In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647–742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain “interkinase region” has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic C. elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signalling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g zebrafish).

Caenorhabditis elegans exhibit avoidance behavior when presented with diverse bacterial pathogens. We hypothesized that exposure to pathogens might not only cause worms to move away but also simultaneously activate pathways that promote resistance to the pathogen. We show that brief exposure to the virulent or avirulent strains of the bacterial pathogen enteropathogenic E. coli (EPEC) “conditions” or “immunizes” C. elegans to survive a subsequent exposure that would otherwise prove lethal. Conditioning requires dopaminergic neurons. Conditioning also requires the p38 MAP Kinase pathway, which regulates innate immunity, and the insulin/IGFR pathway, which regulates lifespan. Our findings suggest that the molecular pathways that regulate innate immunity and lifespan and provide protection may, in nature, be regulated or “conditioned” by exposure to pathogens, and perhaps allow survival in noxious environments.

UNC-89 (obscurin) interacts with MEL-26, a BTB-domain protein/adaptor for cullin-3. MEL-26 colocalizes with UNC-89 at M-lines. Mutations in MEL-26, CUL-3 (cullin-3), and MEI-1 (katanin) result in a muscle phenotype similar to that of unc-89 mutants. The level of MEI-1 is reduced in unc-89 mutants, suggesting that normally UNC-89 inhibits CUL-3/MEL-26 in muscle.

In Caenorhabditis elegans two M-line proteins, UNC-98 and UNC-96, are involved in myofibril assembly and/or maintenance, especially myosin thick filaments. We found that CSN-5, a component of the COP9 signalosome complex, binds to UNC-98 and -96 using the yeast two-hybrid method. These interactions were confirmed by biochemical methods. The CSN-5 protein contains a Mov34 domain. Although one other COP9 signalosome component, CSN-6, also has a Mov34 domain, CSN-6 did not interact with UNC-98 or -96. Anti-CSN-5 antibody colocalized with paramyosin at A-bands in wild type and colocalized with abnormal accumulations of paramyosin found in unc-98, -96, and -15 (encodes paramyosin) mutants. Double knockdown of csn-5 and -6 could slightly suppress the unc-96 mutant phenotype. In the double knockdown of csn-5 and -6, the levels of UNC-98 protein were increased and the levels of UNC-96 protein levels were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Taken together, we found that CSN-5 functions in muscle cells to regulate UNC-98 and -96, two M-line proteins.

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent “needles.” By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel ∼47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.

Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.

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.

C. elegans UNC-112 (kindlin) is required for muscle sarcomere assembly, and is one component of a conserved four-protein complex that associates with the cytoplasmic tail of integrin at the base of integrin adhesion complexes in muscle. The four-protein complex consists of UNC-112 (kindlin), PAT-4 (integrin linked kinase; ILK), PAT-6 (alpha-parvin), and UNC-97 (PINCH). UNC-112 is comprised of 720 amino acid residues and contains FERM and PH domains. The N-terminal half of UNC-112 (1-396 aa) can bind to the C-terminal half of UNC-112 (397-720 aa), and this interaction is inhibited by the association of PAT-4 (ILK) to the N-terminal half of UNC-112. In support of this model, previously, we reported identification of a D382V mutation that results in lack of binding to PAT-4. However, this residue is not conserved in human Kindlins. Here, we report identification of a novel UNC-112 mutation of a conserved residue that cannot bind to PAT-4. UNC-112 E302G cannot bind to PAT-4 and does not localize to integrin adhesion complexes in muscle.