Two LIM domain proteins and UNC-96 link UNC-97/pinch to myosin thick filaments in Caenorhabditis elegans muscle

Abstract

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

Figure 1.

Domain mapping for each interacting pair of proteins by the yeast two-hybrid system. The columns contain the results of testing for interaction. ++, strong growth; +, growth; +/−, poor growth; and −, no growth on the selection media. ND, not done. In A–D, the top lines represent schematics of the indicated proteins in which green bars denote LIM domains, yellow bars denote PDZ domains, and a red box denotes a PET domain. (A) Mapping of binding sites in UNC-97 for UNC-95, LIM-8, and LIM-9. Blue bars represent deletion constructs of UNC-97 as bait. Prey plasmids of UNC-95, LIM-8, and LIM-9 were tested for interaction with UNC-97 deletion series baits. (B) Mapping of the binding site in UNC-95 for UNC-97. Blue bars represent deletion constructs of UNC-95 as prey. Bait plasmids of UNC-97 were examined for interaction with UNC-95 deletion series preys. (C) Mapping of the binding site in LIM-8 for UNC-96, UNC-97, and MHC A. LIM-8a contains 1004 amino acid residues, and LIM-8b contains 868 amino acid residues. Blue bars represent deletion constructs of LIM-8 as bait for UNC-96 and MHC A, and prey for UNC-97. Prey plasmids of UNC-96 and MHC A, and the bait plasmid of UNC-97 were examined for interaction with LIM-8 deletion series baits and preys. (D) Mapping of the binding site in LIM-9 for UNC-96 and UNC-97. Blue bars represent deletion constructs of LIM-9 as bait for UNC-96 and prey for UNC-97. Prey plasmids of UNC-96 and a bait plasmid of UNC-97 were examined for interaction with LIM-9 deletion series baits and preys. (E) Mapping of the binding site in UNC-96 for LIM-8, LIM-9, and MHC A. Top schematic is the UNC-96 protein structure with amino acids numbers displayed. Blue bars represent deletion constructs of UNC-96 as bait for MHC A and prey for LIM-8 and LIM-9. Prey plasmids of MHC A and bait plasmids of LIM-8 and LIM-9 were examined for interaction with UNC-96 deletion series baits and preys. Because some part of UNC-96 can activate transcription in yeast cells, two deletion constructs as baits could not be used to examine binding to MHC A (shown as ND). (F) Mapping of the binding site in MHC A for UNC-96 and LIM-8. Top schematic denotes the C-terminal half of MHC A, in which the black bar represents the coiled-coil domain, the black line the nonhelical tail, and amino acids numbers are shown. Blue bars represent deletion constructs of MHC A as prey. Bait plasmids of UNC-96 and LIM-8 were examined for interaction with MHC A deletion series preys.

Figure 2.

In vitro binding of each interacting pair using purified proteins. In each subfigure, the column of numbers indicates the positions of molecular weight markers in kilodaltons. In A and B, a + means included in assay and a − means not included in assay. (A) Glutathione bead pull down using GST-UNC-97 and MBP-UNC-95, MBP-LIM-8, and MBP-LIM-9. GST or GST-UNC-97–coated glutathione agarose beads were mixed with MBP, MBP-UNC-95, MBP-LIM-8, or MBP-LIM-9. After several washes with buffer, proteins associated with glutathione agarose were eluted by Laemmli buffer. These samples were separated on a 10% SDS-PAGE gel and stained with CBB. The < shows the position of GST-UNC-97. The * shows the positions of MBP-UNC-95, MBP-LIM-8, MBP-LIM-9. (B) Glutathione bead pull down using GST-UNC-96 and MBP-LIM-8 and MBP-LIM-9. GST or GST-UNC-96–coated glutathione agarose beads were mixed with MBP, MBP-LIM-8, or MBP-LIM-9. After several washes with buffer, proteins associated with glutathione agarose were eluted with Laemmli buffer. Eluted samples were separated on a 10% SDS-PAGE gel and stained with CBB. The < shows the position of GST-UNC-96. The * shows the position of MBP-LIM-8 or MBP-LIM-9. (C) Far-Western assay showing interaction between UNC-96 and LIM-8 and between UNC-96 and LIM-9. GST and GST-UNC-96 were separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with MBP or MBP-LIM-8 or MBP-LIM-9, and after washing, bound proteins were visualized by using horseradish peroxidase (HRP)-conjugated anti-MBP antibodies and enhanced chemiluminescence (ECL). The far-Western column shows the ECL film of the anti-MBP antibody reaction. Coomassie staining shows CBB staining of proteins used in this assay. The < shows the position of GST-UNC-96. (D) Far-Western assay showing interaction between purified myosin and UNC-96 and LIM-8. Total myosin II purified from C. elegans was separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with MBP or 96MBP or MBP-LIM-8, and after washing, bound proteins were visualized by using HRP-conjugated anti-MBP antibodies and ECL. The arrows indicate the position of actin that is found in a small amount in this purified myosin preparation.

Figure 3.

lim-8 and lim-9 expression patterns by using promoter-gfp fusions. Approximately 3–4 kb of genomic sequence upstream of the predicted initiation methionines of lim-8 or lim-9 were fused to gfp and used to create transgenic worms. (A and B) lim-8 promoter::gfp expression in the pharynx of adult (A) and in body wall muscle of larvae (B). Arrows indicate positions of body wall muscle cells. (C and D) lim-9 promoter::gfp expression in pharynx (C) and in body wall muscle and neurons (D), all in adults. White bars, 100 μm (A–C) or 50 μm (D).

Figure 4.

Western blot analysis of anti-UNC-95, anti-LIM-8, and anti-LIM-9 antibodies. Each antibody detects a protein of predicted size from wild-type C. elegans lysates (indicated with arrows), and the proteins are missing or in truncated form from mutant lysates. Asterisks denote nonspecific bands that also appeared in wild-type and mutant lysates. (A) Anti-UNC-95 antibodies. (B) Anti-LIM-9 antibodies. (C) Anti-LIM-8 antibodies.

Figure 5.

Localization of UNC-95, LIM-8, and LIM-9 in adult body wall muscle cells. Localization of UNC-95, LIM-8, and LIM-9 are shown together with antibodies to markers for dense bodies (α-actinin) and M-lines (UNC-89). The left column shows UNC-95, LIM-8, or LIM-9 localization. The center column shows dense body and M-line marker localization. The right column shows merged images of left and center columns. Rabbit antibodies were visualized by anti-rabbit IgG conjugated with Alexa 488, and mouse antibodies were visualized by anti-mouse IgG conjugated with Alexa 647. White bar, 10 μm.

Figure 6.

Either a decrease or an increase of UNC-96 results in disorganization of thick filaments. (A) Wild-type or unc-96 (sf18) worms were stained with anti-UNC-97 and anti-MHC A antibodies. (B–D) represent experiments using lines of worms carrying extrachromosomal arrays of plasmids consisting of HA-tagged UNC-96 full-length cDNA under the control of a heat-shock promoter. (B) Laemmli soluble proteins were prepared from these transgenic animals after they were subjected to heat shock (+), or without heat shock (−). Proteins (100 μg) were separated by 10% SDS-PAGE, transferred to a membrane and reacted with anti-HA antibodies. An arrow indicates a protein in the heat shock (+) lane that is expected for HA-UNC-96. (C and D) Worms were stained with anti-HA (rabbit) and anti-MHC A (mouse monoclonal). Results of antibody staining after heat shock on worms lacking (C) or harboring the extrachromosomal array (D). In A, C, and D, rabbit antibodies were visualized by anti-rabbit IgG conjugated with Alexa 488, and mouse antibodies were visualized by anti-mouse IgG conjugated with Cy3. White bars, 10 μm.

Figure 7.

Summary of interactions and working model: Multiple protein clusters link muscle focal adhesions to thick filaments. (A) Summary of protein–protein interactions that were found in this study. Each protein is represented schematically with the indicated domains. Green boxes show LIM domains, a red box shows a PET domain, purple boxes show IQ repeats, a blue box shows the coiled coil region, and a yellow box shows the myosin head region. According to the results of domain mapping, minimum interacting regions are indicated by black bars. (B) Multiple protein clusters link cell adhesion structures to thick filaments. In C. elegans muscle, myofilaments are located close to the surface and anchored by dense bodies and M-lines to the muscle cell membrane. At these attachment structures, UNC-52/Perlecan is located in the ECM. Inside the muscle cell, the cytoplasmic tail of integrin (PAT-3/PAT-2) is associated with a four-protein complex, including UNC-97 (Norman et al., 2007). Among the UNC-97 interactors is UNC-98 (a C2H2 zinc finger protein) that also binds to the C-terminal tail of MHC A, suggesting a protein linkage from integrin-associated proteins to thick filaments (Miller et al., 2006). In this study, we found that UNC-97 binds to two independent LIM domain proteins (LIM-8 and LIM-9), and these two LIM domain proteins bind to UNC-96. UNC-96 has been shown to be an M-line component and to interact with UNC-98 (Mercer et al., 2006). We show that UNC-96 and LIM-8 can also bind to the C terminus of MHC A. Together, we propose that the UNC-97/LIM-8/LIM-9/UNC-96 protein complex functions as one more pathway that links integrin-associated proteins to thick filaments.