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1 Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
2 Electron Microscope Laboratory, RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan
3 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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SFKs are highly conserved in the Animal Kingdom (Suga et al. 2001; Segawa et al. 2006). In Caenorhabditis elegans, two homologs of SFKs have been identified, SRC-1 and SRC-2 (Hirose et al. 2003). SRC-1 functions to control cleavage orientation in early embryo in parallel with Wnt signaling (Bei et al. 2002), and directly regulates migration of distal tip cells (DTCs) and some mechanosensory neurons (Itoh et al. 2005; Lee et al. 2005). In contrast, as loss of src-2 function did not cause any recognizable defects (Itoh et al. 2005), it is considered that src-2 is dispensable for C. elegans development. Like other metazoans examined, C. elegans has a single Csk orthologue (CSK-1) (Hirose et al. 2003). An in vitro study in a yeast expression system demonstrated that the kinase activities of SRC-1 and SRC-2 could be inhibited by CSK-1-mediated tyrosine phosphorylation at their C-terminal regulatory sites. These results are consistent with the hypothesis that the function of SFK/Csk circuit is widely conserved in metazoan (Miller et al. 2000; Song et al. 2001; Segawa et al. 2006). However, if SRC-1 and SRC-2 are indeed controlled by CSK-1 in vivo remains to be examined. However, recent studies suggested that SFKs are not strictly regulated by Csk-mediated phosphorylation in unicellular choanoflagellates (Segawa et al. 2006; Li et al. 2008), and that one SFK family member in multicellular sponges is resistant to negative regulation by its cognate Csk (Segawa et al. 2006). Furthermore, although, the core domains of SFKs are well conserved in metazoans, the C-terminal regulatory region are substantially divergent, especially in invertebrates (Segawa et al. 2006). These findings imply that the mechanisms regulating SFK activity might diverge between evolutional branches, different tissues or SFK family members.
To re-evaluate the in vivo roles of CSK-1, we characterized C. elegans mutant for csk-1. The csk-1 mutants underwent growth arrest at an early larval stage because of defects in pharyngeal muscle organization. However, the growth arrest phenotype in csk-1 mutants cannot be suppressed by src-1 and/or src-2 mutation, or SRC-1 activity is not significantly up-regulated in csk-1 mutants. These observations suggest that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require the C. elegans SFKs.
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Results |
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To isolate a loss-of-function mutation in csk-1, we screened pools of mutagenized C. elegans carrying small chromosomal deletions by PCR genotyping (Jansen et al. 1997). DNA sequence analysis confirmed that animals homozygous for the csk-1 deletion allele ov1 lacked a 396-bp segment of DNA in the indicated region encompassing from intron 4 to a part of exon 5 (Fig. 1A). RT-PCR analysis demonstrated that the mutated transcripts contained an incorrect splicing between the middle of intron 4 and exon 5, which resulted in a frame-shift that removes a large part of the kinase domain (Fig. 1C). These results suggest that this mutant allele, designated csk-1(ov1), is functionally null mutant (Fig. 1C). To confirm the effects of CSK-1 loss of function, we obtained another csk-1 mutant allele, csk-1(tm1916), which has a larger deletion than csk-1(ov1) and a frame-shift in the kinase domain (Fig. 1A).
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Hermaphrodites heterozygous for csk-1(ov1) or csk-1(tm1916) were phenotypically indistinguishable from the wild-type strain N2, indicating that the deletion mutations act recessively. Analysis of terminal phenotypes of these mutant animals showed that a population (approximately 30%) of the csk-1 homozygous embryos (csk-1 mutants) died before hatching (Fig. 1D). The viable embryos completed embryogenesis and hatched successfully. However, approximately 90% of the hatched csk-1 mutant animals underwent growth arrest in the first (L1) larval stage and survived for several days; the remaining larvae (approximately 10%) grew to adulthood (Fig. 1D). Tissue organization appeared grossly normal in developmentally arrested csk-1 L1 larvae (Fig. 2A–a and b). Thus, we considered that nutritional status might affect the viability of the mutant animals. Indeed, the phenotypes of the csk-1 mutant animals were quite similar to those of wild-type animals that has been starved after hatching (Fig. 2A–c), suggesting that the defects in the csk-1 mutants might be associated with impaired feeding (Ohmachi et al. 1999). The L1 arrest phenotype was rescued by the re-expression of a csk-1 cDNA under the control of the csk-1 promoter, but not by a kinase-deficient form of CSK-1, K310M, which has a Lys to Met substitution at the site required for catalytic activity. These results indicate that the expression of csk-1 cDNA can substitute functionally for the endogenous csk-1 gene, the csk-1 promoter can drive expression in tissues that require csk-1 expression in C. elegans development, and that the ability of CSK-1 to rescue the defect of csk-1 mutants is dependent on the kinase activity of CSK-1 (Fig. 2A–d and e, B).
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To ascertain the cause of the defects in the csk-1 mutants, we confirmed the expression profile of CSK-1 in wild-type animals. Previously, the expression profiling of csk-1 using a gfp reporter determined that the csk-1 is expressed at the highest levels in the pharynx (Hirose et al. 2003). To examine the intracellular distribution of CSK-1 protein, we expressed GFP–CSK-1 fusion protein under the control of the csk-1 promoter. Introduction of the gfp::csk-1 gene could rescue the L1 arrest phenotype in the csk-1 mutant (Fig. 2B), indicating that the fusion protein has the physiological function as CSK-1. The expression of GFP–CSK-1 became detectable at around comma stage, when pharynx begins to organize (data not shown). At the loop stage of embryonic development, GFP–CSK-1 expression was widely detectable in embryos, with high levels of expression observed in the pharynx, especially at cell–cell boundaries (Fig. 3A). At the L1 larval stage, GFP–CSK-1 expression was highly concentrated in the pharyngeal muscles (Fig. 3B), although modest expression was detected in body wall muscles, anchor cells and tail region. At adult stages, GFP–CSK-1 was seen in the pharynx, specifically localized along muscle fibers and cell membranes in the procorpus (Fig. 3C) and the terminal bulb (Fig. 3D). The predominant expression of CSK-1 to pharyngeal muscles and the potential association of CSK-1 with muscle filaments suggested that CSK-1 plays roles in the regulation of pharynx-specific function, namely the feeding behavior.
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To examine if the csk-1 mutation causes defects in the feeding behavior, we carried out a semiquantitative assay for feeding. Wild-type and csk-1(ov1) L1 larvae were fed with fluorescent latex beads (
0.5 µm) for 1 h. The beads accumulated in the lumen of the pharynx and in the intestine were detected by fluorescence microscopy (Fig. 4A,B). We scored numbers of wild-type, csk-1(ov1) and the rescued csk-1(ov1);Ex[Pcsk-1::csk-1]
larvae by classifying them into the following four groups: animals that contained fluorescent beads up to (i) the procorpus, (ii) the metacarpus, (iii) the terminal bulb, or (iv) the intestine (Fig. 4C,D). More than 50% of the csk-1(ov1) mutant larvae only contained the beads up to the metacarpus, and the defect was rescued by expression of a csk-1 cDNA under the control of the csk-1 promoter (Fig. 4D). Thus, these indicate that loss of csk-1 function induces defects in feeding.
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Pharyngeal muscles of csk-1 mutant animals are defective in pumping and have defects in muscle fiber organization
To further examine the requirement for csk-1 function in pharyngeal muscles, we then examined the effects of csk-1(ov1) mutation on the pumping activity of the terminal bulb. Caenorhabditis elegans feeding involves two types of pharyngeal muscle action, pumping and peristalsis (Albertson & Thomson 1976; Avery & Horvitz 1989). Terminal bulb pumping facilitates crushing of bacteria by the grinder cells and passage of the debris into the intestine. To quantitate the effect of csk-1 mutation on these processes, we measured the pumping rate of grinder cells using a stereoscopic microscope (Supplementary Movies 1 and 2). The average pumping rate in the csk-1 mutants (50 ± 49) was approximately half of that of the wild-type strain (140 ± 23), and pumping activity was almost completely abrogated in a significant population (approximately 30%) of the mutant animals (Fig. 5A). Pharyngeal muscle contractions that were observed in the csk-1 mutants were frequently defective: the corpus muscles appeared to contract slowly, and the grinder cell muscles were occasionally twitched. These defects were rescued by the expression of CSK-1 under the control of the myo-2 promoter (Fig. 5A).
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CSK-1 function in the pharynx is independent of the Caenorhabditis elegans Src homologs SRC-1 and SRC-2
In vertebrates, it is believed that Csk principally functions by negatively regulating SFKs. To examine if csk-1 functions in the pharynx as a negative regulator of the SFKs, SRC-1 and SRC-2, we tested for the genetic interactions among csk-1, src-1 and src-2. In this study, we analyzed a homozygous src-1(cj293) mutant produced from the balanced heterozygote src-1(cj293)/+, as the src-1(cj293) mutant shows a maternal embryonic lethal phenotype (Bei et al. 2002). The src-1(cj293) mutants exhibited L1 arrest phenotype with a penetrance of approximately 20% (Fig. 6), and the rest of the animals grew into adults with apparent defects in gonad morphogenesis and migration of some neurons (Itoh et al. 2005). The homozygous src-2(ok816) mutants are viable and show no significant defects at any stage of development (Fig. 6). Animals doubly mutant for csk-1 and src-1 (csk-1 src-1) showed an increased penetrance of the L1 arrest phenotype, with nearly all animals affected, showing an additive effect between these genes. Adding a src-2 loss-of-function mutation to the csk-1 mutation (csk-1 src-2) did not affect the csk-1 phenotype. These observations suggest that src-1 or src-2 cannot serve as a suppressor of its putative upstream regulator, csk-1. Similar to the csk-1 src-1 mutants, the triple mutant (csk-1 src-1 src-2) showed effects in the penetrance of L1 arrest phenotype in a manner similar to the csk-1 src-1 mutants. These results suggest that src-1 and src-2 may not be required for the csk-1 phenotype at least in the pharynx. The analysis of the src-1 src-2 mutants confirmed that there is no functional redundancy between these kinases (Fig. 6).
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Discussion |
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Mammalian Csk is ubiquitously expressed throughout the development, although relatively higher Csk expression is observed in the developing nervous system and the immune system (Okada et al. 1991). Studies using csk knockout mice demonstrated that mammalian Csk plays general roles in various cell types by controlling ubiquitous SFKs (Nada et al. 1993; Schmedt et al. 1998; Thomas et al. 2004; Yagi et al. 2007; Takatsuka et al. 2008). In contrast, our studies showed that the expression and function of CSK-1 might be highly specialized during evolution of C. elegans. Another critical feature of C. elegans CSK-1 is that its function may not require the activation of C. elegans SFKs. Analysis of genetic interactions suggested that the requirement for csk-1 function in the pharyngeal muscle is independent of both src-1 and src-2. If SRC-1 or SRC-2 functions downstream of CSK-1, then either src-1 or src-2 or a src-1 src-2 double mutant should serve as a suppressor for csk-1. However, such effects were not observed in any of these multiply mutant strains. Immunostaining and immunoblot analyses using the anti-pY416 antibody that specifically recognizes the activated SRC-1 also demonstrated that SRC-1 activity was not greatly increased in csk-1 mutants. These results were apparently inconsistent with those of the previous in vitro study (Hirose et al. 2003), and raised the possibility that CSK-1 functions independently of SRC-1 in vivo.
In yeast expression system, SRC-1 was inactivated by the phosphorylation at its C-terminal regulatory site by CSK-1 (Hirose et al. 2003). This effect was further confirmed in the expression experiments using HEK293T and COS7 cells (data not shown). However, the inactivation of SRC-1 by CSK-1 was not so strict as that of mammalian SFK by Csk. Mammalian SFKs can be almost completely inactivated by the Csk-mediated phosphorylation (Sicheri & Kuriyan 1997), whereas SRC-1 retained significant activity even when its C-terminus was phosphorylated. Thus, it is possible that the activity retained in the CSK-1-phosphorylated SRC-1 is sufficient for SRC-1 function in C. elegans. Similar situation was observed for SFKs in unicellular choanoflagelates (MoSrc) (Segawa et al. 2006; Li et al. 2008) and one SFK member in multicellular sponge (EfSrc) (Segawa et al. 2006). These primitive SFKs were phosphorylated by their cognate Csk at the C-terminal regulatory sites, but were substantially active even in the phosphorylated forms because of a structural deviation in the N-lobe of the kinase domain. From these findings, it is suggested that the strict negative regulation of SFKs by Csk might be acquired during evolution of particular cell functions in multicellular animals. Although further studies at molecular levels are necessary, C. elegans SRC-1 might conserve the regulatory system depicted for the primitive SFKs (Segawa et al. 2006). Additionally, the previous expression profiling of src-1 showed that, inconsistent with the csk-1 expression profile, src-1 expression is rather ubiquitous and is not necessarily concentrated in the pharynx, although the dispensable src-2 is preferentially expressed in the pharynx (Hirose et al. 2003). These differential expression patterns of csk-1 and src-1 could also account for the independent functions of these kinases especially in pharyngeal muscles.
In summary, we showed that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require the C. elegans SFKs. Further analysis of the mechanisms underlying the function of C. elegans CSK-1 would provide new insights into as-yet-unidentified functions of mammalian Csk.
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Experimental procedures |
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Caenorhabditis elegans strains were cultured at 20 °C as described previously (Brenner 1974). N2 Bristol served as the wild-type strain. The following alleles were used and are listed by linkage groups LGI: csk-1(ov1), csk-1(tm1916), src-1(cj293) and src-2(ok819). The hT2 translocation chromosome was used to balance csk-1. The hT2 balancer chromosome was marked with myo-2::gfp transgene qIs48 (Wang & Kimble 2001), which permitted the identification of csk-1/hT2[qIs48] animals heterozygous for the balancer chromosome. The hT2 translocation chromosome also balances src-1 as described previously (Itoh et al. 2005). Double and triple mutant strains were constructed using standard methods.
PCR screening for deletion mutants
Caenorhabditis elegans library mutagenized with UV and trimethylpsoralen (kindly provided by R. Kuroki and K. Nishiwaki) was screened for deletions in the csk-1 gene. Nested primers were used in two successive rounds of PCR. The first-round primers were D61 (5'-CAG CTG ACC TGT GAC AAG GA-3') and D62 (5'-GAG ACC TCT GCA AAA TTC GG-3'); the second round primers were D63 (5'-TTT GAC ACA GTT GGT CTC G-3') and D64 (5'-CAA ATA ATT TGT GCG CCC ATC-3'). Although a 1.0-kbp fragment was amplified from wild-type genomic DNA, the PCR product from one mutagenized animal consisted predominantly of a 0.6-kbp fragment. Sequencing of this short fragment showed that a 0.4-kbp fragment of genomic DNA had been removed from the csk-1 locus. This deletion allele, csk-1(ov1), was recovered and was backcrossed at least 10 times to N2 strain and balanced with hT2 [qIs48]. Another csk-1 mutant allele, csk-1(tm1916), was obtained from the National Bioresource Project (Tokyo Women's Medical University), and was backcrossed more than five times to N2 strain and balanced with hT2 [qIs48].
Genotyping
Single animals were genotyped by PCR using three primers in a single reaction. For csk-1(ov1) allele, the sequence of the inside primer, csk-delR, was 5'-GTC ACG TTT GTA GTG CTG GA-3'. The sequences of the outside primers were 5'-TTT GAC ACA GTT GGT CTC G-3' (D63) and 5'-CAA ATA ATT TGT GCG CCC ATC-3' (D64). A 0.6-kbp fragment was amplified from the csk-1(ov1)/csk-1(ov1) homozygote, whereas a 0.4-kbp fragment was amplified from the wild-type (+/+) animal. Both the 0.4 and 0.6 kbp fragments were amplified from csk-1(ov1)/+ heterozygote. For csk-1(tm1916) allele, the sequence of the inside primer, csk-delR, was 5'-GTC ACG TTT GTA GTG CTG GA-3'. The sequences of the outside primers were 5'-TTA CTC CGC GGC AAA CCG GA-3' (tm1916 F) and 5'-GAG ACC TCT GCA AAA TTC GG-3' (D62). A 0.8-kbp fragment was amplified from the csk-1(tm1916)/csk-1(tm1916) homozygote, whereas a 0.6-kbp fragment was amplified from the wild-type (+/+) animal. Both the 0.6 and 0.8 kbp fragments were amplified from csk-1(tm1916)/+ heterozygote.
Terminal stage analysis
Parental animals were cultured on NGM plates and allowed to lay eggs for 3 h. The L1 larvae were then recovered and transferred to a new NGM plate. They were cultured at 20 °C for up to 3 weeks, and were occasionally observed using a dissecting microscope (Leica MZ FLIII). Morphologies of the eggs were examined using a confocal laser-scanning microscope (Olympus FV1000).
Feeding assay
To evaluate feeding proficiency, animals were fed with a mixture of bacteria and fluorescent latex beads ( 0.5 µm; Fluoresbrite Carboxylate Microspheres YG, Polysciences, Inc.) (Ohmachi et al. 1999). One drop of the bead concentrate was diluted into 1 mL of M9 solution, and 100 µL of the resulting suspension was spread evenly over a 60-mm NGM agar plate, on which bacteria had been plated and grown. Animals at the L1 stage were transferred to the plate, and 1 h later the amount of ingested beads in the pharynx and intestine was determined using a confocal laser-scanning microscope (Zeiss LSM-510) with a GFP filter.
Transmission electron microscopy
For transmission electron microscopy, animals at the L1 stage were rinsed in M9 buffer and placed into a pre-fixative containing 2.5% glutaraldehyde, 2% formaldehyde and 0.1 M sodium cacodylate buffer (pH 7.2–7.4) at room temperature for 2 h. Samples were then washed with 0.1 M cacodylic acid buffer and post-fixed with 1% OsO4 in 0.1 M cacodylate buffer for 2 h on ice. The samples were rinsed with distilled water and stained with 0.5% aqueous uranyl acetate for 2 h at room temperature, dehydrated with ethanol and embedded in Polybed 812 (Polyscience). Ultra-thin sections were cut, doubly stained with uranyl acetate and lead citrate and viewed using a JEM 1010 transmission electron microscope (JEOL).
Transgenic strains
The csk-1 cDNA was amplified by PCR from the yk657d3 plasmid (a gift from Dr Yuji Kohara) and cloned into pPD49.26 (a gift from Dr Andrew Fire) carrying the csk-1 promoter (a 5.0-kb fragment upstream of the transcription initiation site of the csk-1 gene) to generate Pcsk-1::csk-1. The csk-1 cDNA was fused in frame to the 3' end of gfp cDNA and cloned into pPD49.26 carrying the csk-1 promoter to generate Pcsk-1::gfp::csk-1. The pJM67 vector carrying the elt-2 promoter (5.1 kbp) and N-terminus (92 bp) was kindly provided by Dr J. McGhee and was used to generate Pelt-2::csk-1. The pPD96.48 (a gift from Dr Andrew Fire) vector was used to generate Pmyo-2::csk-1. To generate the kinase-deficient form of CSK-1, the Lys310 residue was substituted with Met to produce csk-1K310M. Transgenic lines were generated using standard techniques (Mello et al. 1991). To rescue growth defects, csk-1 or gfp::csk-1 in pPD49.26 or pPD96.48 was injected at 2 µg/mL into the csk-1 homozygote together with the lin-44::gfp injection marker (50 µg/mL) and csk-1K310M in pPD49.26 or Pelt-2::csk-1 was injected at 5 µg/mL into the csk-1/hT2[qIs48] heterozygote together with the lin-44::gfp injection marker (50 µg/mL). The growth defect was then scored in csk-1 (ov1) and csk-1 (tm1916) homozygotes carrying the extrachromosomal array.
Protein analysis
Caenorhabditis elegans larvae were lysed with Sonifier (Branson) or a tight-fitting Potter–Elvehjem homogenizer in an ice-cold buffer consisting of 25 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.15 M NaCl, 5% glyceorol, 1% NP40, 2% n-octyl-β-D-glucoside, 1 mM sodium orthovanadate, 20 mM NaF, 1 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin and 10 µg/mL trypsin inhibitor. Cell culture of HEK293T cells, gene transfection, immunoblotting, immunoprecipitation and in vitro kinase assays using recombinant cortactin purified from bacteria as a substrate were carried out as described previously (Hirose et al. 2003; Segawa et al. 2006; Yagi et al. 2007). Anti-pY416 antisera were generated by immunizing rabbits with a peptide (KLMEEDIpYEARTGAK) containing phosphorylated Tyr416, and was affinity-purified using a column (Affi-gel 10, Bio-Rad) conjugated with the antigen. Anti-SRC-1 antibody was previously described (Hirose et al. 2003). Antiphosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG were obtained from Zymed Laboratory.
Tissue staining
Larvae were fixed by conventional freeze-cracking and methanol–acetone method on a slide glass (Albertson 1984). The specimens were then rehydrated by passing through an acetone series (90%, 70%, 50% and 30%) at room temperature, and transferred to phosphate-buffered saline containing 0.5% Tween 20. The specimens were incubated with anti-pY416 antibody, followed by incubation with the secondary antibody conjugated with Alexa594.
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Acknowledgements |
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Footnotes |
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* Correspondence: okadam{at}biken.osaka-u.ac.jp
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References |
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Received: 19 September 2008
Accepted: 2 December 2008
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