Michael J. Kimmich

The simple nematode Caenorhabditis elegans has long served as a powerful genetic model system for studying muscle structure and function, with sarcomeres of the striated body wall muscle (BWM) that are considered largely homologous to those in vertebrates. However, we have observed a population of F‐actin in BWM separate from I‐bands and centered at the sarcomere M‐line, something not seen in vertebrate sarcomeres. Here, we investigate whether M‐line‐associated F‐actin is a true feature of worm muscle, or an artifact of sample preparation. Using thin filament components tagged with fluorescent proteins, we confirm that living C. elegans BWM has a traditional sarcomere organization with actin restricted to I‐bands, whereas two methods of fixation resulted in varying degrees of mislocalization of actin as well as barbed end capping protein/CapZ and tropomodulin. Additionally, we observe in live animals that α‐actinin‐rich dense bodies are flanked by discrete patches of CapZ, something we cannot observe in fixed animals, but which provides some support for a model in which dense bodies anchor thin filaments in a manner similar to vertebrate Z‐discs. Overall, our work highlights the susceptibility of thin filaments in worm BWM to fixation artifacts and the potential utility in studying sarcomere structure in live animals.

Tropomyosin is an actin-binding protein that plays roles ranging from regulating muscle contraction to controlling cytokinesis and cell migration. The simple nematode Caenorhabditis elegans provides a useful model for studying the core functions of tropomyosin in an animal, having a relatively simple anatomy, and a single tropomyosin gene, lev-11 , that produces seven isoforms. Three higher molecular weight isoforms (LEV-11A, D, O) regulate contraction of body wall and other muscles, but comparatively less is known of the functions of four lower molecular weight isoforms (LEV-11C, E, T, U). We demonstrate here C. elegans can survive with a single low molecular weight isoform, LEV-11E. Mutants disrupted for LEV-11E die as young larvae, whereas mutants disrupted for all other short isoforms are viable with no overt phenotype. Vertebrate low molecular weight tropomyosins are often considered “nonmuscle” isoforms, but we find LEV-11E localizes to sarcomeric thin filaments in pharyngeal muscle, and co-precipitates from worm extracts with the formin FHOD-1, which is also associated with thin filaments in pharyngeal muscle. Pharyngeal sarcomere organization is grossly normal in larvae lacking LEV-11E, indicating the tropomyosin is not required to stabilize thin filaments, but pharyngeal pumping is absent, suggesting LEV-11E regulates actomyosin activity similar to higher molecular weight sarcomeric tropomyosin isoforms.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from C. elegans results in thin body-wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that actin polymerization by FHOD-1 is required for its function in muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and profilins PFN-2 and PFN-3 to promote body-wall muscle growth. We further demonstrate dense bodies in fhod-1 and pfn-3 mutants are less stable than in wild type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin depolymerization factor/cofilin homolog UNC-60B in body-wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, and may account for the abnormally slow growth and reduced strength of body-wall muscle in fhod-1 mutants. Overall, these results show the importance of FHOD protein-mediated actin assembly to forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.