FHOD formin and SRF promote post-embryonic striated muscle growth through separate pathways in C. elegans

Abstract

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 A expression. However, while a hypomorphic unc-120 allele blunts expression of a set of striated muscle genes, these genes are largely 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.

Keywords: Caenorhabditis elegans; FHOD-1; Formin; Proteasome; Serum response factor; Striated muscle.

Figure 1.. UNC-120 promotes BWM growth in a manner qualitatively similar to FHOD-1.

(A) Fluorescent phalloidin stain showing dorsal views of a portion of single worms of the indicated genotypes, with two BWM quadrants extending horizontally in the field of view. Adult unc-120(hypo) and fhod-1(Δ) worms have BWMs that are narrower than wild type (wt). Double arrows show width of one BWM. (B) Overall body widths, (C) BWM widths, and (D) individual BWM cell widths were measured. (E) The number of striations in individual BWM cells was manually counted. Shown are measures and averages from three independent experiments (n = 60 worms per strain, with measures of one body, two BWMs, and two BWM cells per worm). Error bars indicate one standard deviation. (a) p < 0.001 from wild type; (b) p < 0.05 from fhod-1(Δ); (c) p < 0.05 from unc-120(hypo). Differences in all other comparisons were not statistically significant (p > 0.05).

Figure 2.. UNC-120 and FHOD-1 promote MYO-3 protein expression.

(A) MYO-3 western blot of dilutions (expressed as percentages) of whole adult worm extracts show fhod-1(Δ) worms have less MYO-3 than wild-type worms (wt), and this is partially rescued by a fhod-1::gfp transgene. Sample loads were normalized based on number of worms in each sample. Position of molecular weight standard is indicated. (B) Average band intensities, from 100% and 50% samples from five independently collected lysates, were normalized to wild type, with each lysate being subjected to two independent western blots. Error bars indicate one standard deviation. (C) MYO-3 and tubulin western blots of adult worm extracts show a decrease in MYO-3 in unc-120(hypo) and fhod-1(Δ) worms compared to wild type. Sample loads were normalized based on tubulin signal. Positions of molecular weight standards are indicated. (D) Average ratios of MYO-3 to tubulin band intensities of three independently collected lysates were normalized to wild type, with each lysate being subjected to two independent western blots. Error bars indicate one standard deviation. (a) p < 0.001 from wild type; (b) p < 0.05 from fhod-1(Δ); fhod-1::gfp; (c) p < 0.05 from wild type; (d) p < 0.05 from fhod-1(Δ); (e) p < 0.05 from unc-120(hypo).

Figure 3.. Defects in FHOD-1 and in UNC-120 have differing effects on muscle gene expression.

(A) Venn diagram of differentially expressed genes (FDR step up ≤ 0.05, fold change > 1.5) identified by comparison of mRNA sequencing results for L3 stage fhod-1(Δ), unc-120(hypo), and wild-type worms. Indicated in each sector is the number of differentially expressed genes, and in parentheses, the number of genes expressed predominantly in striated muscle. (*) denotes significant enrichment for striated muscle genes in a gene set, based on the WormBase Enrichment Analysis Tool. Gene identities are provided in Supplementary Table 2. (B) Heat map showing the relative levels of gene expression in four replicate samples of the indicated genotypes, for all genes differentially expressed between unc-120(hypo) and wild-type worms. Red indicates expression was elevated above the average for the entire group, while blue indicates decreased expression. Hierarchical clustering identified four gene groups, with enrichment for tissue expression displayed beneath each group. Only group 2 was enriched for genes expressed primarily in striated muscle and the muscular system. (C) Quantitative RT-PCR values for myo-3 were determined in comparison to reference gene ama-1 by the ΔΔCt method, and normalized to wild type (wt). Shown are qRT-PCR values and the average results from three RNA samples, each analyzed in triplicate, from L2 stage and L4 stage worms. Error bars indicate one standard deviation. (a) p < 0.001 from wild type; (b) p < 0.05 from wild type.

Figure 4.. Inhibition of the proteasome restores MYO-3 to normal levels in fhod-1(Δ) worms.

(A) MYO-3 western blot of dilutions (expressed as percentages) of whole worm extracts of adults treated with 5 μM MG132 or DMSO. Samples were prepared from equal numbers of worms. Position of molecular weight standard is indicated. (B) Shown are the averages of all measured band intensities from 100% and 50% dilutions from six independently collected lysates (normalized to wild type), with each lysate being subjected to two independent western blots. Error bars indicate one standard deviation. Treatment with 5 μM MG132 elevated MYO-3 in fhod-1(Δ) worms to a level statistically indistinguishable from wild-type worms (wt) treated with DMSO or 5 μM MG132. (a) p < 0.001 from wild type DMSO; (b) p < 0.001 from wild type MG132; (c) p < 0.001 from fhod-1(Δ) MG132. Differences in all other comparisons were not statistically significant (p > 0.05).

Figure 5.. Proteasome inhibition in fhod-1(Δ) worms does not restore normal BWM growth.

(A) Fluorescent phalloidin stain shows BWM of wild-type (wt) and fhod-1(Δ) adults grown in the presence of either 5 μM MG132 or DMSO. (B) Overall body width, (C) BWM widths, and (D) individual BWM cell widths were measured. (E) The number of striations in individual BWM cells was manually counted. Shown are measurements and averages from three independent experiments (n = 60 worms per strain, with measures of one body, two BWMs, and two BWM cells per worm). Error bars indicate one standard deviation. BWM measures are identical in fhod-1(Δ) adults grown in the presence of either DMSO 5 μM MG132, but are consistently narrower than wild type grown under either condition. (a) p < 0.05 from fhod-1(Δ) MG132; (b) p < 0.05 from fhod-1(Δ) DMSO; (c) p < 0.001 from wild type DMSO; (d) p < 0.001 from wild type MG132. Differences in all other comparisons were not statistically significant (p > 0.05). (F) Immunostain shows MYO-3 localizes to striations in wild-type and fhod-1(Δ) worms, whether grown in the presence of DMSO or 5 μM MG132.