their well-characterized biochemical properties,
such as binding partners and subcellular localization, CRP3/MLP is the only cytoskeletal LIM protein for which
a loss-of-function phenotypes has been reported so far;
analysis of knockout mice revealed a requirement for
CRP3/MLP in the organization of myofibrillar structures
in cardiomyocytes (Arber et al., 1997
). No physiological role can as yet be ascribed to other LIM-only protein families, such as the SLIM, zyxin, or paxillin families.
The C. elegans UNC-97 protein that we describe here
defines a novel family of LIM-only proteins whose members have been conserved throughout evolution. We find
UNC-97 to be expressed in a tissue restricted manner. It is
present in touch neurons, sex muscles, and body wall muscles and colocalizes in muscle with -integrin to sites of attachment of muscle to the underlying hypodermis. We
show that disruption of UNC-97 function leads to the disruption of these focal adhesion-like structures as well as to
functional defects of the mechanosensory neurons. The
developmental expression profile of UNC-97, as well as its
embryonic lethal null phenotype, is consistent with UNC-97 playing a role late in embryonic development in the
process of terminal differentiation of muscles and mechanosensory neurons.
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Materials and Methods |
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Strains and Genetics
The strains used in this study are: HE110 [unc-97(su110)], mec-3(u298), mec-3(e1338), dpy-20(e1282), and N2 wild-type strain. Double mutant mec-3(u298); unc-97(su110) animals were constructed by first marking unc-97(su110)X with dpy-20(e1282)IV and then repelling dpy-20(e1282) with the closely linked mec-3(u298).
Polarized Light Microscopy
Polarized light images were obtained essentially as described in Waterston
et al. (1980). In brief, single live adult hermaphrodites were placed in a
drop of M9 buffer on a slide and then a coverslip was carefully lowered
onto the animals. Excess liquid was wicked away until the animals were
immobilized and then animals were rolled gently until a good view of
body wall muscle was obtained. Animals were viewed using a Zeiss Axiophot microscope (Carl Zeiss) equipped with polarizing optics.
Sequence Analysis
The unc-97(su110) molecular lesion was identified by PCR amplification of the F14D12.2 gene from unc-97(su110) mutant animals and subsequent DNA sequencing. BLAST searches identified unc-97 cDNAs in Y. Kohara's (Mishima, Japan) cDNA collection (yk460d6, yk457c11, yk455g5, yk457c10, yk403d5, yk267h9, yk114h11, yk184e5). yk184e5 was completely sequenced and found to encode a full-length clone. The nucleotide and protein coding sequence have been submitted to GenBank/EMBL/ DDBJ under accession number AF035583. UNC-97 homologous proteins were identified using the BLAST search algorithm. The cDNA representing the Drosophila expressed sequence tag (EST) sequence was obtained from Genome Systems. The complete nucleotide and protein coding sequence for d-pinch have been submitted to GenBank/EMBL/ DDBJ under accession number AF078907. Sequence alignments and dendograms were constructed using the pileup algorithm in the GCG software package.
Fusion Gene Constructs
All three reporter gene constructs were constructed using a PCR fusion
approach. First, two independent PCRs were performed, one amplifying
the promoter and coding region (in the case of unc-97-prom::GFP just the
promoter) from genomic DNA. The 3' primer contained sequences overlapping with part of the green fluorescent protein (GFP)1 sequences on
pPD95.75; the other PCR amplified GFP and the unc-54-3'-UTR from the
pPD95.75 vector. The 5' primer contained sequences overlapping with the
genomic region targeted for fusion. The two PCR products were then
fused by combining 1 µl of each PCR reaction and subsequent amplification with a set of nested primers, one at the beginning of the promoter, the
other at the end of the unc-54-3'-UTR. For all the PCR reactions a mixture of Taq-polymerase and proofreading PwoI polymerase (Boehringer
Mannheim, Indianapolis, IN) was used to ensure fidelity of the reaction.
The fusion PCR product was injected at ~100 ng/µl into wild-type worms.
The promoter sequence used for both unc-97 reporter constructs starts at
2168 5' of the predicted ATG start codon (reaching almost to the preceding predicted gene). unc-97-prom::GFP is a translational fusion encompassing the first 21 amino acids of UNC-97. unc-97::GFP is a translational fusion encompassing the complete genomic coding sequence of
unc-97 (see Fig. 4). Note that both reporter gene constructs reveal a similar set of expressing cells, arguing that none of the introns contain regulatory sites. The promoter sequence used for pin-2::GFP starts at
2038 5'
of the predicted ATG start codon (reaching almost to the preceding predicted gene) and contains its complete, predicted genomic coding sequence.
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Transgenic Animals and Rescue Analysis
Transgenic strains were constructed by injecting GFP reporter gene constructs as PCR products into N2 wild-type animals. No coinjection marker was used; transgenic animals were identified by their green fluorescence. The extrachromosomal GFP reporter gene arrays were integrated using a Stratalinker 1800 UV light source (Stratagene) at 300 J/m2. The transgenic strains used are: N2 Ex[unc-97::GFP], N2 Ex[unc-97-prom::GFP]; mgIs25 (integrated Ex[unc-97::GFP]), mgIs26 (integrated Ex[unc-97-prom:: GFP]); N2 Ex[pin-2::GFP, rol-6]. Rescue of unc-97(su110) was performed by making use of (a) the X-linkage of unc-97 and (b) an autosomally integrated, full-length unc-97::GFP copy: mgIs25 heterozygous males were mated with unc-97(su110) hermaphrodites. There are two types of F1 male cross progeny from this cross. One type contains a chromosome from the father that has no integrated unc-97::GFP. This type of F1 male cross progeny is not fluorescent and display the uncoordinated (unc) phenotype, since it is hemizygous for unc-97(su110). The other type received a chromosome from the father that does contain an integrated unc-97:: GFP (identified as fluorescent animals). If unc-97::GFP rescues, these fluorescent males should be non-unc. Indeed we found male animals of both phenotypes: nonfluorescent unc as well as fluorescent non-unc, thus demonstrating rescue specific for the animals that carry the unc97::GFP containing array.
Antibody Staining and GFP Expression Analysis
Fixation of animals for antibody staining was performed as described by
Finney and Ruvkun (1990). In brief, animals were freeze-cracked by two
rounds of freezing and thawing in dry-ice/ethanol and fixed in 3%
paraformaldehyde, 80 mM KCl, 20 mM NaCl, 10 mM EGTA, 5 mM spermidine, 15 mM Pipes, pH 7.4, 25% methanol for 3 h at 4°C. Animals were
washed twice in Tris/Triton buffer (100 mM Tris/HCl, pH 7.4; 1% Triton
X-100, 1 mM EDTA), antigens reduced in Tris/Triton buffer + 1%
-mercaptoethanol for 1 h at 37°C. After washing once in borate buffer (33.3 mM boric acid, 16.6 mM NaOH, pH 9.5), antigens were oxidized in borate
buffer + 0.3% hydrogen peroxide for 15 min at room temperature. After
washing once in borate buffer, samples were blocked in antibody buffer
(1× PBS, pH 7.4, 1% BSA [fraction V, 96-99%; Sigma, St. Louis, MO],
0.5% Triton X-100, 0.05% sodium azide, 1 mM EDTA) for at least 15 min. Antibodies used were: monoclonal mouse anti-pat-3 antibody MH25
(Francis and Waterston, 1985
), monoclonal mouse anti-vinculin antibody MH24 (Francis and Waterston, 1985
), and mouse polyclonal anti-unc-52 antibody GM1 (Moerman et al., 1996
). MH24 and MH25 were used at 1:250, GM1 at 1:50, and incubated overnight at 4°C. Secondary antibodies
(FITC- or TRITC-coupled) were incubated for 6-12 hours at 4°C. DNA
was stained using 4',6-diamidino-2-phenylindole (DAPI) at 10 µg/ml.
The expression of the unc-97::GFP reporter gene was either monitored in live animals or by fixing animals as described below for antibody staining. The fixation procedure used does not interfere with the fluorescent signal of GFP. We found that in several instances subcellular structures (such as the nuclear staining) could be better observed in fixed animals, although they were also clearly visible in the live animals, thus excluding fixation artifacts.
RNA Interference
The probable null phenotype for the unc-97 gene was generated using RNA
interference as described by Fire et al. (1998). The template for the sense
and antisense unc-97 RNA transcripts was the complete unc-97 cDNA,
subcloned into the Bluescript vector, which was amplified using Bluescript
specific primers flanking the T3 and T7 primer sites. Sense and antisense
RNA was produced from this PCR product in separate transcription reactions using T3 and T7 primers and commercially available RNA synthesis
kits. The RNA was resuspended in water, the antisense and sense templates were mixed to a final concentration of ~1-5 µg/µl and injected into
the gonad. Scoring of the phenotype was done by allowing single injected
animals to lay eggs at 20°C from hour 24 to hour 48 postinjection (see figure). The progeny derived from the hour 0 to hour 24 postinjection egg lay
(which usually has not received as much RNA as the later progeny) also
showed lethality; however, we occasionally observed the presence of entirely immobile and uncoordinated animals that grow to adulthood. The
control RNA was derived from the COOH-terminal half of the cDNA of
a predicted, nonessential homeobox gene on the CO4F1 cosmid.
Mechanosensory Assays
For each genotype 30-100 individual animals were scored by touching the animals with a thin hair 10 times, five times at the anterior and five times at the posterior. Normally, animals respond by backing up away from the touch. The responsiveness of every single animal was recorded so that, for example, six responses out of 10 touches were recorded as 60% responsiveness. Note that although unc-97(su110) display an uncoordinated phenotype associated with an almost immobile appearance, those mutant animals are still capable of responding to touch by backing up.
Whole Mount In Situ Hybridization of Drosophila Embryos
w1118 embryos were collected over a 17-h period and then prepared for
whole mount in situ hybridization as described in Lehmann and Tautz
(1993). The hybridization reaction proceeded for 12 h at 65°C in the following buffer, pH 4.5, 50% formamide (UltraPure; GIBCO BRL, Gaithersburg, MD), 5× SSC, 100 µg/ml tRNA, 100 ug/ml sonicated salmon-sperm DNA (denatured), 50 µg/ml heparin (Sigma), and 0.1% Tween-20,
plus the appropriate riboprobe at a concentration of 0.25 ng/ml. After hybridization, samples were washed several times over a 2-h period at 65°C.
For detection of probe, samples were incubated with a 1:5,000 dilution
of anti-digoxigenin Fab fragments (Boehringer Mannheim), presorbed
against fixed Drosophila embryos, either overnight at 4°C or 2 h at room
temperature, and then processed according to the manufacturer's instructions.
Full-length digoxigenin-labeled RNA probes, corresponding to the sense and antisense strands of the d-pinch cDNA, were prepared using the Boehringer Mannheim Genius kit according to the manufacturer's instructions. The full-length probes were hydrolyzed to a size of ~100 bp by treatment with carbonate buffer (60 mM Na2CO3, 40 mM NaHCO3, pH 10.2) at 60°C for 1 h.
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Results |
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unc-97 Is Required for the Maintenance of C. elegans Adherens Junctions
The X-linked unc-97 gene was identified by Zengel and
Epstein (1980) and is defined by a single recessive allele,
su110. Adult animals homozygous for the su110 mutation
are limp, egg laying-defective, and show movement defects that vary from slow moving to paralyzed animals.
This probably reflects the degree of disorganization of the
vulval and body wall muscles. Zengel and Epstein (1980)
reported that unc-97 mutants have shallow, easily disrupted muscle sarcomeres. Using polarized light microscopy we also find that mutant muscle cells are fragile, but
if animals are handled carefully intact sarcomeres can be
observed (Fig. 2 A). As can be seen in Fig. 2 A (top left
panel), when handled gently, unc-97(su110) muscle largely
resembles wild-type muscle. Sarcomere dimensions and
overall organization are similar between unc-97(su110)
and wild-type muscle, but the dense bodies in su110 animals are never as clearly defined as in wild-type animals.
Fig. 2 A (bottom four panels) illustrates the range of defects than can be observed within individual muscle cells
of su110 animals. These panels are all of cells where the
sarcomeres had already become disorganized. The combination of coverslip pressure and slight rolling of an unc-97(su110) animal will also lead to the collapse of the sarcomere structure. Under similar conditions sarcomeres
within wild-type muscle cells do not collapse. Anchorage
of the sarcomere complex to the plasma membrane is
therefore weak and easily disrupted in unc-97(su110) animals.
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The lability of the sarcomeres as observed by polarized
light microscopy prompted us to analyze the structural integrity of the muscle attachment structures in more detail.
To this end, we determined the subcellular localization of
two defined components of these muscle-hypodermal adherens junctions. Immunhistochemistry with a -integrin/
PAT-3 monoclonal antibody (Gettner et al., 1995
) revealed that the dense bodies do not for the most part show
up as rows of discrete spots in unc-97 mutants, but instead appear primarily as diffuse stripes running parallel to the
M line (Fig. 2 B). Similarly, vinculin, which normally localizes exclusively to dense bodies (Barstead and Waterston,
1991
), shows a diffuse stripe-like appearance in unc-97
mutants (Fig. 2 B); the diffuse stripes do not always run in
parallel, but occasionally fuse to one another. Due to the
presence of M lines in the anti-PAT-3-stained animals,
the fusion of these stripes can more easily be observed using anti-vinculin stained antibodies. These observations
are consistent with UNC-97 playing a role in determining
the structural integrity of adherens junctions, perhaps
through clustering integrin or other dense body-associated proteins.
The C. elegans proteoglycan perlecan, encoded by the
unc-52 gene, localizes to the basement membrane underlying the muscle (Francis and Waterston, 1991; Rogalski et al.,
1993
). Staining with anti-UNC-52 antibodies reveals staining at periodicities corresponding to the sites of the dense
bodies and M-line structures (Fig. 2 B) (Francis and Waterston, 1991
). In unc-97(su110) mutant animals, the periodicity of the staining is abrogated; instead, UNC-52 appears more diffuse (Fig. 2 B). The effect of the intracellular UNC-97 protein on the localization of the extracellular
UNC-52 protein demonstrates that the correct localization
of extracellular matrix components depends on the structural integrity of the muscular integrin complexes.
unc-97 Defines a New Family of LIM Domain Proteins
unc-97(su110) was mapped to linkage group X in the lon-2
dpy-7 interval by Zengel and Epstein (1980). The cosmid
F14D12 maps to this interval and contains a predicted LIM
domain protein (F14D12.2; Fig. 3, A and B). Sequencing
of this predicted gene from the mutant strain revealed that
unc-97(su110) harbors a G
A splice site mutation at the
last intron-exon boundary, which is predicted to disrupt
the structural integrity of the last LIM domain of the
UNC-97 protein (Fig. 3 A). A genomic PCR fragment
containing the predicted unc-97 gene rescues the uncoordinated phenotype of unc-97(su110) (Materials and Methods). Taken together, these results demonstrate that the
unc-97 gene is encoded by the predicted LIM domain containing protein F14D12.2.
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Eight independent unc-97 cDNA clones were present in
Y. Kohara's EST collection library. We completely sequenced the largest cDNA clone and found it to represent
a likely full-length clone containing a poly-A tail and an
in-frame stop codon 5' of the putative ATG start codon.
The open reading frame (Fig. 3 C) was similar to the predicted gene structure; no evidence for alternative splice
forms was detected in the other 7 cDNA clones. The
UNC-97 protein encoded by the open reading frame
(GenBank/EMBL/DDBJ accession number AF035583)
consists entirely of 5 LIM domains (Fig. 3, B and C). Database searches revealed the presence of highly related proteins from other species, including the human PINCH
protein (Rearden, 1994), another predicted C. elegans protein, which we termed pin-2 (for PINCH-related gene 2)
and several ESTs from mice, humans, and Drosophila (Fig.
4 D). To address cross-species conservation in more detail,
we obtained the cDNA corresponding to the Drosophila
EST, and after fully sequencing the transcript, found it to
contain the entire open reading frame (GenBank/EMBL/ DDBJ accession number AF078907). Like the other family members, Drosophila PINCH is a LIM-only protein
comprised of five LIM domains and contains a significant
sequence identity (on the order of 60%) to UNC-97 in each
of its LIM modules (Fig. 3 C).
We propose to term this new family of LIM proteins
the PINCH family after its founding member, PINCH
(Rearden, 1994; renamed to h-PINCH-1 in Fig. 3). The
PINCH family can be clearly distinguished from other
multiple LIM domain-containing subfamilies (Fig. 3 D)
and is defined by the following features: (a) all sequenced members consist exclusively of five LIM domains; (b) the
spacing between the Zn-coordinating amino acids, a variable parameter in different LIM domain proteins, is with
two exceptions invariant in the PINCH family; and (c) the
Zn-coordinating amino acids in each LIM domain are with
two exceptions invariant. Most notably, the first Zn finger
of the last LIM domain of all PINCH family members is
characterized by the highly unusual replacement of a His
in the Cys-Cys-His-Cys LIM domain consensus motif by a
Cys residue.
Expression of C. elegans unc-97 and Its Drosophila Homologue d-pinch in Muscle
To monitor the expression pattern of UNC-97, we fused
the transcriptional regulatory regions of the unc-97 gene to
GFP and analyzed its expression in transgenic C. elegans.
A translational fusion protein, encoding all exons and introns of unc-97 reveals a similar set of expressing cells (see
below). In both larvae and adult animals, unc-97 is expressed in two different cell types, muscles and neurons
(Fig. 4). Strong expression is detectable in body wall muscle cells and vulval muscle cells, whereas no expression can
be detected in pharyngeal muscles, intestinal muscle, or
the anal depressor muscle. Weak expression can be observed in the anal sphincter muscle (data not shown). In
the nervous system, unc-97 is expressed in the six mechanosensory receptor neurons ALML/R, PLML/R, AVM,
and PVM (Fig. 4), which are responsible for sensing light
touch (Chalfie et al., 1985). Intriguingly, a common theme
of all the unc-97-expressing cell types is their attachment via
the extracellular matrix to the hypodermis. We next examined unc-97 expression at earlier developmental stages to
determine its onset of expression in development. We
found that in embryos, unc-97 expression can first be observed at mid-embryogenesis at ~300 min of development
(Fig. 4).
In an effort to deduce some conserved function among PINCH family members, we characterized the expression pattern of the Drosophila homologue of UNC-97, d-pinch. We generated a developmental profile and expression pattern for d-pinch by whole mount RNA in situ hybridization on 0-17-h embryos (Fig. 5). D-pinch transcripts were first detected in stage 10 embryos, where it is expressed in the visceral and body wall muscle. By stage 13, the expression has increased, and some pharyngeal muscle staining is also detected. Of particular interest is the intense expression of the transcripts at the sites of gut constriction. In late-stage 16 embryos, when the myotendinous junction is just beginning to form, we detect d-pinch transcripts in the epidermal tendon cells, as well as the aforementioned muscle lineages (Fig. 5). At this time of development, the heart musculature has differentiated as well; however, no d-pinch expression is seen in this muscle lineage.
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Like unc-97-expressing cells, d-pinch-expressing cells
are attached to extracellular matrix components via integrin receptors. The timing of d-pinch expression in these
cells is consistent with the expression of integrin subunits
as well. A very striking example of coexpression is the
temporal pattern of d-pinch and the integrin subunit PS1
in tendon cells. By late stage 16, high levels of
PS1 are detected in the tendon cells with no other epidermal expression of the transcript (Wehrli et al., 1993
); it is at this embryonic stage that we first detect d-pinch transcript in
tendon cells.
Adherens Junction and Nuclear Localization of UNC-97 in Muscle
The complete coding sequence of unc-97 was fused to
GFP to reveal the subcellular localization of UNC-97. The
unc-97::GFP fusion gene rescues the unc-97(su110) mutant phenotype (Materials and Methods) and is thus likely
to reveal a functionally relevant subcellular localization. In
adult body wall muscles UNC-97 is located in discrete
spots and lines along the body wall muscle (Fig. 6). Double
labeling with an antibody directed against the C. elegans
-integrin PAT-3 (Gettner et al., 1995
) revealed a colocalization of UNC-97 with PAT-3 at dense bodies and along the M lines (Fig. 6, see also Fig. 1). Costaining with anti-vinculin antibodies, which also stain dense bodies, confirms that UNC-97 is part of the integrin complex at muscle-hypodermal attachment sites (data not shown). Taken
together with the requirement of unc-97 for the integrity
of dense bodies shown in Fig. 2, the localization of UNC-97 to dense bodies suggests that UNC-97 acts at the dense
bodies to ensure the integrity of this type of adherens junction.
In the vulval muscles UNC-97 also localizes at sites of muscle attachment to the hypodermis (Fig. 6). 56% (n = 32) of the unc-97(su110) animals display a protruding vulva (p-vul) phenotype (Fig. 7), which could be ascribed to defects of these attachment sites in the absence of full unc-97 function. Structural defects of the vulval muscle attachments could explain the egg laying-defective phenotype of unc-97(su110), which is manifested by an accumulation of eggs in the gonad (data not shown).
Surprisingly, we also found UNC-97 to localize to the nuclei of muscle cells (Fig. 6). UNC-97 appears in discrete dots in muscle nuclei. We have not analyzed these dots in detail, however, their subnuclear localization appears to be somewhat variable. In many cases, these spots do not appear to be confined to submembraneous structures of the nuclear envelope, but appear to localize throughout the nucleoplasm (Fig. 6). In younger animals, however, particularly in embryos, we observe these subnuclear dots to be more concentrated towards the periphery of the nucleus (Fig. 7, A-C).
RNA Interference of unc-97 Causes an Embryonic Arrest Phenotype
Using the full-length UNC-97::GFP fusion protein, we analyzed the developmental progression of UNC-97 expression and localization. In accordance with results from the
promoter fusion (see above), we observe UNC-97 expression from ~300 min of embryonic development onward
(Fig. 7, A-C). This is about the time when myoblasts adopt their final identity, as judged by the onset of expression of structural muscle components, but is before the positioning
of muscles into quadrants (Epstein et al., 1993; Coutu-Hresko et al., 1994
). Upon formation of the muscle quadrant and the onset of muscle elongation, UNC-97 expression becomes stronger and its localization into discrete
nuclear spots becomes visible (350-420 min; comma stage).
This is about the stage when muscle components are first
assembled into sarcomeres. Due to the strong punctate nuclear pattern it is hard to discern whether UNC-97 localizes to the newly formed dense bodies at this early stage.
Discrete punctate UNC-97::GFP localization to dense
bodies, which is clearly discernible from the punctate nuclear localization, can be observed around the time of
hatching. Although most of the embryonic UNC-97::GFP signals can be accounted for by body wall muscle localization, some additional signals appear in the head and tail,
which presumably are touch neurons that form late in embryogenesis (Sulston et al., 1983
).
The early embryonic expression of UNC-97 in muscle
prompted us to study its involvement in embryonic muscle
development. Early defects in muscle development caused
by the loss of the dense body components -integrin/pat-3
or vinculin/deb-1 function have been shown to cause a specific developmental arrest phenotype, termed Pat phenotype (paralyzed, arrested elongation at twofold) (Barstead
and Waterston, 1991
; Williams and Waterston, 1994
). unc-97(su110) mutant animals do not display such a phenotype; however, the molecular nature of the (su110) allele
(Fig. 3) strongly suggests that this mutation does not lead
to a complete loss of unc-97 function. To address this issue,
we thus determined the probable loss-of-function phenotype of unc-97 using RNA interference (RNAi). This technique has been shown to effectively decrease the expression of both maternally and zygotically expressed genes and, in many cases, has been shown to phenocopy the null
phenotype of a gene (Fire et al., 1998
). We found that unc-97 RNAi causes a characteristic developmental arrest phenotype, which shows all the hallmarks of the Pat phenotype (Fig. 7, D and E). Embryos grow to the twofold stage,
but arrest body elongation. The pharynx occasionally develops, but the embryo is paralyzed. Several embryos hatch, but are misshapen and then die (Fig. 7, D and E).
None of these phenotypes could be observed upon injection of control double-stranded (ds) RNA. The Pat phenotype of unc-97 RNAi is similar to the loss-of-function phenotype of
-integrin/pat-3 and vinculin/deb-1, both factors
which colocalize with UNC-97 at adherens junctions.
Taken together, these data indicate that unc-97 is essential
for early stages of muscle development.
UNC-97 expression is maintained in muscles throughout the postembryonic life of the animal (data not shown). Also, its dual subcellular localization in muscle nuclei and muscle attachment sites is not altered in development. These observations suggest that UNC-97 is continuously required to maintain the structural integrity of muscle attachment sites during growth and in the adult. The sarcomere fragility of unc-97(su110) described above clearly supports this notion. Conditional alleles of unc-97 will be required to address this issue in more detail.
unc-97 Functions in Mechanosensation
The subcellular localization of UNC-97::GFP in touch
neurons is more diffuse than in muscles (Fig. 8 A). Although UNC-97 appears to be concentrated in submembraneous regions, it can be found throughout the cytosol
as well as throughout the nucleus (Fig. 8 A). Occasionally
we observe a concentration of UNC-97::GFP in the periphery of the touch neuron nuclei in what appear to be ring like structures (data not shown). The nuclear staining
profile of UNC-97::GFP in neurons thus appears to be distinct from that in muscles, suggesting that UNC-97 associates with cell type-specific and nonubiquitous subnuclear
domains. We investigated whether unc-97 is required for
the function of mechanosensory neurons. In behavioral
touch assays we found that the partial loss-of-function allele unc-97(su110) displays no mechanosensory touch defectiveness on its own (Fig. 8 B). However, we find that
this partial loss of unc-97 function strongly enhances the
mechanosensory defect of a partial loss-of-function mutation in mec-3, a LIM homeobox gene required for mechanosensory function (Way and Chalfie, 1988). Although the
strong mec-3 allele e1338 is completely touch insensitive, mec-3(u298) mutant animals retain some responsiveness
to touch (Fig. 8 B). Examining a unc-97(su110); mec-3(u298) double mutant, we found that this partial responsiveness is almost completely abolished by the partial reduction unc-97 gene activity (Fig. 8 B). This experiment
reveals that unc-97 contributes to the function of mechanosensory neurons. Due to the arrested lethal phenotype of a complete loss of unc-97 function as determined by
RNAi (Fig. 7), we could not address the null phenotype of
unc-97 in touch neurons. Genetic mosaic experiments will
address this issue in the future.
The UNC-97-related LIM protein PIN-2 Is Expressed in Neurons and Intestine
The now completely sequenced genome of C. elegans reveals the presence of a single other PINCH family member in C. elegans, which we termed PIN-2 (Fig. 3). Although it represents the most diverged member of the PINCH family, it clearly shares all the features of this family including the presence of five LIM domains and a characteristic signature of Zn-coordinating residues of the LIM domains (Fig. 4; PIN-2 shows 55.7% amino acid similarity to UNC-97 and 58.5% amino acid similarity to h-PINCH-1). pin-2 maps on LGIV, ~7 kb to the left of the cloned unc-31 gene; no candidate mutations have been mapped to this region.
We were motivated to look for the expression and subcellular localization of PIN-2 to address whether PIN-2 might perform a role in muscle development similar to UNC-97 or whether this diverged PINCH family member might be used in distinct cellular processes. A pin-2::GFP fusion gene construct (Fig. 9), which contains the full coding region of pin-2 and presumably reflects the authentic subcellular localization of PIN-2, is not expressed during early stages of embryogenesis; expression is first detectable in embryos shortly before hatching (data not shown). In early larval stages, PIN-2::GFP is expressed in two major tissue types, neurons and intestinal cells (Fig. 9). It does not reveal any overlap of expression with UNC-97. Notably, however, PIN-2 expression in the intestine might be somewhat related to the expression of Drosophila d-pinch in the visceral mesoderm that envelopes the gut. Neural expression of PIN-2::GFP is restricted to few neurons, arguing for a cell type-specific role of PIN-2. Moreover, whereas PIN-2::GFP expression fades in intestinal cells and is almost undetectable in adults, its expression is maintained in neurons throughout adulthood (data not shown), suggesting a role for PIN-2 in neural maintenance. PIN-2::GFP localizes uniformly throughout the cytoplasm and nucleus. In neurons, it localizes uniformly along axonal processes. PIN-2 appears to label variscosities of the ventral cord axon where it is expressed. The functional significance of this observation is unclear at the moment.
|
In summary, both the tissue type expression and the subcellular distribution of the only two PINCH family members in C. elegans, UNC-97 and PIN-2 is strikingly different, thus arguing for a divergence in function of the genes.
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Discussion |
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PINCH Family Members across Phylogeny
The invertebrate PINCH-like genes described here as well
as several as yet uncharacterized vertebrate PINCH genes
define a new family of LIM proteins that have been highly
conserved across phylogeny. No other LIM domain family
contains the same LIM domain architecture as the PINCH
family. Considering the involvement of LIM domains in
protein-protein interactions (see introduction), we propose that PINCH proteins represent adapter proteins capable of assembling multiprotein complexes. Within C. elegans, we identified two PINCH-like genes, unc-97 and
pin-2. unc-97 is more closely related to the vertebrate
PINCH genes. The function of unc-97 at focal adhesion
sites appears to be conserved as well, since vertebrate
PINCH-1 colocalizes with integrins to sites of cell-matrix contact (Tu et al., 1999).
Additional evidence that PINCH family members may
function at focal adhesion sites comes from the analysis
of d-pinch expression during Drosophila embryogenesis.
D-pinch transcripts are expressed in both the body wall
muscles and epidermal tendon cells, coincident with integrin subunit expression in those tissues (Bogaert et al.,
1987; Leptin et al., 1989
; Wehrli et al., 1993
). The timing and tissue-specific expression of d-pinch suggests that it is involved in the terminal differentiation of muscles and
tendon cells; one common feature of these cell types is
their formation of adherens junctions. Integrin complexes
are crucial for the stability of the junctions, as demonstrated by the dramatic muscle detachment phenotype
seen in myospheroid/
PS-integrin mutants (Wright, 1960
;
Leptin et al., 1989
; Volk et al., 1990
). Based on the observation that the null unc-97 phenotype is very severe and
phenocopies mutations in the pat-3/
-integrin gene, we
predict that loss of d-pinch will also result in a severe disruption of muscle function.
The PIN-2 protein, which displays a high degree of similarity to UNC-97 in architectural composition, as well as
primary sequence, is expressed in entirely different tissue
types and, unlike UNC-97, displays a uniform subcellular
distribution. Nevertheless, it is conceivable that, although
deployed in different tissue types, distinct PINCH gene
paralogues (such as UNC-97 and PIN-2) might fulfill a
similar function. For example, integrin complexes in C. elegans are not only present in muscle cells but also in neurons, where they do not appear to localize in discrete spots
(Baum and Garriga, 1997). It is possible that PIN-2 might
be involved in neural integrin complex assembly and/or
signaling. The expression of PIN-2 in the intestine could
potentially indicate a similarity to the expression of Drosophila d-pinch in the visceral mesoderm that envelopes
the gut.
UNC-97 and Muscle Development
The structural components that anchor myofibers to the
extracellular matrix, such as integrin, vinculin, talin, and
-actinin are remarkably conserved in C. elegans and resemble the protein composition of adherens junctions in
other systems, such as focal adhesions in tissue culture
cells (Waterston et al., 1980
; Francis and Waterston, 1985
;
Waterston, 1988
; Moerman and Fire, 1997
). It is thus likely
that the fundamental mechanisms required to assemble
these structure are conserved as well. Based on our functional analysis of UNC-97 and the presence of highly related homologous molecules in flies and vertebrates, we
suggest that members of the PINCH family of LIM-only
proteins are a critical component of muscle attachment
and adherens junction assemblies across phylogeny. Indeed, the mouse UNC-97 homologue PINCH-1 has recently been shown to colocalize with
1-integrins at focal
adhesions (Tu et al., 1999
). This interaction appears to be
mediated by the ankyrin-repeat containing serine/threonine kinase ILK, which by direct association with both
1-integrins (Hannagan et al., 1996) and the mouse UNC-97 homologue PINCH-1 (Wu, C., personal communication)
appears to serve as a bridging molecule.
Cellular attachment sites contain several LIM domain
proteins, such as zyxin, paxillin, CRP proteins, and others
(Sadler et al., 1992; Turner et al., 1990
; Crawford et al.,
1994
; Arber and Caroni, 1996
). In muscles, the CRP3/
MLP and ALP proteins localize to Z-discs, attachment
points of myofibers in vertebrates (Arber et al., 1997
; Xia
et al., 1997
). Drosophila CRP homologues also localize to
muscle attachment sites (Stronach et al., 1996
). However, the functional requirement for any LIM containing protein at these subcellular sites has until now only been reported for the CRP3/MLP protein, whose loss-of-function
causes disorganization of cardiac myofibers (Arber et al.,
1997
). We demonstrated here a similar requirement for
the LIM-only protein UNC-97 in body wall muscles in C.
elegans. Since the C. elegans genome contains a gene
highly related to the CRP proteins (T04C9.4; data not
shown), we speculate that other LIM proteins besides
UNC-97 will also be involved in adherens junction assembly.
The su110 allele indicates that UNC-97 has an important role in maintaining sarcomere organization in growing and adult animals. Moreover, the RNAi (probable loss-of-function) experiment indicates an important regulatory role for UNC-97 during embryonic muscle development. However, it is unclear so far as to whether the embryonic defects caused by unc-97 RNA interference reflect a requirement for UNC-97 in the initial stages of assembly of the adherens junctions or reflects a requirement for UNC-97 in stabilizing adherens junctions after they are formed to resist the mechanical stress they are exposed to upon elongation of the animal. Since UNC-97 expression clearly coincides with the onset of expression of adherens junction components and their assembly into these structures, we favor a model in which UNC-97 is involved in the initial assembly of the adherens junction components and keeps residing in these structures to ensure their stability. We will address these issues in more detail in the future by isolating deletion alleles of unc-97, which will provide a better source for a detailed characterization of the embryonic phenotype than the dsRNA injected animals described here.
Although it is unclear how UNC-97 cooperates with
-integrins and other focal adhesion components such as
vinculin to determine adherens junction integrity, the
modular organization of UNC-97 into five LIM domains
implicates UNC-97 in binding to multiple proteins. UNC-97 might be a scaffold or adapter protein onto which various different components assemble to form a functional
muscle attachment site. Since defective myofibrils cause as
a secondary consequence the destabilization of muscle attachment sites (Epstein, 1986
), it is for example possible
that UNC-97 serves as an anchor between myofibrils and
membrane-anchored integrin components. In this model,
dense body defects in unc-97(su110) mutant animals would
not arise from direct defects in the dense body structure per se, but would represent a secondary consequence of
the myofibril disorganization.
A Nuclear Function for UNC-97?
Our analysis of UNC-97 localization in a living, nonfixed
animal, using a rescuing, i.e., functionally intact unc-97::
GFP reporter gene, lends support for an authentic in vivo
dual subcellular localization of this LIM protein. However, at this point it is unclear whether the nuclear localization of UNC-97 is indeed functionally significant. In
contrast to the localization of UNC-97 to adherens junctions, which correspond to the site of action of UNC-97 as inferred from the unc-97 mutant phenotype, no such clear
correlation exists to a possible nuclear function of UNC-97. However, our demonstration of a genetic interaction of
unc-97 with the LIM homeodomain transcription factor
mec-3 could be explained on the basis of a physical interaction between these proteins and could thus reflect a
functional requirement for UNC-97 in the nucleus. LIM-
LIM interaction have been previously described (Sadler et al., 1992) and the direct interaction of UNC-97 with MEC-3
could affect the transcriptional activity of the MEC-3 transcription factor. Alternatively, it is also entirely possible
that the genetic interaction of unc-97 and mec-3 reflects an
independent requirement for these genes in mechanosensory processes.
Although further experiments will need to address the
physiological significance of UNC-97 in the nucleus, there
are several attractive hypotheses regarding a potential nuclear function of UNC-97. As mentioned above, UNC-97
could be directly involved in gene regulatory events by interacting with specific transcription factors. The vertebrate
LIM-only proteins CRP3/MLP and SLIM1/KyoT have
been directly implicated in gene regulation via interaction with the transcription factors MyoD and RBP-J, respectively (Kong et al., 1997; Tanigushi et al., 1998), whereas
the LIM-only protein LMO2 assembles higher order transcriptional activation complexes by bridging other transcription factors that are directly involved in DNA binding
(Wadman et al., 1997
). Alternatively, but not necessarily mutually exclusive, UNC-97 could represent a structural
component of specific subnuclear domains. The localization of UNC-97 to discrete dots in muscle nuclei supports
this hypothesis, although the nature of these dots is entirely unclear. Subnuclear domains of different types, such
as speckles, coiled bodies, gems, and Kr bodies/nuclear domains have been described in various systems. Factors localizing to these domains are involved in distinct nuclear processes such transcriptional silencing and RNA processing (for review see Lamond and Earnshaw, 1998
).
Lastly, in regard to the dual subcellular localization of
UNC-97 it is also tempting to speculate that UNC-97 transmits a signal from attachment sites to the nucleus. Although
we have not addressed the question whether UNC-97 dynamically shuttles between attachment sites and the nucleus, focal adhesion-nuclear shuttling has been recently
demonstrated for the LIM protein zyxin (Nix and Beckerle,
1997). Recently, the mouse UNC-97 homologue PINCH-1
was shown to interact specifically with, and might serve as a
substrate of, the integrin-linked kinase ILK (Tu et al., 1999
), a serine-threonine kinase implicated in integrin signal transduction (Hannagan et al., 1996). This observation
might point to a potential role for UNC-97 in integrin-mediated signal transduction.
![]() |
Footnotes |
---|
Address correspondence to O. Hobert, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114. Tel.: (617) 726-5973. Fax: (617) 726-6893. E-mail: hobert{at}molbio.mgh.harvard.edu
Received for publication 25 August 1998 and in revised form 27 October 1998.
We wish to thank Y. Liu for expert technical assistance in DNA and RNA microinjection, Y. Kohara for providing cDNA clones, R. Waterston (Washington University, St. Louis, MO) for his gift of MH24 and MH25 antibodies, H. Epstein (Baylor College of Medicine, Houston, TX) for helpful discussions on the unc-97 phenotype, the Caenorhabditis Genetics Center (funded by the National Institutes of Health Center for Research Resources) for providing strains, B. Reinhart for critical reading of the manuscript, and C. Wu (University of Alabama, Birmingham, AL) for sharing his results on PINCH function in vertebrates.
We acknowledge support from Hoechst AG to G. Ruvkun, the National Institutes of Health to M.C. Beckerle (R01 HL60591), and National Science and Engineering Research Council (963226), and Medical Research Council of Canada (962565) grants to D. Moerman. M.C. Beckerle is a recipient of a faculty research award from the American Cancer Society. O. Hobert was supported by a postdoctoral fellowship from the Human Frontiers in Science Organization.
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Abbreviations used in this paper |
---|
EST, expressed sequence tag; GFP, green fluorescent protein; LIM, LIN-11, ISL-1, MEC-3; MEC, mechanosensory; PAT, paralyzed and arrested at twofold stage; RNAi, RNA interference; UNC, uncoordinated.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Arber, S., and P. Caroni. 1996. Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Dev. 10: 289-300 [Abstract]. |
2. | Arber, S., J.J. Hunter, J. Ross Jr., M. Hongo, G. Sansig, J. Borg, J.C. Perriard, K.R. Chien, and P. Caroni. 1997. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell. 88: 393-403 |
3. | Barstead, R.J., and R.H. Waterston. 1991. Vinculin is essential for muscle function in the nematode. J. Cell Biol. 114: 715-724 [Abstract]. |
4. | Baum, P.D., and G. Garriga. 1997. Neuronal migrations and axon fasciculation are disrupted in ina-1 integrin mutants. Neuron. 19: 51-62 |
5. | Bogaert, T., N. Brown, and M. Wilcox. 1987. The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell. 51: 929-940 |
6. | Chalfie, M., J.E. Sulston, J.G. White, E. Southgate, J.N. Thomson, and S. Brenner. 1985. The neural circuit for touch sensitivity in C. elegans. J. Neurosci. 5: 956-964 [Abstract]. |
7. | Clark, E.A., and J.S. Brugge. 1995. Integrins and signal transduction pathways: the road taken. Science. 268: 233-239 |
8. | Coutu-Hresko, M., B.D. Williams, and R.H. Waterston. 1994. Assembly of body wall muscle and muscle cell attachment structures in C. elegans. J. Cell Biol. 124: 491-506 [Abstract]. |
9. | Crawford, A.W., J.D. Pino, and M.C. Beckerle. 1994. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton. J. Cell Biol. 124: 117-127 [Abstract]. |
10. | Dawid, I.B., J.J. Breen, and R. Toyama. 1998. LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet. 14: 156-162 |
11. | Epstein, H.F. 1986. Different roles of myosin isoforms in filament assembly. In Molecular Biology of Muscle Development. Vol. 29. C. Emerson, D. Fischman, B. Nadal-Ginard, and M.A.Q. Siddiqui, editors. Liss, New York. 653- 666. |
12. | Epstein, H.F., D.L. Casey, and I. Ortiz. 1993. Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multifilament assemblages. J. Cell Biol. 122: 845-858 [Abstract]. |
13. |
Feuerstein, R.,
X. Wang,
D. Song,
N.E. Cooke, and
S.A. Liebhaber.
1994.
The
LIM/double zinc-finger motif functions as a protein dimerization domain.
Proc. Natl. Acad. Sci. USA.
91:
10655-10659
|
14. | Finney, M., and G. Ruvkun. 1990. The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell. 63: 895-905 |
15. | Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391: 806-811 |
16. | Francis, G.R., and R.H. Waterston. 1985. Muscle organization in C. elegans: Localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101: 1532-1549 [Abstract]. |
17. | Francis, G.R., and R.H. Waterston. 1991. Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114: 465-479 [Abstract]. |
18. |
Gettner, S.N.,
C. Kenyon, and
L.F. Reichardt.
1995.
Characterization of ![]() |
19. |
Hannigan, G.E.,
C. Leung-Hagesteijn,
L. Fitz-Gibbon,
M.G. Coppolino,
G. Radeva,
J. Filmus,
J.C. Bell, and
S. Dedhaer.
1996.
Regulation of cell adhesion and anchorage-dependent growth by a new ![]() |
20. | Kong, Y, M.J. Flick, A.J. Kudla, and S.F. Konieczny. 1997. Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol. Cell Biol. 17: 4750-4760 [Abstract]. |
21. |
Lamond, A.I., and
W.C. Earnshaw.
1998.
Structure and function in the nucleus.
Science
280:
547-553
|
22. | Lehmann, R., and D. Tautz. 1993. In situ hybridization to RNA. In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology. L.S.B. Goldstein and E.A. Fyrberg, editors. Academic Press, San Diego, CA. 575-598. |
23. | Leptin, M., T. Bogaert, R. Lehmann, and M. Wilcox. 1989. The function of PS integrins during Drosophila development. Cell. 56: 401-408 |
24. |
Louis, H.A.,
J.D. Pino,
K.L. Schmeichel,
P. Pomies, and
M.C. Beckerle.
1997.
Comparison of three members of the cysteine-rich protein family reveals
functional conservation and divergent patterns of gene expression.
J. Biol.
Chem.
272:
27484-27491
|
25. | Moerman, D.G., and A. Fire. 1997. Muscle: structure, function and development. In C. elegans II. D.L. Riddle, T. Blumenthal, B.J. Meyer, and J.R. Priess, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 417-470. |
26. | Moerman, D.G., H. Hutter, G.P. Mullen, and R. Schnabel. 1996. Cell autonomous expression of perlecan and plasticity of cell shape in embryonic muscle of Caenorhabditis elegans. Dev. Biol. 173: 228-242 |
27. |
Moulder, G.L.,
M.M Huan,
R.H. Waterston, and
R.J. Barstead.
1996.
Talin requires ![]() |
28. |
Nix, D.A., and
M.C. Beckerle.
1997.
Nuclear-cytoplasmic shuttling of the focal
contact protein, zyxin: a potential mechanism for communication between
sites of cell adhesion and the nucleus.
J. Cell Biol.
138:
1139-1147
|
29. | Rearden, A.. 1994. A new LIM protein containing an autoepitope homologous to "senescent cell antigen." Biochem. Biophys. Res. Commun. 1201: 1124-1131 . |
30. | Rogalski, T.M., B.D. Williams, G.P. Mullen, and D.G. Moerman. 1993. Products of the unc-52 gene in Caenorhabditis elegans are homologous to the core protein of the mammalian basement membrane heparan sulfate proteoglycan. Genes Dev. 7: 1471-1484 [Abstract]. |
31. | Sadler, I., A.W. Crawford, J.W. Michelsen, and M.C. Beckerle. 1992. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J. Cell Biol. 119: 1573-1587 [Abstract]. |
32. | Schmeichel, K.L., and M.C. Beckerle. 1994. The LIM domain is a modular protein-binding interface. Cell. 79: 211-219 |
33. | Stronach, B.E., S.E. Siegrist, and M.C. Beckerle. 1996. Two muscle-specific LIM proteins in Drosophila. J. Cell Biol. 134: 1179-1195 [Abstract]. |
34. | Sulston, J.E., E. Schierenber, J.G. White, and J.N. Thomson. 1983. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100: 64-119 |
35. |
Taniguchi, Y.,
T. Furukawa,
T. Tun,
H. Han, and
T. Honjo.
1998.
LIM protein
KyoT2 negatively regulates transcription by association with the RBP-J
DNA-binding protein.
Mol. Cell Biol.
18:
644-654
|
36. | Tu, Y., F. Li, S. Goicoechea, and C. Wu. 1999. The LIM-only protein PINCH directly interacts with the integrin-linked kinase (ILK) and is recruited to integrin-rich sites in spreading cells. Mol. Cell. Biol. In press. |
37. | Turner, C.E., J.R. Glenney Jr., and K. Burridge. 1990. Paxillin: a new vinculin-binding protein present in focal adhesions. J. Cell Biol. 111: 1059-1068 [Abstract]. |
38. | Volk, T., L.I. Fessler, and J.H. Fessler. 1990. A role for integrin in the formation of sarcomeric architecture. Cell. 63: 525-536 |
39. |
Wadman, I.A.,
H. Osada,
G.G. Grütz,
A.D. Agulnick,
H. Westphal,
A. Forster, and
T.H. Rabbitts.
1997.
The LIM-only protein Lmo2 is a bridging molecule
assembling an erythroid DNA-binding complex which includes TAL1, E47,
GATA-1 and Ldb1/NLI proteins.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
3145-3157
|
40. | Warren, A.J., W.H. Colledge, M.B. Carlton, M.J. Evans, A.J. Smith, and T.H. Rabbitts. 1994. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erthroid development. Cell 78: 45-57 |
41. | Waterston, R.H. 1988. Muscles. In The Nematode Caenorhabditis elegans. W.B. Wood, editor. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 281-336. |
42. | Waterston, R.H., J.N. Thomson, and S. Brenner. 1980. Mutants with altered muscle structure in C. elegans. Dev. Biol. 77: 271-302 |
43. | Way, J.C., and M. Chalfie. 1988. mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell. 54: 5-16 |
44. |
Wehrli, M.,
A. DiAntonio,
I.M. Fearnley,
R.J. Smith, and
M. Wilcox.
1993.
Cloning and characterization of ![]() |
45. | White, J. 1988. The anatomy. In The Nematode Caenorhabditis elegans. W.B. Wood, editor. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 81-122. |
46. | Williams, B.D., and R.H. Waterston. 1994. Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J. Cell Biol. 124: 475-490 [Abstract]. |
47. | Wright, T.R.F.. 1960. The phenogenetics of the embryonic mutant, lethal myospheroid in Drosophila melanogaster. J. Exp. Zool. 143: 77-99 . |
48. |
Xia, H.,
S.T. Winokur,
W.L. Kuo,
M.R. Altherr, and
D.S. Bredt.
1997.
Actinin-associated LIM protein: identification of a domain interaction between PDZ
and spectrin-like repeat motifs.
J. Cell Biol.
139:
507-515
|
49. | Zengel, J.M., and H.F. Epstein. 1980. Identification of genetic elements associated with muscle structure in the nematode Caenorhabditis elegans. Cell Motil. 1: 73-97 |