CPNA-1, a copine domain protein, is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans

Abstract

We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1-null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.

FIGURE 1:

Gene model for cpna-1 and alignment of CPNA-1 with human and mouse homologues. (A) Exon–intron organization of the cpna-1 gene; thin lines, introns; thick black boxes, exons. The last row includes all the exons, with labeling of key features of the gene or protein. cpna-1 has four predicted splice isoforms, with the largest isoform (isoform b) spanning 21,834 nucleotides. cpna-1b codes for a protein of 1107 amino acids and contains a predicted amino-terminal transmembrane domain and a carboxy-terminal copine domain. The gk266 allele has a 9–base pair insertion at the 3′ end of intron 5, followed by a 393–base pair deletion that extends into the 5′ end of exon 6. Regions used for antibody production and coding for the predicted transmembrane domain (TM) and copine domain are indicated. The black bar at the bottom right represents 1 kb. (B) When aligned with human and mouse homologues, CPNA-1 does not align at its amino terminus due to its longer length, but there is strong conservation among all of the proteins in the copine domain and surrounding region. In addition, key residues of a MIDAS site are conserved in each aligned protein and are indicated by open rectangles. Asterisks indicate identical amino acids, colons indicate conserved substitutions, and periods indicate semiconserved substitutions.

FIGURE 2:

Copine family proteins in C. elegans. (A) The C. elegans genome has seven genes (1–7) that encode proteins containing copine domains. The protein names shown are based on gene names (e.g., CPNA-1) and sequence names (e.g., F31D5.3). Percentages refer to the percentage of identical amino acid residues in each copine domain as compared with the copine domain of CPNA-1. The numbers in parentheses after the total number of amino acid residues in each protein denote the positions of copine domains. Note that only NRA-1 and GEM-4 are “typical,” in that they also contain C2 domains. Only CPNA-1 also has a predicted transmembrane domain. In addition, some isoforms of CPNA-1 and CPNA-2 are not shown. These isoforms, predicted on WormBase (CPNA-1c, CPNA-1d, CPNA-2b, and CPNA-2d), lack copine domains. The rightmost column represents results of yeast two-hybrid assays in which UNC-89 Ig1–5 was tested for interaction with copine domains from each of the seven C. elegans genes. +, interaction; –, no interaction. (B) Multisequence alignment of the copine domains in the seven C. elegans copine domain proteins. Green shading indicates residues present in all seven copine proteins, and yellow shading indicates residues present in four or more copine proteins. “Consensus” represents the green (uppercase) and yellow (lowercase) shaded residues. Asterisks denote conserved residues that were mutated in the copine domain of CPNA-1 and tested for binding to UNC-89 and UNC-96 (Figure 7).

FIGURE 3:

Immunolocalization of CPNA-1 to adult muscle M-lines and dense bodies. (A) Wild-type adults were costained with anti-CPNA-1 and anti-UNC-89 (first row) or anti-CPNA-1 and anti–α-actinin (second row); a transgenic line carrying UNC-112::GFP was also stained with anti–CPNA-1 (third row); costaining with CPNA-1 and PAT-6 is shown in the fourth row. CPNA-1 clearly localizes to both M-lines (colocalizing with the M-line protein UNC-89) and dense bodies (colocalizing with the dense body protein α-actinin). At least some CPNA-1 localizes near the muscle cell membrane, since some CPNA-1 colocalizes with the membrane-proximal proteins UNC-112 and PAT-6. Colocalization is indicated by white (overlap of green and magenta). Bar, 10 μm. (B) Major components of the muscle cell are labeled in the TEM image (left). Thin (small dots) and thick (larger dots) filaments can be observed in the Z-plane to the sides of the dense body. Gold particles (dark black dots) show CPNA-1::YFP within (arrow) and surrounding the dense body (arrowhead; middle). A control TEM section incubated with complete chicken IgY and secondary antibody conjugated with gold particles (right) has random gold localization at much lower levels (arrow). Bar, 100 nm.

FIGURE 4:

Characterization of the cpna-1–null phenotype and localization of CPNA-1::YFP in body-wall muscle. Wild-type C. elegans proceeds through embryogenesis to hatch as a first-larval-stage animal (A), whereas a cpna-1–null animal of the same age arrests elongation at the twofold stage of embryogenesis (B). CPNA-1::YFP is localized to dense body (arrows) and M-line integrin adhesion complexes (arrowheads) in adult body-wall muscle (C). Colocalization of CPNA-1::YFP is observed (E) in worms immunostained for PAT-3 (β-integrin; D), a key constituent of the same integrin adhesion structures. In E note that CPNA-1 is also localized to areas directly adjacent to the dense body, observed by the green localization pattern around the dense body; stronger M-line staining by PAT-3 (red) overshadows that of CPNA-1 (arrowhead). Compared to wild-type staining (F, H, J, L), when cpna-1 embryos are immunostained with antisera for PAT-3 (G), DEB-1 (I), PAT-4 (K), and PAT-6 (M), only slight disorganization is seen and each of the four proteins is still localized to the appropriate integrin adhesion sites (arrowheads). In pre–1.5-fold embryos, UNC-89 is able to localize to M-lines in both wild-type embryos (N) and cpna-1–null embryos (O), but after the 1.5-fold stage, wild-type embryos (P) show normal UNC-89 localization, whereas cpna-1–null embryos show UNC-89 mislocalized into large foci within muscle cells (Q; arrows). Similarly, MYO-3 is able to organize into nascent thick filaments in both pre–1.5-fold wild-type (R) and cpna-1–null (S) embryos, but after the 1.5-fold stage, wild-type embryos (T) show normal MYO-3 localization to nascent thick filaments, whereas in cpna-1–null embryos, MYO-3 is disorganized and mislocalized into large accumulations within the muscle cells (U; arrows). Bars, (A and B) 20 μm, (C–U) 2 μm.

FIGURE 5:

Immunolocalization of CPNA-1 in the null background of other Pat genes. In late-stage, wild-type embryos, CPNA-1 is organized into bands of dense bodies and M-lines in the muscle cell quadrants (A; arrowheads). In null pat-3 embryos, however, CPNA-1 is mislocalized into large aggregates within body-wall muscle cells (B; arrows). Similarly, large accumulations of CPNA-1 are also seen in null embryos for pat-4 (C) and pat-6 (D; arrows), although in some pat-6 embryos, CPNA-1 mislocalized to a lesser extent than for pat-3 and pat-4 embryos (E; arrow). In arrested twofold embryos lacking MYO-3, however, CPNA-1 is organized properly into bands of dense bodies and M-lines (F), as it is in wild-type embryos (A, arrowheads). Bar, 2 μm.

FIGURE 6:

CPNA-1 interacts with M-line proteins UNC-89, PAT-6, LIM-9, SCPL-1b, and UNC-96. (A) Schematic representation of domains within the largest isoform of UNC-89, and indication that Ig1-5 was used as bait to screen a yeast two-hybrid library. Two positive preys representing CPNA-1 were recovered, as indicated. CPNA-1, isoform b, has a predicted transmembrane domain (TM) and a copine domain (copine). (B) When CPNA-1 was used to test for interaction with the other 16 clones that fully cover UNC-89, interaction was only found with Ig1–5. (C) Domain mapping of UNC-89 Ig1–5 shows that Ig1–3 are minimally required for interaction with CPNA-1 (173–1107). (D–H) Proteins found to interact with CPNA-1 by screening a collection of known M-line and dense body proteins using the two-hybrid method. (D–G) Depiction of the results of domain mapping to determine the minimal region (indicated as a blue bar) of each protein required for interaction with the indicated regions of CPNA-1. (H) Summary of the results showing protein domains of each protein and which regions are involved in the interactions.

FIGURE 7:

Point mutations in the copine domain of CPNA-1 affects its binding to UNC-89 and UNC-96; evidence for a ternary complex containing PAT-6 and portions of CPNA-1 and UNC-89. (A) The copine domain of CPNA-1 has distinct binding sites for UNC-89 and UNC-96. Three of the 12 conserved residues of copine domains (indicated with asterisks in Figure 2B) were changed: G922 was changed to V; Y985 was changed to A and F, respectively; S1015 was changed to A. The G922V mutation eliminates binding to UNC-89 but not UNC-96. Similarly, the S1015A mutation eliminates the binding to UNC-96 but not UNC-89. (B) UNC-89 Ig1-5 as bait was coexpressed with HA-tagged CPNA-1 (residues 173–1107; or empty vector as control) and PAT-6 (full length) as prey. +, growth on –Ade plates; –, no growth on –Ade plates. Right, the yeast growth on –Ade plates from each experiment from three independent colonies.

FIGURE 8:

In adult muscle, PAT-6 is required for localization of CPNA-1. RNAi was used to knock down pat-6 beginning at the L1 larval stage, and the resulting adults were immunostained for PAT-6 and CPNA-1. Top and bottom, portions of body-wall muscle from two such animals. In muscle cells in which PAT-6 was knocked down, CPNA-1 is found in abnormal accumulations (arrowhead) or mislocalized to the edge of the muscle cell near the muscle cell membrane (arrows). Bar, 10 μm.

FIGURE 9:

Analysis of mutants places the M-line proteins UNC-96, LIM-9, and SCPL-1 downstream of CPNA-1 in late larval or adult muscle. The indicated loss-of-function mutants or RNAi animals were coimmunostained with anti–CPNA-1 and anti–α-actinin. The localization of CPNA-1 at M-lines (indicated by yellow arrows) is unaffected by the absence or reduced levels of UNC-89, SCPL-1, UNC-96, or LIM-9. Note that the unc-89–mutant allele su75, which lacks all large UNC-89 isoforms, lacks CPNA-1–binding sites. Bar, 10 μm.

FIGURE 10:

Model suggested by this study to explain the role of CPNA-1 in assembly of integrin adhesion complexes. UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), and PAT-6 (actopaxin) form a four-protein complex associated with the cytoplasmic tail of β-integrin. Lin et al. (2003) showed that the second CH domain of PAT-6 interacts with PAT-4. Here we show that PAT-6 (actopaxin) is needed to localize CPNA-1 to the muscle integrin adhesion complexes. CPNA-1, in turn, is needed for proper localization of UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96 to M-lines. We have not yet identified CPNA-1–interacting proteins that are specific for dense bodies. Yellow indicates proteins that are located at both M-lines and dense bodies and whose loss of function result in Pat embryonic lethality. Red indicates proteins that are located at M-lines and whose loss of function results in adult muscle phenotypes.