Article |
Address correspondence to Ze'ev Ronai, Ruttenberg Cancer Center, Mount Sinai School of Medicine, Box 1130, New York, NY 10029. Tel.: (212) 659-5571. Fax: (212) 849-2425. email: zeev.ronai{at}mssm.edu; or Limor Broday, email: limor.broday{at}mssm.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: UNC-95; RNF-5; LIM; RING; E3 ligase
Abbreviations used in this paper: dsRNA, double-stranded RNA; Pat, paralyzed arrest at embryonic two-fold stage; RNAi, RNA interference.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The dense bodies and M-lines establish the adhesion sites of the muscle cells to the basal lamina (ECM), and are essential both for the organization of the actinmyosin contractile structure into sarcomeres and the maintenance of the muscles in their correct position. The assembly process of the myofilament lattice during embryogenesis initiates at the basal lamina, proceeds through the cell membrane (sarcolemma) into the cell to form the dense bodies and M-lines, and ends with the formation of thin and thick filaments of the myofilament lattice (Francis and Waterston, 1985, 1991; Hresko et al., 1994; Williams and Waterston, 1994). On the basis of mutant analysis, genetic pathways have been described for dense bodies and M-line formation (Lin et al., 2003). UNC-52/perlecan, a basement membrane heparan sulfate proteoglycan, is initially deposited in the basal lamina (Francis and Waterston, 1991; Rogalski et al., 1993; Williams and Waterston, 1994). Later on, integrin is polarized to the basal sarcolemma to initiate myofilament assembly (Francis and Waterston, 1985, 1991; Hresko et al., 1994). After this step, cytoplasmic dense bodies and M-line proteins such as talin, vinculin, and -actinin are recruited from the cytosol and enter into the nascent attachment sites. Finally, actin- and myosin-containing filaments are recruited into I and A bands, respectively. By hatching, the mature body wall muscle cells are polarized such that all the contractile filaments are located on the basal surface of the cell associated with the hypodermis (Barstead and Waterston, 1991). Proteins that have been shown to be required for the proper assembly of muscle attachments are DEB-1/vinculin, which has a critical role in thin filament organization (Francis and Waterston, 1985; Barstead and Waterston, 1991),
-actinin (Francis and Waterston, 1985), the UNC-112/Mig-2 FERM domain protein (Rogalski et al., 2000), PAT-4/ILK (Mackinnon et al., 2002), PAT-6/actopaxin(Lin et al., 2003), talin (Moulder et al., 1996), UNC-89 (Benian et al., 1996), the zinc finger protein UNC-98 (Mercer et al., 2003), and the LIM domain protein UNC-97/PINCH (Hobert et al., 1999).
Studies of vertebrate focal adhesion plaques and C. elegans muscle adhesion structures have recently demonstrated the important role of LIM domain proteins in the assembly of these structures (Labouesse and Georges-Labouesse, 2003). LIM domain proteins are double zinc fingerlike structures that mediate proteinprotein interactions (Schmeichel and Beckerle, 1994). Interactions of LIM domains with specific protein partners influence subcellular localization and mediate the assembly of multimeric protein complexes (for review see Dawid et al., 1998; Bach, 2000). In C. elegans, UNC-97, a LIM domain protein of the PINCH family, has been shown to have a central role in the assembly of muscle attachment structures (Hobert et al., 1999).
Here, we describe a new LIM domain protein, UNC-95, that is required for the proper assembly of muscle attachment sites. As first described by Zengel and Epstein (1980), adult animals homozygous for the unc-95(su33) and unc-95(su106) mutation are very slow to paralyzed (UNCoordinated) and have altered body wall muscle cell structure. Lack of striations and birefringent structures of varying size were observed in these animals by polarized light microscopy. The altered structures correspond to disorganized thick and thin filaments as observed by EM of the su106 allele (Zengel and Epstein, 1980). We originally isolated UNC-95 (Y105E8A.6) as a positive interactor of the C. elegans RNF-5 in a yeast two-hybrid screen (Didier et al., 2003) and here, by searching neighboring candidates on the genetic map, we identified it as the unc-95 gene. RNF-5 is a RING finger protein with high homology to the human gene Rnf5 (Kyushiki et al., 1997; 36% identity). RING finger proteins have been demonstrated to function as ubiquitin protein ligases (E3s) in the ubiquitin modification system (for review see Fang et al., 2003). RNF-5 contains a RING finger domain (C3HC4 type) followed by a proline/serine-rich domain. We demonstrated previously that C. elegans RNF-5 exhibits E3 ligase activity (Didier et al., 2003; unpublished data). Through its E3 ligase activity, RNF-5 affects the cellular localization and abundance of its associated proteins, as was demonstrated for human paxillin. Overexpression of human Rnf5 causes exclusion of paxillin from focal adhesions, resulting in inhibition of cell motility (Didier et al., 2003). Here, we characterize the role of UNC-95 in the assembly of muscle attachment structures and identify RNF-5 as a regulator of the localization and expression of UNC-95 within the C. elegans body wall muscle cells.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The UNC-95 protein is a 350-aa LIM-only protein. It includes a single LIM domain at the COOH terminus that is highly homologous to the LIM domain of Drosophila and vertebrate focal adhesion protein, paxillin (for review see Turner, 2000). High homology was detected specifically to LIM1 (30%) and LIM4 (43%) of human paxillin. The LIM domain of UNC-95 is located at the COOH terminus, as in the paxillin family proteins. The UNC-95 sequence also contains a predicted coiled-coil motif characteristic of muscle proteins and NLS (Fig. 3 E). Comparison with the predicted UNC-95 protein sequence from the closely related species Caenorhabditis briggsae revealed complete conservation of the LIM domain and 97% homology along the entire protein sequence. Blast search of the genome did not identify clear UNC-95 relatives in C. elegans.
UNC-95 is required for the initial assembly of muscle attachment sites
To determine whether UNC-95 is required for the assembly of muscle attachment sites (dense bodies and M-lines) during embryogenesis, we stained homozygous unc-95(su33) embryos with mAbs to UNC-52/perlecan, PAT-3/ß-integrin, and DEB-1/vinculin. Staining of unc-95(su33) embryos with UNC-52/perlecan antibody demonstrated that perlecan is concentrated in the muscle basal lamina in a pattern similar to that of wild-type embryos (Fig. 4, AF). The MH27 antibody for the hypodermal cell junction protein, AJM-1 (Francis and Waterston, 1991) was used to follow the body wall muscles in the context of the embryo shape. Staining with the PAT-3/ß-integrin antibody revealed that UNC-95 is not required for the deposition of integrin in the muscle attachment sites as well, and the pattern is very similar to the wild-type pattern. At the 1.5-fold stage, staining is detected as a band along the anteriorposterior axis of the embryo, whereas at the two- and threefold embryos the structural elements of the muscle (dense bodies and M-lines) can be detected in both wild-type and unc-95(su33) embryos (Fig. 4, GL). Only after hatching does the organization of ß-integrin begin to deteriorate, as shown by the disorganized pattern of staining in the already mature but defective dense bodies (Fig. 4, M and N). In contrast, the dense body protein DEB-1/vinculin is not recruited to the nascent muscle attachments. In 1.5-fold and twofold embryos, staining is much more diffuse than in the wild-type embryos. At the threefold stage, DEB-1/vinculin staining remains diffuse and does not form the regular pattern of the mature dense bodies as shown in the wild-type threefold embryos (Fig. 4, OT; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200401133/DC1). Therefore, during embryonic muscle development UNC-95 is neither required for the normal localization of UNC-52/perlecan in the muscle basement membrane nor for the recruitment of ß-integrin to the basal sarcolemma and the formation of nascent muscle attachments. However, UNC-95 is necessary for the subsequent recruitment of vinculin to these focal adhesion-like sites.
|
|
RNF-5 colocalizes with UNC-95 in muscle dense bodies
Immunostaining with a pAb that we raised against RNF-5 revealed its expression in the larval muscle dense bodies (Fig. 6 A), the apical cell junctions of the spermatheca (septate junctions), and the junctions between the gonadal sheath cells and the gonadal basal lamina (unpublished data). During embryogenesis, RNF-5 is not expressed specifically in the developing muscle and is localized to the nucleus of most of the embryo cells (unpublished data). The interaction between RNF-5 and UNC-95 identified by the yeast two-hybrid system (Didier et al., 2003), together with their similar pattern of expression in muscle dense bodies suggest that RNF-5 may have a regulatory role in the muscle and that UNC-95 is likely to be one of its substrates. To test this hypothesis, we first performed rnf-5(RNAi) analysis on N2 animals. Motility and viability of the RNAi animals were not affected. However, when the dense body structure was analyzed in adult animals using the DEB-1/vinculin antibody, we found that the dense bodies are not organized in linear stripes as in control animals; in addition, the cellcell boundaries were abnormal (Fig. 6, AD; n > 100). Next, we analyzed the motility and muscle structure of the RNF-5 RING-deleted mutant rnf-5(tm794) that is expected to express the RNF-5 form that lacks the E3 ligase activity (see Materials and methods). Although the motility of the mutant is normal, an irregular pattern of staining of the DEB-1/vinculin antibody was detected, especially in the musclemuscle cell contacts. The contacts are thicker than normal, suggesting higher levels of vinculin accumulation at these sites (Fig. 6 E; n > 100).
|
Next, we analyzed the relative localization of RNF-5, UNC-95, and DEB-1/vinculin in the muscle dense bodies. RNF-5 does not colocalize with DEB-1/vinculin at the muscle dense bodies (Fig. 6 G). However, staining of the transgenic lines harboring the extrachromosomal array of the UNC-95::GFP translation fusion construct with both anti-GFP and anti-RNF-5 antibodies revealed that RNF-5 and UNC-95 are partially colocalized (Fig. 6 H, yellow region between the red and green area), and thus may be part of the same protein complexes within the muscle attachment sites.
RNF-5 regulates UNC-95 levels and subcellular localization
To determine whether UNC-95 level of expression is affected by RNF-5 E3 ligase activity, we analyzed the pattern of UNC-95::GFP (translational fusion construct) expression when RNF-5 was overexpressed under a heat shock promoter. When the wild-type form of RNF-5 was overexpressed in adult animals, we detected a marked decrease in the intensity of GFP labeling after heat shock, especially in the muscle cell body, as compared with control animals (Fig. 7, A and B; n > 100; see Materials and methods). However, upon overexpression of RNF-5, which harbors a mutation within the RING finger domain (see Materials and methods), there was a less pronounced effect on the GFP levels (Fig. 7 C; n > 40). These findings suggest that RNF-5 affects the stability of UNC-95 and requires an intact RING domain for this activity. These observations are in line with our finding that the RING finger domain is necessary for the E3 ligase activity of RNF-5 (Didier et al., 2003; unpublished data).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is interesting that unc-95(su33) and unc-95(RNAi) animals (likely null phenotype) are not embryonic lethal, exhibiting the Pat phenotype, but develop into adults with reduced motility and some degree of paralysis. The Pat phenotype is characteristic of mutants in dense bodies and M-line components (Williams and Waterston, 1994; Moerman and Fire, 1997). We suggest that the reason is the capability of the actin- and myosin-containing filaments to be recruited to the aberrant dense bodies and M-lines of the mutant. Although the A and I bands are aberrant, they do assemble into the myofilament lattice, which contains linkages to the basement membrane that are stable enough to proceed past the twofold stage, complete elongation, and allow larval development. It is possible that other muscle LIM domain proteins (for example UNC-97) contribute either redundantly or additively and function as molecular adaptors to enable the recruitment of essential structural components as vinculin to the muscle adhesion sites, and in this way substitute for the lack of UNC-95 activity.
UNC-95 belongs to the LIM-only protein group
A central role in the assembly of multiprotein complexes at mammalian focal adhesions in cultured cells was shown for LIM-only proteins such as paxillin, zyxin, enigma, and others. These proteins are primarily cytoplasmic and are associated with the actin cytoskeleton (Dawid et al., 1998; for review see Turner, 2000). UNC-95 is a LIM-only protein and may similarly function as an adaptor protein, allowing the recruitment of various structural and signaling molecules to the nematode muscle focal adhesionlike structures. This possibility is supported by the analysis of transgenic animals harboring the UNC-95::GFP translational fusion construct, which is expressed not only in the dense bodies and M-lines, but also in the muscle cell body. The cytoplasmic and nuclear localization of UNC-95 suggests that it shuttles between the cytoplasm, nucleus, and attachment sites, and by its association with specific proteins may affect both structural and signaling processes. Shuttling between cytoplasmic and nuclear compartments was shown for mammalian focal adhesion LIM domain proteins such as paxillin, zyxin, and Hic5, and is thought to facilitate signaling between adhesion sites and the nucleus (for review see Wang and Gilmore, 2003). In parallel to the focal adhesion proteins, a common feature of a variety of LIM domain proteins that have a role in muscle differentiation or assembly is their bimodal subcellular distribution. Examples are the muscle LIM-proteins of the CRP/MLP/TLP family (for review see Weiskirchen and Gunther, 2003), and the four-and-a-half LIM (FHL) proteins (Li et al., 2001). UNC-95 may also have a dual role in the nematode body wall muscles. In C. elegans muscle, UNC-97 (PINCH) and UNC-98 (zinc finger) are required for the assembly of muscle attachment sites and, like UNC-95, are localized to the nucleus in addition to muscle attachment sites (Hobert et al., 1999; Mercer et al., 2003). Because UNC-95, -97, and -98 exhibit a similar pattern of expression, it is possible that they interact to form a multidomain adaptor complex.
Using the COOH-terminal truncated form of UNC-95 in the translation fusion unc-95(su33)::GFP, which lacks the LIM domain, we demonstrated that the LIM domain of UNC-95 is not required for the primary localization of the truncated protein to the dense bodies and M-lines, but for its accumulation in these sites. This is in contrast to paxillin localization in focal adhesions, which requires the LIM domain (for review see Turner, 2000). The truncated unc-95(su33)::GFP construct enabled nuclear localization, although the putative NLS (aa 177193) is not included. Most important, the truncated transgene was not expressed in muscle cell boundaries, which are the regions of contact between adjacent muscle cells. Thus, the COOH-terminal region of UNC-95 (from aa 144), which includes the LIM domain, is essential for the localization of UNC-95 to the musclemuscle cell boundaries. This suggests that additional signaling pathways and protein interactions are involved in the recruitment of UNC-95 to the cellcell contacts. The "mosaic-like" pattern of expression of the su33::GFP allele could be a dominant-negative effect caused by overexpression of the mutant form of UNC-95 in the wild-type background expressing the wild-type UNC-95 protein.
It was shown previously that paxillin binds to vinculin through its LD motifs (Turner, 2000). It is possible that UNC-95 also binds vinculin directly to recruit it to the dense bodies and musclemuscle cell boundaries and that the vinculin-binding domain lies in the COOH-terminal part of the UNC-95 protein, which is missing in the unc-95(su33) mutant embryo. Along these lines, aa 234265 of UNC-95 show high similarity to the LD4 region of vertebrate paxillin (see alignment in Didier et al., 2003).
RNF-5 and UNC-95
RNF-5 is detected in muscle dense bodies at the L1 larval stage. This observation suggests that RNF-5 is not required for the assembly of the I bands in the body wall muscle cells, but probably for subsequent regulatory events that maintain and stabilize the thin filaments within the I bands during contraction of the mature muscle.
Overexpression of wild-type RNF-5 through the heat shock promoter caused a marked decrease in the UNC-95::GFP expression levels. This decrease was detected as soon as 68 h following induction of RNF-5, which is not likely to be a result of transcriptional repression of the transgene. Therefore, we suggest that RNF-5 plays a key role in the regulation of UNC-95 stability in the mature muscle. Accumulation of UNC-95 (as shown when RNF-5 was depleted) may induce inadvertent signaling pathways and cause damage to the muscle. This is prevented by the specific E3 ligase activity of RNF-5 that limits the levels of UNC-95 in the mature muscle. In addition, an RNF-5 RING mutant exhibits a substantially weaker effect on the UNC-95::GFP expression. In accordance with this observation, we demonstrated earlier that mutation within the conserved cysteines in the RING finger domain or RNF-5 that is deleted of its RING domain were no longer capable of exhibiting E3 ligase activity (Didier et al., 2003), further indicating that the RING finger domain is required for RNF-5 ability to elicit its E3 ligase activity. Along these lines, rnf-5(RNAi) or the RING-deleted mutant rnf-5(tm794) caused a major increase in the level of UNC-95::GFP predominantly in the muscle cell body, and lethality in the rnf-5(tm794) homozygous background. Of interest to note is that considerable efforts to detect association between RNF-5 and UNC-95 did not succeed, either due to the localization of these proteins within insoluble membrane fractions, which results in their dissociation by methods used for extraction, or because RNF-5 mediates its effect on UNC-95 via an intermediate protein/ligase, as commonly seen for RING finger proteins. Consistent with this observation is the weak association seen for RNF-5 with paxillin in vitro (Didier et al., 2003).
The COOH-terminal tail of the Arabidopsis RNF-5 has been implicated in membrane association (Matsuda and Nakano, 1998). In addition, the COOH-terminal tail of human RNF-5 was shown to be required for paxillin ubiquitination (Didier et al., 2003). Together with the confocal analysis presented in this paper, it is likely that RNF-5 is localized through its COOH terminus to the muscle cell membrane at the base of the dense bodies where it regulates UNC-95 localization and stability. Although UNC-95 is expressed both in the muscle attachment sites and the muscle cell body, we suggest that RNF-5 regulates the levels and distribution of UNC-95 between these muscle cell compartments.
Finally, because the homology of UNC-95 to paxillin is limited to the LIM domain and the LD4 region, it is possible that RNF-5 regulates the stability and localization of additional LIM/LD domain proteins that participate in the assembly of muscle attachment sites in C. elegans.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
unc-95(su33) was mapped by Zengel and Epstein (1980) to linkage group I and appeared to be near and to the left of the unc-54 gene. The cosmid Y105E8A maps to this area and contains a predicted LIM domain protein, Y105E8A.6, identified by the C. elegans genome sequencing consortium. The complete gene structure (exonintron) was determined by RT-PCR. The unc-95 gene mRNA includes 1,053 nucleotides and is encoded by five exons.
The unc-95(su33) allele is affected by temperature. The decrease in motility and paralysis is less severe in animals raised in 15°C compared with animals raised at 20°C.
Rescue of the unc-95(su33) mutant phenotype was performed by mating males carrying the extrachromosomal array Ex[unc-95::GFP; rol-6] with the unc-95(su33) hermaphrodite. F2 fluorescent rol animals homozygous to the unc-95(su33) allele were analyzed. Homozygosity to unc-95(su33) was confirmed by the non-rol progeny, which all displayed the slow/paralyzed phenotype.
Electron microscopy
Transmission EM was performed as described by Hall (1995).
Sequence analysis
The unc-95(su33) amber mutation was identified by PCR amplification of the Y105E8A.6 sequence from DNA purified from unc-95(su33) mutant animals. Subsequent DNA sequencing of the amplified region was performed on the two strands. In addition, single worm PCR and DNA sequencing of five independent animals was performed to verify that this was indeed a molecular lesion and not an error introduced randomly by the PCR.
RT-PCR
RNA from N2 and unc-95(su33) mutant animals was purified from mixed stage cultures using the RNeasy kit (QIAGEN). Nonquantitative RT-PCR was performed using the OneStep RT-PCR kit with primers designed for the amplification of the full-length unc-95 cDNA.
Transcriptional and translational unc-95::GFP fusion constructs
The transcriptional GFP construct, Ex[Punc-95::GFP; rol-6], included a 2.5-Kb fragment upstream of the predicted initiator methionine of Y105E8A.6 fused to the GFP reporter that includes the unc-54 3' untranslated region at the 3' end (a gift from A. Fire, Stanford University, Stanford, CA). The translational GFP construct, Ex[unc-95::GFP; rol-6], which rescues the unc-95(su33) mutant phenotype, includes the same 2.5-Kb upstream sequences fused to the entire genomic sequence of Y105E8A.6 (unc-95), including all exons and introns and fused in the COOH terminus to the GFP reporter that includes the unc-54 3' untranslated region at the 3' end. The mutated construct that mimics the su33 allele, Ex[unc-95(su33)::GFP; rol-6], included the first three exons and two introns of Y105E8A.6 until position 429 and fused at the COOH terminus to the same GFP reporter. An irregular pattern of staining (as shown in Fig. 5 L) could be observed in few cells of the body wall muscle in most of the transgenic animals of this fusion construct in all lines generated (n = 4 independent lines).
The GFP reporter constructs were generated by PCR fusion according to Hobert (2002). The template was N2 (wild-type) genomic DNA. Transgenic arrays were constructed through DNA microinjection into the N2 background with the dominant marker pRF4/rol-6. The expression of the above constructs was monitored in live animals.
RNF-5 overexpression constructs
Full-length rnf-5 cDNA and the rnf-5 RING mutant were cloned into vectors containing the heat shock promoters hsp16-2 and hsp16-41 (Stringham et al., 1992). In the RING-mutated form of RNF-5, both cysteine 26 and cysteine 29, which are part of the RING finger domain (C3HC4 type), were mutated to arginine. This mutagenesis disrupts the structure of the RING. Each form was cloned into both vectors and the two vectors were mixed before injection. Transgenic lines were crossed to the Ex[unc-95::GFP; rol-6] strains. Two independent lines were constructed for each RNF-5 construct. Heat shock was performed for 1 h at 32°C. Five independent experiments were performed for the RNF-5 wild-type construct and in each experiment 20 animals were scored (n > 100). Three independent experiments were performed for the RNF-5 mutant construct (n > 40). Confocal analysis of live animals was done 68 h after heat shock. Images shown were chosen as representative examples. Two control experiments were performed: first, identical heat shock treatment and analysis of the Ex[unc-95::GFP; rol-6] parental strain (n > 50), and second, no heat shock of the examined strain Ex[unc-95::GFP; rol-6]; Ex[hsp::rnf-5; rol-6] (n > 100; served as the control group for t test). Measurement of GFP fluorescence (mean pixel values) was performed using the Laser Sharp processing program (Bio-Rad Laboratories) and the Confocal Assistant program on n = 10 animals of each group. t tests were performed on the GFP fluorescence values.
RNAi
For RNAi experiments (Fire et al., 1998) of the unc-95 gene, we used the first 600 bp of the unc-95 cDNA, which does not include the conserved LIM domain. The gene-specific primers included T7 and Sp6 sites at their 5' end and were used for PCR from the unc-95 cDNA. RNA was synthesized from the amplified fragment with either T7 or Sp6 RNA polymerase (Riboprobe®; Promega). Equal amounts of sense and antisense RNA were annealed to create dsRNA. dsRNA was injected into the gonad of wild-type hermaphrodites (n = 40) or to the Ex[unc-95::GFP; rol-6] transgenic animals (n = 10) at a concentration of 0.51.0 mg/ml. Scoring of the phenotype was done by allowing single injected animals to lay eggs at 20°C starting at 24 h after injection and analysis of the progeny (n > 100). For RNAi experiments of the rnf-5 gene, we used the spliced form that does not contain the RING domain. dsRNA was synthesized as above and injected to wild-type animals or to the Ex[unc-95::GFP; rol-6] transgenic animals. At least five independent experiments (n = 20 injected animals per experiment) were performed for each strain. GFP pattern of expression was analyzed in the progeny (n = 85). Images shown are representative examples.
Antibody staining and confocal microscopy
Fixation of larvae and adult animals for antibody staining was performed as described by Finney and Ruvkun (1990). Permeabilization of embryos was done by freeze-cracking and fixation by methanol acetone. Incubation times were overnight at 4°C with the primary antibodies, and 2 h at RT with the secondary antibodies. Antibodies used were: monoclonal mouse anti-vinculin antibody MH24 (used at 1:150; Francis and Waterston, 1985), monoclonal mouse anti-ß-integrin antibody MH25 (1:100; Francis and Waterston, 1985), monoclonal mouse anti-perlecan antibody MH2 (1:100; Francis and Waterston, 1985), monoclonal mouse anti-myosin myoA (1:100; Miller et al., 1983), monoclonal mouse anti-GFP antibody (1:100; MBL International Corporation), and rabbit polyclonal anti-RNF-5 antibody (1:150; this paper).
Staining of the rnf-5(RNAi) animals and rnf-5(tm794) was repeated three independent times in parallel to staining of wild-type animals in order to verify that the moderate phenotype observed was not a technical artifact.
Confocal microscopy images were captured as a stacked series using a confocal scanning microscope (MRC 1024; Bio-Rad Laboratories) as described in Kolotuev and Podbilewicz (2004), and were processed using the Confocal Assistant (CAS) program and Adobe Photoshop®.
Online supplemental material
Online supplemental material contains enlarged insets from Fig. 4 (Q and T), and is available at http://www.jcb.org/cgi/content/full/jcb.200401133/DC1.
![]() |
Acknowledgments |
---|
Support by National Institutes of Health grant (CA97105 to Z. Ronai) and the Fund for Promotion of Research at the Technion (to B. Podbilewicz) is gratefully acknowledged.
Submitted: 26 January 2004
Accepted: 10 May 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bach, I. 2000. The LIM domain: regulation by association. Mech. Dev. 91:517.[CrossRef][Medline]
Barstead, R.J., and R.H. Waterston. 1991. Vinculin is essential for muscle function in the nematode. J. Cell Biol. 114:715724.[Abstract]
Benian, G.M., T.L. Tinley, X. Tang, and M. Borodovsky. 1996. The Caenorhabditis elegans gene unc-89, required for muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains. J. Cell Biol. 132:835848.[Abstract]
Dawid, I.B., J.J. Breen, and R. Toyama. 1998. LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet. 14:156162.[CrossRef][Medline]
Didier, C., L. Broday, A. Bhoumik, S. Israeli, S. Takahashi, K. Nakayama, S.M. Thomas, C.E. Turner, S. Henderson, H. Sabe, and Z. Ronai. 2003. RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization. Mol. Cell. Biol. 23:53315345.
Fang, S., K.L. Lorick, J.P. Jensen, and A.M. Weissman. 2003. RING finger ubiquitin protein ligases: implications for tumorigenesis, metastasis and for molecular targets in cancer. Semin. Cancer Biol. 13:514.[CrossRef][Medline]
Finney, M., and G. Ruvkun. 1990. The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell. 63:895905.[Medline]
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. 391:806811.[CrossRef][Medline]
Francis, G.R., and R.H. Waterston. 1985. Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101:15321549.[Abstract]
Francis, G.R., and R.H. Waterston. 1991. Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114:465479.[Abstract]
Geiger, B., and A. Bershadsky. 2001. Assembly and mechanosensory function of focal contacts. Curr. Opin. Cell Biol. 13:584592.[CrossRef][Medline]
Hall, D.H. 1995. Electron microscopy and three-dimensional image reconstruction. Methods Cell Biol. 48:395436.[Medline]
Hobert, O. 2002. PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques. 32:728730.[Medline]
Hobert, O., D.G. Moerman, K.A. Clark, M.C. Beckerle, and G. Ruvkun. 1999. A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans. J. Cell Biol. 144:4557.
Hresko, M.C., B.D. Williams, and R.H. Waterston. 1994. Assembly of body wall muscle and muscle cell attachment structures in Caenorhabditis elegans. J. Cell Biol. 124:491506.[Abstract]
Kolotuev, I., and B. Podbilewicz. 2004. Pristionchus pacificus vulva formation: Polarized division, cell migration, cell fusion and evolution of invagination. Dev. Biol. 266:322333.[CrossRef][Medline]
Kyushiki, H., Y. Kuga, M. Suzuki, E. Takahashi, and M. Horie. 1997. Cloning, expression and mapping of a novel RING-finger gene (RNF5), a human homologue of a putative zinc-finger gene from Caenorhabditis elegans. Cytogenet. Cell Genet. 79:114117.[Medline]
Labouesse, M., and E. Georges-Labouesse. 2003. Cell adhesion: parallels between vertebrate and invertebrate focal adhesions. Curr. Biol. 13:R528R530.[CrossRef][Medline]
Li, H.Y., E.K. Ng, S.M. Lee, M. Kotaka, S.K. Tsui, C.Y. Lee, K.P. Fung, and M.M. Waye. 2001. Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET). J. Cell. Biochem. 80:293303.[CrossRef][Medline]
Lin, X., H. Qadota, D.G. Moerman, and B.D. Williams. 2003. C. elegans PAT-6/actopaxin plays a critical role in the assembly of integrin adhesion complexes in vivo. Curr. Biol. 13:922932.[CrossRef][Medline]
Mackinnon, A.C., H. Qadota, K.R. Norman, D.G. Moerman, and B.D. Williams. 2002. C. elegans PAT-4/ILK functions as an adaptor protein within integrin adhesion complexes. Curr. Biol. 12:787797.[CrossRef][Medline]
Matsuda, N., and A. Nakano. 1998. RMA1, an Arabidopsis thaliana gene whose cDNA suppresses the yeast sec15 mutation, encodes a novel protein with a RING finger motif and a membrane anchor. Plant Cell Physiol. 39:545554.[Medline]
Mercer, K.B., D.B. Flaherty, R.K. Miller, H. Qadota, T.L. Tinley, D.G. Moerman, and G.M. Benian. 2003. Caenorhabditis elegans UNC-98, a C2H2 Zn finger protein, is a novel partner of UNC-97/PINCH in muscle adhesion complexes. Mol. Biol. Cell. 14:24922507.
Miller, D.M., III, I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Differential localization of two myosins within nematode thick filaments. Cell. 34:477490.[Medline]
Moerman, D.G., and A. Fire. 1997. Muscle: structure, function and development. C. Elegans II. D.L. Riddle, T. Blumenthal, B.J. Meyer and J.R. Priess, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 417470.
Moulder, G.L., M.M. Huang, R.H. Waterston, and R.J. Barstead. 1996. Talin requires ß-integrin, but not vinculin, for its assembly into focal adhesion-like structures in the nematode Caenorhabditis elegans. Mol. Biol. Cell. 7:11811193.[Abstract]
Pulak, R., and P. Anderson. 1993. mRNA surveillance by the Caenorhabditis elegans smg genes. Genes Dev. 7:18851897.[Abstract]
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. 7:14711484.[Abstract]
Rogalski, T.M., G.P. Mullen, M.M. Gilbert, B.D. Williams, and D.G. Moerman. 2000. The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane. J. Cell Biol. 150:253264.
Schmeichel, K.L., and M.C. Beckerle. 1994. The LIM domain is a modular protein-binding interface. Cell. 79:211219.[Medline]
Stringham, E.G., D.K. Dixon, D. Jones, and E.P. Candido. 1992. Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans. Mol. Biol. Cell. 3:221233.[Abstract]
Turner, C.E. 2000. Paxillin and focal adhesion signalling. Nat. Cell Biol. 2:E231E236.[CrossRef][Medline]
Wang, Y., and T.D. Gilmore. 2003. Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear. Biochim. Biophys. Acta. 1593:115120.[CrossRef][Medline]
Waterston, R.H. 1988. Muscle. The Nematode Caenorhabditis elegans. W.B. Wood, editor. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 281335.
Weiskirchen, R., and K. Gunther. 2003. The CRP/MLP/TLP family of LIM domain proteins: acting by connecting. Bioessays. 25:152162.[CrossRef][Medline]
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. 124:475490.[Abstract]
Zengel, J.M., and H.F. Epstein. 1980. Identification of genetic elements associated with muscle structure in the nematode Caenorhabditis elegans. Cell Motil. 1:7397.[Medline]