The sole essential low molecular weight tropomyosin isoform of Caenorhabditis elegans is essential for pharyngeal muscle function

Figures

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  Figure 1.. Structure of the C. elegans tropomyosin gene, lev-11, and exon usage for LEV-11 splice isoforms. Exons are numbered and shown with protein-coding sequences in green and non-coding sequences in grey. At top are indicated positions of nonsense mutation alleles generated during this study, and the location of sequences encoding peptides used to generate anti-LEV-11(Long) and anti-LEV-11(Short) in orange. Transcripts for long LEV-11 isoforms (A, D, and O) initiate from Promoter 1 upstream of Exon 1, while transcripts for short LEV-11 isoforms (C, E, T, and U) initiate from Promoter 2 located between exons 3a and 3b. Scale bar, 1 kb.

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  Figure 2.. LEV-11 isoform detection and expression. Whole worm extracts (designated by worm genotype) and purified recombinant LEV-11 proteins (long isoform LEV-11A and short isoforms LEV-11C and E) were probed by immunoblot using antibodies specific for long or short isoforms. (A) Anti-LEV-11(Long) recognizes purified LEV-11A but not LEV-11C or E, and recognizes endogenous protein of similar molecular weight to LEV-11A, and exogenous GFP-tagged LEV-11A, but not endogenous or GFP-tagged LEV-11C or E. Conversely, anti-LEV-11(Short) recognizes purified LEV-11C and E but not A, and recognizes endogenous proteins of similar molecular weight to LEV-11C/E, and exogenous GFP-tagged LEV-11C and E, but not endogenous or GFP-tagged LEV-11A. (B) Immunoblotting whole worm extracts and purified recombinant LEV-11 proteins with anti-LEV-11(Short) shows worms bearing CRISPR-generated mutations simultaneously targeting LEV-11E, T, and U, or simultaneously targeting LEV-11C, E, T, and U lack most or all endogenous low molecular weight LEV-11, but express exogenous mCherry-tagged LEV-11E.

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  Figure 3.. LEV-11E is essential for larval development and pharyngeal muscle function. (A) Eggs laid by worms of the indicated genotypes were counted on day 1, and the resultant progeny were examined over 4 days. For worms from three independently isolated lev-11(C-) strains, essentially all eggs hatched by day 2, and resultant larvae grew to adulthood (n = 9 wild type animals, 10 animals for each lev-11(C-) strain). (B) The number of eggs laid was counted over the lifetime of worms of the indicated genotypes, showing lev-11(C-) mutants lay the same number of eggs as wild type animals (n = 10 worms per strain). (C, D) Viability of progeny was monitored as in (A), showing ~ 100% of eggs hatch to larvae, but only ~75% of larvae from (C) heterozygous lev-11(ETU-)/(+) mutants or (D) heterozygous lev-11(CETU-)/(+) mutants grew beyond the L1 stage (n = 10 wild type animals, 19 lev-11(+) segregants of lev-11(ETU-)/(+) animals, 19 lev-11(ETU-)/(+) animals in C, n = 10 wild type, 18 lev-11(CETU-)/(+) animals in D). (E) lev-11(ETU-) and lev-11(CETU-) worms with mosaic inheritance of the Ex[mCH::lev-11E] ECA were assayed for expression of mCherry-tagged LEV-11E in the pharyngeal muscle of L1 larvae…

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  Figure 4.. LEV-11E associates with but is not required to organize thin filaments in pharyngeal muscle sarcomeres. (A) Deconvolved maximum intensity projections of the XY plane show the pharynx, with YZ cross sections through pharyngeal muscle pm3, in L1 larvae immunostained for short LEV-11 isoforms and for mCherry. Anti-LEV-11(Short) similarly stains endogenous LEV-11 and mCherry-tagged LEV-11E in pharyngeal muscle, but does not stain pharyngeal muscle in lev-11(CETU-) worms lacking all short LEV-11 isoforms. The green channel signal observed in body wall muscle of Ex[mCH::lev-11E]-bearing animals (arrowheads) reflects the presence of a body wall muscle-expressing GFP transgene that is coinherited as part of the Ex[mCH::lev-11E] array. (B) Single deconvolved XY confocal plane images showing pharyngeal muscles pm3–5, with YZ cross sections through pm4, in L1 larvae stained for F-actin (green) and expressing or not expressing mCH::LEV-11E (magenta) from Ex[mCH::lev-11E]. Shown in enlarged XY and YZ views (right), mCH::LEV-11E colocalizes along F-actin structures except at the ends of filament bundles near the basal and apical muscle cell surfaces (arrows). Absence of mCH::LEV-11E in worms…

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  Figure 5.. FHOD-1::GFP localizes near the barbed ends of thin filaments in pharyngeal muscle independently of LEV-11E. Single deconvolved XY confocal plane images showing pharyngeal muscles pm3–5, with YZ cross sections through pm4, in L1 larvae show FHOD-1::GFP is concentrated at the apical and basal surfaces of pharyngeal muscle cells, particularly at the ends of mCH::LEV-11E-positive filament bundles (arrows). Localization of FHOD-1::GFP is not significantly changed in lev-11(ETU-) mutants that express no LEV-11E.

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  Figure 6.. Absence of FHOD-1 and short LEV-11 isoforms do not substantially disturb actin organization in pharyngeal muscle. (A) Single deconvolved XY confocal slices showing the pharynx (all pms), with YZ cross sections through pm4, in L1 larvae stained for F-actin. Thin filament organization is not significantly altered in pharyngeal muscle in animals lacking FHOD-1 or short LEV-11 isoforms. Additionally, mCH::LEV-11E organization is not significantly altered in the pharyngeal muscle of fhod-1(Δ) larvae. (B) Single deconvolved XY confocal slices showing pm3–5, with a YZ cross sections through pm4, in day 1 adults stained for F-actin. Even in absence of FHOD-1 in fhod-1(Δ) animals, mCH::LEV-11E localizes along sarcomeric thin filaments except immediately adjacent to sarcomere Z-lines at the apical and basal membranes (arrows).

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  Figure 7.. Short LEV-11 isoforms bind thin filaments in body wall muscle when ectopically expressed, but are not endogenously present in body wall muscle sarcomeres. (A) Single deconvolved confocal slices of body wall muscle of day-1 adults expressing mCherry-tagged ATN-1 and the indicated GFP-tagged LEV-11 isoforms were stained for F-actin. All three LEV-11 isoforms localize to thin filaments based on strong overlapping signal with F-actin, but are not present immediately adjacent to mCH::ATN-1-positive dense bodies. (B) Single deconvolved confocal slices of body wall muscle of a wild type day-1 adult immunostained with anti-LEV-11(Long) and anti-ATN-1 shows a striated distribution of LEV-11 flanking ATN-1-positive dense bodies. (C) Single deconvolved confocal slices of day-1 adult worms immunostained for ATN-1, GFP (a co-expression marker for the mCH::LEV-11E-expressing array), and short LEV-11 isoforms shows anti-LEV-11(Short) immunostain along striations that intersect dense bodies in wild type animals and in lev-11(CETU-) mutants, regardless of whether body wall muscle cells inherited the mCH::LEV-11E + GFP-expressing array.

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  Figure 8.. Mutations disrupting short LEV-11 isoforms do not result in body wall muscle defects. (A, B) Adult worms were stained for F-actin and (A) body wall muscle cell widths were measured, and (B) the number of striations per body wall muscle cell were counted (n = 15–20 animals per strain, 2 – 4 muscle cells per animal). (**) p = 0.0016; (***) p < 0.0001; (n.s.) not significant, p ≥ 0.05. (C) Single deconvolved confocal slices of body wall muscle in worms immunostained for dense body marker ATN-1 and for GFP, a marker for the presence of an ECA bearing an mCH::LEV-11E-expressing transgene. Dense bodies have relatively uniform appearance and spacing along striations in both strains, including in GFP-negative cells of lev-11(CETU-) animals that lack all short LEV-11 isoform expression.