- Research article
- Open Access
The C. elegansEMAP-like protein, ELP-1 is required for touch sensation and associates with microtubules and adhesion complexes
© Hueston et al; licensee BioMed Central Ltd. 2008
Received: 31 July 2008
Accepted: 17 November 2008
Published: 17 November 2008
The founding member of the EMAP-like protein family is the Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs. The EMAP-like protein family has five members in mammals (EML1 through EML5) and only one in both Drosophila (ELP-1) and C. elegans (ELP-1). Biochemical studies of sea urchin EMAP and vertebrate EMLs implicate these proteins in the regulation of microtubule stability. So far, however, the physiological function of this protein family remains unknown.
We examined the expression pattern of C. elegans ELP-1 by means of transgenic gene expression in living embryos and adults, and by immunolocalization with an ELP-1-specific antibody in fixed tissues. In embryos, ELP-1 is expressed in the hypodermis. In larvae and adults, ELP-1 is expressed in the body wall, spermatheca and vulval muscles, intestine, and hypodermal seam cells. In muscle, ELP-1 is associated with adhesion complexes near the cell surface and is bound to a criss-crossing network of microtubules in the cytoplasm. ELP-1 is also expressed in a subset of mechanoreceptor neurons, including the ray neurons in the male tail, microtubule-rich touch receptor neurons, and the six ciliated IL1 neurons. This restricted localization in the nervous system implies that ELP-1 plays a role in mechanotransmission. Consistent with this idea, decreasing ELP-1 expression decreases sensitivity to gentle touch applied to the body wall.
These data imply that ELP-1 may play an important role during the transmission of forces and signals between the body surface and both muscle cells and touch receptor neurons.
Microtubule networks are necessary for a variety of essential processes including cell polarity, migration, division and mechanotransduction [1–3]. Microtubule formation and function is regulated by a variety of proteins that mediate the structural and regulatory interactions between these microtubules and their cargo [4, 5], and catalyze their assembly and disassembly . In this report we examine the tissue-specific expression and the subcellular location of ELP-1, an EMAP-like protein from C. elegans and demonstrate that ELP-1 contributes to touch-sensitivity in a subset of mechanoreceptor neurons.
The founding member of the EMAP-like protein family is the 75 kDa Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs . Genes that code for EMAP-like proteins are conserved in the genomes of nematodes, chordates, insects, echinoderms, and platyhelminthes and several EMAP-like proteins have been shown to bind to microtubules in vitro and in vivo [8–13].
The in vivo function of EMAP and EMAP-like proteins is unknown. However there are indications that loss or alteration of EMAP function may lead to human disease. Human EML2 RNA is abundant in cancer cell lines including chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), colorectal adenocarcinoma (SW480), and lung carcinoma (A549) . Furthermore, in certain patients with T-cell acute lymphoblastic leukemia (TALL), the gene encoding the nonreceptor protein kinase (c-ABL1) is fused to the EML1 gene on chromosome 14, which causes expression of an EML1-ABL1 fusion protein, that functions as a dysregulated tyrosine kinase . The EML1-ABL1 fusion protein constitutively activates the ERK, Stat5, and Src signalling pathways. The N-terminal coiled-coil domain of EML1 is required for kinase activation, which suggests that oligomerization of EML1 is required for the function of EML1-ABL1 fusion proteins.
Kinase oncogenes are not restricted to fusions with EML1. EML4 fusions with the anaplastic lymphoma kinase (ALK) occur in a subset of non-small cell lung cancers and adenocarcinomas of the lungs [15, 16]. The amino portion of EML4 (residues 1–496) is fused to residues 1058–1620 of the intracellular domain of the ALK tyrosine kinase. The basic amino terminal domain of EML4 is critical to the catalytic activity of the ALK fusion . These results imply that EML-kinase fusions and rearrangements may underlie other acquired solid tumours and blood related cancers.
The conservation of the EMAP-like protein family amongst metazoans and the direct correlation between EML translocations and cancer indicates that this novel protein family may perform an important function in cells and tissues. To begin to understand the function of EMAP and EMAP-like proteins we undertook a molecular and cytological analysis of the elp-1 gene encoded by the ORF F38A6.2 in Caenorhabditis elegans. We determined whether ELP-1 bound to microtubules in vitro and in vivo. Furthermore, we took advantage of the transparency of the worm to examine the expression pattern of an ELP-1::GFP fusion protein in embryos and in adults. Our results indicate that ELP-1 is expressed in cells that make productive interactions with the extracellular matrix, including but not limited to the hypodermis, body wall muscles, male-specific sex muscles, and the microtubule-rich touch receptor neurons. In addition, our behavioural studies show that wild-type levels of ELP-1 are needed for touch sensation in the worm.
elp-1 encodes the sole C. elegansmember of the EMAP-like protein family
Exon 5 is conserved in nematodes but not detected in other EMAP-like proteins. Twenty-four of the twenty-seven predicted amino acids (88%) are identical in Caenorhabditis briggsae and C. elegans. All three substitutions are conservative (V15I, I16V, and S19N; numbers refer to the 27 amino acids coded by this exon). This domain shows a slightly higher level of sequence conservation when compared to the HELP domain protein sequence (81% identical) and the full-length protein sequence (80% identical) of C. elegans and C. briggsae. Although the function of this 27 amino acid region, rich in potential phosphorylation sites, is unknown, these observations indicate that exon 5 may be important for a nematode-specific function.
To learn more about the function of elp-1, we obtained a deletion allele, ok347, from the C. elegans Knockout Consortium. The ok347 allele deletes 1301 nucleotides, spanning intron 1 through intron 4 (Figure 2A, 2D). Starting from the ATG of the ELP-1a open reading frame, ok347 is missing base pairs 421 to 1721. This genome deletion does not remove ELP-1 function however, since elp-1(ok347) worms retain two nearly full-length transcripts (Figure 2E). These transcripts are predicted to encode two proteins that include both the HELP and WD repeat domains, a finding which implies that these proteins could retain partial function.
Expression patterns of the elp-1gene in embryos and adults
To learn how ELP-1 contributes to development, physiology and behaviour, we examined the expression pattern of the elp-1 gene. Three GFP reporter constructs were generated: an ELP-1 promoter construct [pKA99-3], a truncated ELP-1 with an NLS [pKA99-2]; and a full-length ELP-1 [pKA99-1] (Figure 2B). All three constructs were expressed in the same cells and tissues. For clarity, the protein expressed from the pKA99-1 construct will be noted as ELP- 1Δ::NLS::GFP and the full-length protein expressed from the pKA99-2 construct will be written as ELP-1::GFP.
ELP-1 is expressed in a subset of mechanoreceptor neurons
As indicated above ELP-1::GFP and ELP-1Δ::NLS::GFP were expressed in cells that were identified as mechanoreceptor neurons (Figures 4E &4F). Although the NLS did not restrict expression to the nucleus, it was generally easier to identify neurons with this construct than with the full-length ELP-1::GFP construct. Specifically, ELP-1Δ::NLS::GFP was found in the six touch receptor neurons (ALML/R, AVM, PVM, and PLML/R) responsible for detecting light touch applied to the body surface (Figure 4E, F) . These cells were identified by the position of their cell bodies and the long nerve cell processes that extend anteriorly or posteriorly over half of the body length. Touch receptor neurons (TRNs) are not ciliated or organized into sensilla, but extend neurites along the midline and lateral line of the worm with their dendritic receptors lying within 150 nm of the inner border of the cuticle .
Quantification of IL1 neurons in a deg-1 mutant background.
Total number of worms
5.4 + 0.7
deg-1(u38); lkEx4[Pelp-1::elp-1(Δ11–16)::nls::gfp; rol-6(su1006)]
1.9 + 1.2
In addition to the mechanoreceptor neurons described above, ELP-1::GFP was associated with the nine pairs of rays of the male tale (Figure 4H). The male tale is a sensory apparatus used to find the hermaphrodite vulva during mating. The relatively diffuse and broad band of fluorescence appears to originate from the hypodermal cells (hyp7) and one or more of the neuronal cells of the ray (RnA, RnB).
ELP-1 is associated with adhesion sites
Figure 5B shows a male with ELP-1::GFP associated with repeating focal attachment points in the sex-specific muscles of the worm. These muscles are located in the posterior end of the worm and function in various phases of male mating behaviour . ELP-1 expression was prominent at the adhesion sites located at the sarcolemma. Unlike the dense body adhesion sites described below, these sites occur at the muscle ends in a plane perpendicular to the long axis of the sarcomere.
Body wall muscle cells are anchored along their length to the basement membrane by the dense bodies, finger-like projections analogous to the vertebrate Z-lines. Anchorage of actin in the myofilament lattice to the dense bodies is necessary for force transduction in body wall muscle. Dense body puncta are distinctive because they are aligned in a row that runs at a 6-degree pitch from the longitudinal axis of the muscle cell .
In Figure 6E, individual dense bodies are shown as phase-dark puncta. The fluorescent image of ELP-1::GFP was examined at the same focal plane (Figure 6F) and these micrographs were overlaid and shown in Figure 6G. The ELP-1::GFP fluorescence slightly overlaps the edges of the dense bodies but is not superimposable with the dense bodies at this angle. These results indicate that ELP-1 is unlikely to be a component of the dense bodies.
To further examine the association of ELP-1 with dense bodies, we double-stained muscle cells with anti-ELP-1 antibodies and a monoclonal antibody MH25 against β integrin (PAT-3), an integral membrane component that anchors the dense bodies to the sarcolemma. Figure 6 H-J shows that integrin and ELP-1 have overlapping staining patterns at this level of resolution. Because of the relatively impenetrable cuticle that surrounds the nematode, these worms were frozen and cracked open prior to antibody staining. Occasionally a portion of the muscle cell membrane was removed carrying along its complement of dense bodies (see white outline in Figure 6J). In areas lacking dense bodies, ELP-1::GFP was sometimes lost. However most of the ELP-1::GFP was retained in a linear pattern. These results indicate that the membrane and dense bodies can be separated from the ELP-1::GFP.
ELP-1 is associated with microtubules
ELP-1 is needed for normal touch-sensitivity
EMAP and EMAP-like proteins are conserved in nematodes, insects, echinoderms and vertebrates [8, 26–31]. Most members of the superfamily associate with microtubules [8, 9, 11–13] and several are implicated in regulating microtubule stability during mitosis [8, 9, 11, 27]. Their expression is not limited to dividing cells, however. The function of EMAP-like proteins in post-mitotic cells such as neurons is poorly understood. To learn more, we combined molecular and behavioural genetics with cell biological approaches to analyze ELP-1, the sole EMAP-like protein in the C. elegans genome. Because all 959 somatic cells in each adult hermaphrodite are post-mitotic, this analysis offers new insight into the cell biology and physiology of ELP-1 in post-mitotic cells and tissues.
We show here that ELP-1 is prominently expressed in cells that make productive interactions with the cuticle. In males and hermaphrodites, these include mechanoreceptor neurons such as the TRNs and diverse muscle cell types. In males, ELP-1 is found in the hypodermal cells and neurons of the male sensory rays. Additionally, ELP-1 is found in the cell bodies, throughout the neuronal processes, and in the ciliated endings of all six IL1 neurons. The IL1 neurons are putative mechanoreceptor neurons needed for wild-type foraging movements and for sensing touch applied to the nose .
ELP-1 is a promising candidate for regulating microtubule function in mechanoreceptor cells. For example, the TRNs are unique in that their sensory processes contain 15-protofilament microtubules rather than 11-protofilament microtubules observed in the majority of C. elegans cells . Approximately 450 of these large-diameter microtubules are bundled together and constrained to move as a single structure by distinct 10 nm filaments [19, 32, 34]. The bundles are also linked to the plasma membrane through a series of 14 nm filaments . ELP-1 is an attractive candidate to bundle microtubules. In addition, the HELP motif, rich in hydrophobic amino acids, could link the bundles to the plasma membrane. In either scenario, ELP-1 would contribute to TRN function by amplifying the forces generated at the cuticle. Consistent with this idea, reducing ELP-1 levels decreased touch-sensitivity.
In addition to mechanoreceptor neurons, ELP-1 is expressed in cells that utilize cadherin-based apical junctions and or fibrous organelles for cell adhesion and attachment to the basal lamina. These include the cells of the intestine, body wall, vulval, and male-specific sex muscles, hypodermis and seam cells . The hemidesomosome-like fibrous organelles, formed by the hypodermis, transmit cuticle deformation to the touch receptor neurons and muscle tension to the cuticle [36, 37]. Microtubules are interspersed with actin filaments near these fibrous organelles, however it is not known how they might be anchored to the plasma membrane . We speculate that ELP-1 is involved in the anchoring or bundling of microtubules to the plasma membrane and perhaps the transmission of forces therein.
It is intriguing that ELP-1 is associated with adhesion sites similar to mammalian focal adhesions. It has been known for some time that microtubules can locate or target focal adhesions  and that depolymerization of microtubules enhance actomyosin-based cell contractility . The increase in cell contractility is mediated by GEF-H1 [43, 44], a microtubule-associated guanine nucleotide exchange factor that activates the small G-protein, RhoA . Upon microtubule depolymerization, GEF H1 is released and activates the Rho-associated kinase (ROCK) that phosphorylates the myosin regulatory light chain (MLC) resulting in increased contractility [46, 47]. Whether a similar pathway is functional in C. elegans muscle remains to be determined.
In C. elegans, ELP-1 is the sole member of a highly conserved family of metazoan microtubule-binding proteins. In adults, ELP-1 is expressed in post-mitotic cells that make productive interactions with the cuticle such as body wall muscle and mechanoreceptor neurons, suggesting a role in force generation and sensing. In support of this idea, disrupting ELP-1 expression by mutation and RNAi renders animals insensitive to gentle body touch. This study is a critical first step toward elucidating the function of EMAP-like proteins in post-mitotic cells, including many mechanoreceptor neurons.
Stock solutions of levamisole, DiD (1,1'-dioctadecyl-3, 3,3', 3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt, Invitrogen-Molecular Probes, Carlsbad, CA), paclitaxel (Taxol™, Merck-Calbiochem, San Diego, CA), and nocodazole were stored at -20°C and prepared as follows (solvent, concentration): levamisole (M9 buffer, 100 mM); DiD (1 mg/ml, dimethyl formamide); paclitaxel (10 mM, dimethylsulfoxide); and, nocodazole (10 mM, dimethylsulfoxide).
C. elegansstrains and culture
Wild-type worms (N2, Bristol) were grown on NGM plates seeded with the E. coli strain OP50 at 20°C, unless otherwise indicated [48, 49]. MT8189 lin-15(n765ts) X, KP3948 eri-1(mg366) IV; lin-15B(n744) X, TU38 deg-1(u38) X, RB639 elp-1(ok347) V, and BS518 ozDf1/sdc-3(y52y180) unc-76(e911)V animals were supplied by the Caenorhabditis Genetics Center, Minneapolis. Genotypes of new strains generated to examine tissue specific expression include lkEx4[P elp-1 ::elp-1(exon Δ11–16)::nls::gfp (pKA99-1); rol-6(su1006)] (KA15-17), (lin-15(n765); lkEx1[P elp-1 ::elp-1::gfp (pKA99-2); lin-15(+)]) (KA6-8), (lin-15(n765); lkEx3[P elp-1 ::gfp (pKA99-3); lin-15(+)]), (KA14) and deg-1(u38)X;lkEx4[P elp-1 ::elp-1::gfp (pKA99-2); rol-6(su1006)] (KA37).
The elp-1(ok347) strain, RB639, was obtained from the C. elegans Gene Knockout Consortium at the Oklahoma Medical Research Foundation (Oklahoma City, OK), out-crossed four times and the exact location of the deletion was confirmed by sequencing a PCR product that spanned the deletion (Figure 2D).
Non-complementation of elp-1(ok347) allele and ozDf1deficiency
To test whether elp-1(ok347) was a null or hypomorphic allele of elp-1, the elp-1(ok347) allele was placed in trans to ozDf1. The deficiency ozDf1 maps to linkage group V (left end: 10.318; right end 25.1193) and deletes dpy-21, pro-3, srf-4, and unc-51  and covers elp-1. The absence of elp-1 in strain BS518 was confirmed via PCR.
Western blotting and ELP-1 antibodies
A 2595-bp cDNA clone for elp-1b (yk209e10) was kindly provided by Y. Kohara at the DNA Data Bank of Japan http://www.ddbj.nig.ac.jp. The insert was sub-cloned into a pET14b expression vector (Novagen, Madison, WI). The 6-His-tagged ELP-1b (aa1-864) was expressed in E. coli (BL21(DE3)) and purified under denaturing conditions via chromatography on an iminodiacetic acid Sepharose column . Antibodies were generated in rabbits against the purified 6-His-tagged ELP-1b immunogen and affinity-purified as specified .
Proteins were separated on 8% acrylamide mini-gels, transferred to nitrocellulose, probed with affinity-purified anti-ELP-1 antibodies (1:1000), and visualized with alkaline phosphatase-conjugated secondary antibodies and chemiluminescence.
Vectors that express ELP-1 fused to GFP were created from the Fire Lab Vectors http://www.addgene.org by PCR amplification or restriction digestion of the genomic cosmid, F38A6.2. Standard molecular techniques were used and all PCR products were cloned and sequenced to be certain that no errors were introduced during amplification. The P elp-1 ::elp-1(Δ11–16)::nls::gfp construct (pKA99-1) was generated by ligating a SphI-BamHI genomic fragment that contains 4 kb of the elp-1 5'UTR region (P elp-1 ) and 3 kb of the elp-1 gene (exons 1–10) into the pPD95.67 vector upstream of a nuclear localization sequence (NLS) and the gfp gene. The construct expresses a truncated ELP-1 protein fused to GFP with an NLS under the endogenous elp-1 promoter. The P elp-1 ::elp-1::gfp construct (pKA99-2) contains 9 kb of the elp-1 gene with the endogenous elp-1 promoter (P elp-1 ) inserted into the SphI-XbaI site in the pPD95.75 vector and expresses the full-length ELP-1 protein (exons 1–16) fused to GFP. elp-1 was amplified from the F38A6 cosmid with the Expand Long Template PCR system (Roche-Boehringer Mannheim; Alameda, CA) with the primers, EMAPUSGFP1-5', corresponding to sequence upstream of the SphI genomic restriction site (5'-AACACCGAACTTGATGAAATATTCGGTGCAAC-3') and EMAPSTP2GFP2-3' (5'CCTCTCTAGATCCGCTTCCAGGCACCATTCAAAAACCGAATTATCAG-3'), corresponding to the sequence at the 3' end of the coding region of the gene, and substituting an XbaI restriction site for the stop codon. The P elp-1 ::gfp construct (pKA99-3) expresses GFP under the control of the 4 kb elp-1 promoter region. For this construct the promoter region was amplified with primer, EMAPUSGFP1-5' and primer, EMAPSTRTGFP1-3' (5'-CGGGGATCCTCCATTTTTTTGAAGAATTTTTGCAAATTTTCTCCTGCAAC-3'), corresponding to sequence at the start site of the gene, with a BamHI tag (GGGATCC) used to ligate into the pPD95.75 vector.
Transgenic strains carrying each of these extrachromosomal arrays (200 ng/μl) were established with rol-6(su1006) (100 ng/μl) as a transformation marker in N2 worms or with the rescue reporter lin-15(+) (100 ng/μl) injected into a lin-15(n765ts) mutant strain. Three independent array-bearing lines were isolated for each array with each line showing the same tissue expression pattern.
DiD dye-filling protocol
The inner labial 2 (IL2) sensory neurons were labelled with 10 μg/ml DiD in M9 buffer containing 50 mM calcium acetate (personal communication from Elizabeth Ryder, Worcester Polytechnic Institute). After incubation for one hour, the animals were washed twice with dH2O and put onto an OP50 feeding plate for 1 hr to allow the excess DiD to pass through the intestines. Worms were washed off the plate with M9 and placed on 2% agar pads in a drop of M9 buffer containing 10 mM sodium azide, covered with a coverslip, and examined via fluorescence light microscopy.
Microtubules were disrupted in adult worms via a protocol adapted from a previous study of the microtubule binding protein ZYG-9 . ELP-1::GFP expressing worms were gently flattened between a coverslip and slide to extrude a portion of the intestine. Squashed worms were perfused three times with a small volume (10 μl) of M9 buffer containing levamisole (25 mM) and observed for ~15 minutes. We disrupted microtubules in this preparation using nocodazole, an antagonist of microtubule polymerization. Nocodazole (10 μM) was applied by superfusing squashed worms with 10 μl of solution.
Immunofluorescent staining of worms was carried out following the methods of Miller and Shakes (1995) . A mixed population of worms was freeze-cracked by immersion in liquid nitrogen, fixed in ice-cold methanol (15 min) and in ice-cold acetone (10 min). Animals were washed twice in PBT [PBS containing 0.1% Triton-X (v/v) and 0.1% BSA (w/v)] and stained with primary antibodies overnight at 4°C. The primary antibodies used were the affinity-purified anti-ELP-1 antibodies (diluted 1:50 in PBST with BSA) and a mouse monoclonal anti-PAT-3 antibody MH25 (a gift from Michelle Hresko, Washington University School of Medicine, St. Louis, ) (diluted 1:250 in PBST). The worms were washed three times in PBST and then incubated with the secondary antibodies: Cy2-conjugated donkey anti-rabbit [1:250] or Cy3-conjugated donkey anti-mouse [1:250] in PBT buffer overnight at 4°C. Lastly, the slides were washed three times in 1× PBT and mounted in PBT buffer.
C. elegans were examined by means of bright field, differential interference contrast (DIC), and fluorescence microscopy . Worms were anesthetized with a drop of 1% 1-phenoxy-2-propanol in M9 buffer and placed on 2% agar pads on glass slides. The highest resolution images were taken with a 60× 1.4 NA Plan-Apochromat Nikon objective and captured with a Hamamatsu Orca-ER camera (Hamamatsu City, Japan) driven by the OpenLab software V3 (Improvision, Lexington, MA). Individual files were compiled and contrast-balanced when necessary using Adobe PhotoShop. All the images for each dataset were treated the same.
RNAi-mediated gene knockdown
Recently hatched larvae of the RNAi-hypersensitive mutant, lin-15b;eri-1, were fed RNAi-expressing bacteria from the Ahringer RNAi feeding library .
Young adult animals were alternately touched on the anterior and posterior region of the body a total of ten times with an eyebrow hair glued to a toothpick . Animals that failed to respond to all ten touches were considered touch-insensitive. Three independent assays of 30–50 animals were tested. Tests were conducted blind to RNAi treatment or to genotype.
We thank G. Moulder for the elp-1(ok347) allele, Y. Kohara for the cDNA clones, Wendy Picking for help with affinity chromatography, and Michelle Hresko for MH25 antibodies. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. This research supported by grants from the National Science Foundation (MCB-9982377 to KAS), the National Institutes of Health (NS047715 to MBG; NS40945 to EAL; DK55526 to MJB), and a fellowship from the McKnight Foundation (MBG). JLH was the recipient of a NIH predoctoral traineeship (GM08545).
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