Hueston JL et al. (2008), The C. elegans EMAP-like protein, ELP-1 is requ...

ECB-ART-40953

BMC Dev Biol 2008 Nov 17;8:110. doi: 10.1186/1471-213X-8-110.

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The C. elegans EMAP-like protein, ELP-1 is required for touch sensation and associates with microtubules and adhesion complexes.

Hueston JL , Herren GP , Cueva JG , Buechner M , Lundquist EA , Goodman MB , Suprenant KA .

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BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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Species referenced: Echinodermata
Genes referenced: emap LOC100887844 LOC100888955 LOC100893907 LOC105443877 LOC115919910 LOC594261 tubgcp2

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	Figure 1. ELP-1 domain structure. All members of the EMAP-like protein family are constructed with a Hydrophobic ELP (HELP) motif (PF03451) preceding a WD domain (PF00400). A COILED domain (PF05710) is predicted in C. elegans ELP-1 as well as in three of the five human EMLs. An additional doublecortin (DCX) domain (PF 03607) is predicted in fruit fly ELP-1. Accession numbers for the domains/motifs (parentheses) are provided from the Protein Family (PFAM) database [57]. The accession numbers for the human EMLs are as follows: EML1 [GenBank: NM_001008707], EML2 [GenBank: NM_012155], EML3 [GenBank: NM_153265], EML4 [GenBank: NM_019063], and EML5 [GenBank: NM_183387].
	Figure 2. Genetic and molecular characterization of elp-1. (A) The physical map of elp-1 is drawn in the reverse orientation. Two alternatively spliced transcripts, elp-1a and elp-1b, are shown with exons as solid boxes and introns as lines. We confirmed the identity of the PCR products by a diagnostic restriction digest with EcoR1 and BglII (Panel A, right). (B) Genetic structures of the elp-1::gfp expression constructs pKA99-1, pKA99-2 and pKA99-3. Exons are indicated by black boxes. The proteins encoded by these constructs are indicated as Pelp-1::GFP, ELP- 1Δ::NLS::GFP, and ELP-1::GFP, respectively in the Text. (C) Amino acid sequence of ELP-1a showing a potential alternative start site for elp-1(ok347) (black arrow), exon 5 aa sequence (magenta), HELP motif (green) and the beginning of the WD domain (red arrow). (D) PCR reaction with a forward primer in the deletion site and a reverse primer in exon 11. There is no detectable wild-type copy of the elp-1 gene in the deletion strain as evidenced by the absence of the 1105 bp band. (E) The elp-1(ok347) strain retains two mRNAs of approximately 2200 and 2100 bp.
	Figure 3. ELP-1::GFP is expressed in hypodermal cells at the 1 1/2-fold stage of development. Two focal planes within the same embryo are examined by differential interference contrast (DIC) (A, C) and fluorescent light microscopy (B, D). The bar represents 10 μm.

	Figure 5. ELP-1 is expressed in diverse muscle cellular junctions. (A) A triplet of dots surrounds the fusiform-shaped body wall muscle cell. ELP-1::GFP is located throughout the muscle arms (arrowheads) that make junctions with the ventral nerve cord. The nucleus is shown in this plane of focus. (B) ELP-1::GFP is located at the adhesion sites of the male-specific sex muscles (boxed insert). The auto-fluorescent spicules of the male tail are visible. A body wall muscle cell is observed below the sex-muscle adhesion sites. The cell is outlined with triplet dots. Dotted arrows show a linear expression pattern in this body wall muscle cell. Bar represents 10 μm.
	Figure 6. ELP-1 localizes to body wall muscle in hermaphrodites. (A) Near the muscle cell membrane, ELP-1::GFP is prominent in lines (small arrows) oriented with the longitudinal axis of the cell. ELP-1::GFP is also associated with an array of fluorescent filaments (jagged arrow). (B) Affinity-purified antibodies against ELP-1 also stain a linear array near the cell surface. Bar represents 10 μm. Within these "lines" there often is a repeating unit of fluorescence that is apparent in both transgenic animals (C) and in antibody-stained animals (D). Bar represents 2.5 μm. (E) Dense bodies (arrowheads) are shown at the muscle cell surface by phase contrast microscopy. ELP-1::GFP expression was examined via fluorescence light microscopy (F) and the two images taken at the same focal plane were overlaid in Panel G. Bar represents 1 μm. Muscle cells that were double-stained with antibodies against ELP-1 (H, J) and PAT-3/β-integrin (MH25) (I, and J) show overlapping staining patterns. The integrin puncta are superimposed upon the more linear staining pattern of ELP-1. When the membrane is removed by the freeze-cracking procedure (outlined in white in panel J), integrin is lost and the ELP-1 antigen remains. Bar represents 10 μm.
	Figure 7. Analysis of subcellular filaments in a single body wall muscle cell of a hermaphrodite. These three panels (A-C) were part of a Z-series of images that were taken every 0.2 μm from the muscle cell membrane towards the interior of the muscle cell. The first image in Panel A was taken approximately 0.8 μm from the cell surface (4 sections down from the surface). At this focal plane, ELP-1::GFP is associated with oblique linear striations overlap with the anti-β integrin (MH25) staining pattern shown in Figure 6. In successive focal planes (B and C) the filaments are no longer in linear arrays and have branched off into a criss-crossing array. Bar represents 10 μm.

	Figure 9. ELP-1 antigens co-pellet with paclitaxel-stabilized C. elegans microtubules. Paclitaxel-stabilized microtubules (Mt) were prepared from a mixed-stage worm extract (Ex) as described in the Materials and Methods. The panel on the left shows the protein complexity in a C. elegans extract (Ex, 25,000 g supernatant) and in a Paclitaxel-stabilized microtubule preparation (Mt, 25,000 g pellet). An ~100 kDa polypeptide and α and β tubulin are the most prominent proteins visualized in the microtubule preparation. Proteins were separated on an 8% acrylamide mini-gel (left panel) and probed with affinity-purified anti-ELP-1 antibodies on a nitrocellulose blot (right panel). Asterisk is next to the ~100 kDa band that cross-reacts with the anti-ELP-1 antibodies. Molecular masses × 10-3 (Mr) are shown to the left. Approximately 15 μg of protein was loaded in each lane of a 7% polyacrylamide SDS-page mini-gel.
	Figure 10. ELP-1 is needed for normal touch sensation. Bars are mean ± s.d. for at least three independent assays of 30–50 animals each. RNAi knockdown of elp-1 in the lin-15b;eri-1 background induces a stronger touch-insensitive phenotype (66 ± 11%, mean ± s.d.) than does partial deletion of elp-1(ok347) (24 ± 5%) and is similar to the defect found in ok347/ozDf1 heterozygotes (82 ± 6%). Percentage of touch-insensitive animals for the remaining genotypes and RNAi treatments are as follows: wild-type (12 ± 0%) and lin-15b;eri-1 without RNAi (19 ± 6%). Tests were conducted blind to genotype or RNAi treatment. *P < 0.001 Fisher's Exact test.
	Figure 11. Three-dimensional sketch illustrating the relationship of ELP-1-associated microtubules and the dense bodies (green). A transverse section through a single body wall muscle cell is shown. The thin filaments, which are attached to the dense bodies, and the thick filaments attached to the M-lines, have been eliminated from the sketch for clarity. ELP-1 associated microtubules (magenta) are associated with the apical portion of the dense body. Neighbouring dense bodies are linked by microtubules giving rise to the linear staining patterns in Figure 6. The microtubules extend into the cytosol that contains the nucleus, mitochondria, and other membranes (not shown).

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