David Pruyne

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.

Profilin is a small actin-monomer binding protein that inhibits pointed-end elongation and spontaneous nucleation, but promotes formin-mediated assembly of actin onto barbed-ends. In Caenorhabditis elegans , the profilin PFN-3 , in conjunction with the formin FHOD-1 , promotes sarcomere formation and dense body stability in the worm striated body-wall muscle (BWM). As a tool for further study of PFN-3 , we have expressed mScarlet-tagged PFN-3 (mSc::PFN-3) under pfn-3 promoter sequence at a single exogenous locus. We demonstrate here that mSc::PFN-3 is expressed in multiple muscles, partially colocalizes with FHOD-1 in BWM, and rescues BWM defects associated with deletion of pfn-3 .

Background: Actin filament organization in cardiomyocytes critically depends on the formin Fhod3, but a role for Fhod3 in skeletal muscle development has not yet been described. Results: We demonstrate here that in zebrafish mutated for one of two fhod3 paralog genes, fhod3a , skeletal muscle of the trunk appears normal through 2 days post-fertilization, but afterward exhibits myofibril damage, including gaps between myofibrils and myofibril fragmentation. Despite the apparent progressive nature of myofibril damage, fhod3a mutant embryos differ from muscular dystrophy models in that damage is exacerbated by inhibition of muscle activity, and fhod3a mutants show no evidence of sarcolemma disruption. Rather, our results suggest myofibril damage coincides with growth of the skeletal muscle fiber contractile apparatus. We find that neither the second fhod3 paralog, fhod3b , nor the related fhod1 contribute to embryonic skeletal muscle development, but simultaneous mutation of fhod3a and fhod3b was associated with pericardial edema suggestive of cardiac dysfunction. Conclusions: Taken together, these results indicate fhod3- encoded formins are dispensable for initial myofibril assembly in skeletal muscle, but promote myofibril stability during muscle fiber growth. This is the first demonstration that Fhod3 contributes to skeletal muscle development in a vertebrate.

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 regulate the 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 that C. elegans can survive with a single low molecular weight isoform, LEV‐11E. Mutants disrupted for LEV‐11E die as young larvae, whereas mutants lacking 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 that 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.

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 Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 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 and actin depolymerization factor/cofilin homologue UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein–mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

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.

The striated body wall muscles of Caenorhabditis elegans are a simple model for sarcomere assembly. Previously, we observed deletion mutants for two formin genes, fhod‐1 and cyk‐1, develop thin muscles with abnormal dense bodies (the sarcomere Z‐line analogs). However, this work left in question whether these formins work in a muscle cell autonomous manner, particularly since cyk‐1(∆) deletion has pleiotropic effects on development. Using a fast acting temperature‐sensitive cyk‐1(ts) mutant, we show here that neither postembryonic loss nor acute loss of CYK‐1 during embryonic sarcomerogenesis cause lasting muscle defects. Furthermore, mosaic expression of CYK‐1 in cyk‐1(∆) mutants is unable to rescue muscle defects in a cell autonomous manner, suggesting muscle phenotypes caused by cyk‐1(∆) are likely indirect. Conversely, mosaic expression of FHOD‐1 in fhod‐1(Δ) mutants promotes muscle cell growth and proper dense body organization in a muscle cell autonomous manner. As we observe no effect of loss of any other formin on muscle development, we conclude FHOD‐1 is the only worm formin that directly promotes striated muscle development, and the effects on formin loss in C. elegans are surprisingly modest compared to other systems.

Previous work with cultured cells has shown transcription of muscle genes by serum response factor (SRF) can be stimulated by actin polymerization driven by proteins of the formin family. However, it is not clear if endogenous formins similarly promote SRF-dependent transcription during muscle development in vivo . We tested whether formin activity promotes SRF-dependent transcription in striated muscle in the simple animal model, Caenorhabditis elegans . Our lab has shown FHOD-1 is the only formin that directly promotes sarcomere formation in the worm’s striated muscle. We show here FHOD-1 and SRF homolog UNC-120 both support muscle growth and also muscle myosin II heavy chain expression. However, while a hypomorphic unc-120 allele blunts transcription of a set of striated muscle genes, these genes are upregulated or unchanged by absence of FHOD-1. Instead, pharmacological inhibition of the proteasome restores myosin protein levels in worms lacking FHOD-1, suggesting elevated proteolysis accounts for their myosin deficit. Interestingly, proteasome inhibition does not restore normal muscle growth to fhod-1(Δ) mutants, suggesting formin contributes to muscle growth by some alternative mechanism. Overall, we find SRF does not depend on formin to promote muscle gene transcription in a simple in vivo system.