Using reverse transcription-polymerase chain reaction and cDNA cloning from C. elegans RNA, we identified a cDNA clone encoding a new low-molecular-weight Tpm isoform containing an exon 7 sequence that has not been recognized as an exon in previous studies or any sequence databases including WormBase (Davis et al., 2022). This is the third alternative exon 7 and designated as exon 7c (E7c) (Figure 1). The new Tpm isoform, which we designated as LEV-11U (GenBank accession number: OQ473578), contained an exon combination of E3b-E4a-E5a-E6-E7c-E8-E9b (Figure 1A). Therefore, LEV-11U is identical to LEV-11T (E3b-E4a-E5a-E6-E7b-E8-E9b) (GenBank accession number: LC215398) (Watabe et al., 2018) except for the exon 7 sequence (Figure 1A). The accumulated RNA-seq data in WormBase (release WS289) indicate that E7b is the most frequently selected exon, but E7a (∼1/200 of E7b) and E7c (∼1/5000 of E7b) are rarely selected exons (Figure 1B). Tissue and cell distribution of the E7c selection is currently under investigation.

Alignment of the exon 7-encoded sequences showed that the E7c-encoded sequence was distinct from E7a- and E7b-encoded sequences (Figure 2; Table 1). Although E7a and E7b encode 78.7% identical amino acid sequences, the E7c-encoded sequence is only 17.0% and 19.2% identical to E7a- and E7b-encoded sequences, respectively (Figure 2; Table 1). By comparing with the equivalent sequence of human Tpm1.8 (also known as Tpm5 or Tpm1.8cy) (residues 188–234; NCBI Reference Sequence: NP_001288218.1), E7a- and E7b-encoded sequences are 55.3% and 59.6% identical, respectively, but E7c-encoded sequence is only 17.0% identical (Figure 2; Table 1), suggesting that the E7c-encoded polypeptide is biochemically different from other equivalent polypeptides.

Further comparison of the E7c-encoded sequence with the equivalent sequences of Tpm isoforms from various species (Figure 2) also suggested its unique properties. The three exon 7-encoded sequences from the C. elegans lev-11 gene were aligned with equivalent sequences of Tpm isoforms from human (Homo sapiens), fruit fly (Drosophila melanogaster), pacific oyster (Crassostrea gigas), and ascidian (Ciona intestinalis). The sequence comparison showed that eight basic (R, K, and H) and fourteen acidic (D and E) residues were evolutionarily conserved among the examined species (Figure 2), because many of these conserved charged residues are important for actin binding or actomyosin regulation (Barua et al., 2011; Barua et al., 2012; Barua et al., 2013). Although most of these charged residues were conserved in the E7a- and E7b-encoded sequences, four basic and nine acidic residues were not conserved in the E7c-encoded sequence (Figure 2, asterisks). Since charged amino acids are often involved in regulating the structure and function of proteins, the E7c-encoded polypeptide might have distinct biochemical properties from other Tpm isoforms.

The biophysical properties of the E7-encoded sequences were analyzed using in silico approaches. Tropomyosins are generally 100% α-helical polypeptides forming coiled-coil dimers (Hitchcock-DeGregori and Barua, 2017). Prediction of coiled-coil formation by COILS (Lupas et al., 1991) indicated nearly 100% probability for the E7b-encoded sequence but medium and low probabilities for the E7a- and E7c-encoded sequences, respectively, in a heptad at residues 190–196 (Figure 2; Figure 3A). To examine the structural stability of the E7-encoded polypeptides, a coiled-coil dimer of each E7-encoded sequence was analyzed by molecular dynamics (MD) simulations (Figures 3B,C). The E7a- and E7b-encoded polypeptides maintained relatively stable coiled-coils (Figure 3B), whereas the E7c-encoded polypeptide was readily unwound at around E190 (in the LEV-11U sequence) resulting in a separation of the two strands (Figure 3B). Quantitative analysis of root-mean-square deviation (RMSD) values indicates that residue 190 of LEV-11U (E7c) was more significantly displaced from the initial position than the equivalent residues of LEV-11O (E7a) and LEV-11T (E7b) (Figure 3C). It should be noted that our simulations of 40 ns may be too short, and that longer simulations of the E7-encoded sequences and full-length proteins may be needed to predict how the E7-encoded sequences contribute to the biophysical properties of the Tpm isoforms. These results suggest that LEV-11U has a region that is unfavorable to maintain a coiled-coil within the E7c-encoded sequence.

As suggested by the sequence alignment (Figure 2), the E7c-encoded polypeptide was predicted to be distinct from the E7a- and E7b-encoded polypeptides in the pattern of electrostatic surface potentials (Figure 4). After the MD simulations, electrostatic surface potentials of each polypeptide were estimated using Adaptive Poisson-Boltzmann Solver (APBS) (Jurrus et al., 2018). Although the E7a- and E7b-encoded polypeptides were dominated by negative charges with scattered small patches of positive charges on their surfaces (Figures 4A,B), the E7c-encoded polypeptide had a large patch of positive charges at the N-terminal portion at K160–R170 (Figure 4C, underlined in Figure 2). This region corresponds to a second half of the fifth actin-binding period (Barua et al., 2011; Barua, 2013), but how this region contributes to actin binding is not understood. Nonetheless, the difference in the surface charges suggest that the E7c-encoded polypeptide may have unique properties in interacting with other proteins.

Previously, we have reported that the choice of E7a or E7b in high-molecular-weight Tpm isoforms does not significantly alter their actin-binding properties (Barnes et al., 2018). To determine the effects of the E7c-encoded sequence on the biochemical properties of Tpm isoforms, we compared LEV-11T and LEV-11U, the two low-molecular-weight isoforms that differ only at the E7-encoded regions (Figure 5A). Both isoforms were bacterially expressed with extra Ala-Ser after the initiator Met to mimic N-terminal acetylation (Monteiro et al., 1994), purified to homogeneity (Figure 5B), and tested for actin-filament binding using F-actin co-sedimentation assays. Typically, we perform the F-actin co-sedimentation assays by using a constant concentration of F-actin and varying concentrations of Tpm, and quantify Tpm in the pellets (Ono and Ono, 2002; Barnes et al., 2018). Instead, we performed the assays using a constant concentration of a LEV-11 isoform and varying concentrations of F-actin and quantified depletion of a LEV-11 isoform from the supernatants to determine the bound portions, because LEV-11U bound poorly to F-actin and non-specifically precipitated portions of LEV-11U obscured its specific co-sedimentation with F-actin. Constant concentrations (2 μM) of LEV-11T or LEV-11U were incubated with increasing concentrations (0–60 μM) of F-actin and fractionated into supernatants and pellets after ultracentrifugation. Under these conditions, LEV-11T, containing the E7b-encoded sequence, was increasingly shifted from the supernatant to pellet fractions as F-actin concentration was increased (Figure 5C, top), indicating that LEV-11T specifically bound to F-actin. Binding of LEV-11T to F-actin was saturable with an estimated dissociation constant (K _d ) of 3.7 ± 0.64 μM (n = 3) (Figure 5D). In contrast, the majority of LEV-11U, containing the E7c-encoded sequence, remained in the supernatants regardless of the F-actin concentrations (Figure 5C, bottom), indicating that LEV-11U bound poorly to F-actin with no measurable affinity (Figure 5D). Since LEV-11T and LEV-11U differ only in the E7-encoded sequences, these results demonstrate that the choice of alternative E7s confers a major difference in the actin affinity of the Tpm isoforms.

To determine whether LEV-11T and LEV-11U can bind to actin in vivo, these Tpm isoforms were tagged with green fluorescent protein (GFP) and expressed transgenically in the body wall muscle cells (Figure 6). Although low-molecular-weight Tpm isoforms are typically not enriched in muscle cells, highly ordered sarcomeric actin organization allows us to determine colocalization of the Tpm isoforms with F-actin in vivo. In live worms without fixation, GFP-LEV-11T localized in a striated pattern (Figure 6A), whereas GFP-LEV-11U was present in the diffuse cytoplasm and often concentrated in aggregates of various sizes at random locations within the cytoplasm (Figure 6B). These aggregates are likely the result of overexpression because such structures have not been detected by immunofluorescent staining of endogenous Tpm using a polyclonal antibody that was raised against the whole Tpm protein (Ono and Ono, 2002; Ono et al., 2022). When these worms were fixed and stained with fluorescently labeled phalloidin, GFP-LEV-11T colocalized with F-actin in a striated manner (Figures 6C–E), but GFP-LEV-11U was diffusely present in the cytoplasm and did not exhibit a striated pattern (Figures 6F–H). Furthermore, when GFP-LEV-11U was overexpressed and aggregated, F-actin was not accumulated in the aggregates of GFP-LEV-11U, and striated organization of F-actin was not significantly affected (Figures 6I–K), suggesting that association of GFP-LEV-11U with F-actin was negligible in the muscle cells. Therefore, these results indicate that in vivo localization of LEV-11T and LEV-11U can be significantly different, which could be due to the difference in their affinity for F-actin.

To determine in vivo significance of E7c, we generated a mutant, lev-11(syb4266), in which a STOP-IN cassette containing premature stop codons was inserted in E7c (Figure 7A). The STOP-IN cassette contains stop codons in all three reading frames (Wang et al., 2018) and is likely to cause nonsense-mediated decay of the transcripts containing premature stop codons. Therefore, lev-11(syb4266) should not express a LEV-11 Tpm isoform containing the E7c-encoded sequence. The STOP-IN cassette contains a unique Nhe I site (Figure 7A). Therefore, a PCR-amplified genomic DNA fragment could be cut by Nhe I only when the STOP-IN cassette was present (Figure 7B). The lev-11(syb4266) homozygotes were viable and showed no gross phenotypes. Sarcomeric organization of actin filaments in the body wall muscle was indistinguishable between wild-type and lev-11(syb4266) (Figure 7C). However, lev-11(syb4266) showed slightly slower worm motility (Figure 7D) and reduced brood size (Figure 7E) as compared with wild-type. Although the cellular basis of these phenotypes is currently unknown, these data suggest that the E7c-encoded sequence is required for optimal health of the worms.

The worms were cultured following standard methods (Stiernagle, 2006). The N2 wild-type C. elegans strain was obtained from the Caenorhabditis Genetics Center (Minneapolis, MN).

Clones for LEV-11U were discovered in the process of reverse-transcription PCR, cloning, and sequencing of low-molecular-weight Tpm isoforms as described previously (Watabe et al., 2018). The LEV-11U cDNA was subcloned into a pET-3d vector with additional codons for Ala-Ser (GCTAGC) after the initiator Met and used for expression of recombinant LEV-11U in Escherichia coli. Sequences were verified by DNA sequencing.

CCBuilder Mk.2 (Wood and Woolfson, 2018) was used to generate structural models of parallel coiled-coil dimers of full-length LEV-11O, LEV-11T, and LEV-11U, which were then truncated into the E7-encoded regions: residues 188–234 for LEV-11O (E7a) and residues 160–206 for LEV-11T (E7b) and LEV-11U (E7c). Using Visual Molecular Dynamics (VMD) (Humphrey et al., 1996), each of these structures was solvated by a rectangular box of water particles and ionized at 0.1 M KCl. The layer of the box was set to 15 Å. Nanoscale Molecular Dynamics (NAMD) was used to perform simulations with the Chemistry at Harvard Macromolecular Mechanics 36 (CHARMM36m) force fields (Huang et al., 2017), the TIP3 water model (Jorgensen et al., 1983), and the Beglov and Roux ion parameters (Beglov and Roux, 1994). The switchdist, cutoff, and pairlistdist were set to 9, 10, and 11 Å, respectively. The energy of each model was first minimized by 5,000 steps, followed by three 40-ns production runs at 310 K under the periodic boundary condition and Langevin piston. For every 20 ps, residues 214–225 of LEV-11O (E7a) and residues 186–197 of LEV-11T (E7b) and LEV-11U (E7c) of the simulation products were compared with the corresponding portions of the initial energetically minimized structures along alpha carbons. Then, the average root mean squared deviations (RMSD) of E218 of E7a and E190 of E7b and E7c were calculated. The data were tested by one-way analysis of variance with the Holm-Sidak method for multiple pairwise comparison using SigmaPlot 14.5 (Systat Software). The APBS-PDB2PQR software was used to construct the electrostatic surfaces of E7a, E7b, and E7c at pH 7 and with CHARMM27 force fields using the automatically configured finite difference Poisson-Boltzmann calculations (Jurrus et al., 2018). The electrostatic surface potentials were visualized with red-white-blue coloring, ranging from −10 to 10 kT/e, with an offset of 0.1 and a midpoint of 0.5 by VMD.

Actin was purified from rabbit muscle acetone powder as described (Pardee and Spudich, 1982). Recombinant LEV-11T and LEV-11U were expressed in E. coli with extra Ala-Ser after the initiator Met to mimic N-terminal acetylation (Monteiro et al., 1994) using a pET-3d vector with no other tag sequence and purified in the same procedure as described for other LEV-11 isoforms (Barnes et al., 2018). F-actin co-sedimentation assays were performed essentially as described (Ono and Ono, 2020) in F-buffer (0.1 M KCl, 2 mM MgCl₂, 20 mM HEPES-KOH, pH 7.5, 1 mM dithiothreitol), except that the actin-bound portions of LEV-11 isoforms were calculated from depletion of a constant concentration (2 μM) of the LEV-11 isoforms from the supernatants by sedimentation of varying concentrations of F-actin. Ultracentrifugation was performed at 42,000 rpm (200,000 × g) for 20 min using a Beckman 42.2Ti rotor. Only the top halves of the supernatants were taken and analyzed as supernatant fractions. The rest of the supernatants were carefully removed, and remaining pellets were analyzed as pellet fractions. Proteins were analyzed by SDS–PAGE (12% acrylamide gel) with molecular weight markers (Nacalai United States, catalog no. 29458–24) and staining with Coomassie Brilliant Blue R-250 (National Diagnostics). Percentages of bound LEV-11T or LEV-11U (P _bound ) were determined densitometrically as (1-D _x /D ₀ )x100, where D _x is band density in the supernatants at x μM actin and D ₀ is band density in the supernatants in the absence of actin. Dissociation constants (K _d ) were calculated using SigmaPlot 14.5 by fitting the data to an equation: P _bound = (P _bound-Max x [actin])/(K _d + [actin]), where P _bound-Max is a maximum P _bound value and [actin] is an actin concentration.

The cDNA for LEV-11T or LEV-11U was cloned in-frame at the 3’ end of the GFP sequence of pPD-118.20 (provided by Dr. Andrew Fire, Stanford University) containing the myo-3 promoter that is active in the body wall muscle (Okkema et al., 1993), and injected into the syncytial gonad to generate transgenic animals with extrachromosomal arrays as described (Mohri et al., 2006). The strains generated and used in this study are ON378 ktEx257 [myo-3p::GFP::LEV-11T] and ON375 ktEx254 [myo-3p::GFP::LEV-11U]. Live worms were anesthetized in M9 buffer containing 0.1% tricaine and 0.01% tetramisole for 30 min, mounted on 2% agarose pads for observation. Staining of worms with ATTO594-phalloidin (Millipore Sigma) was performed as described (Ono, 2001; 2022). Samples were observed by epifluorescence using a Nikon Eclipse TE2000 inverted microscope (Nikon Instruments, Tokyo, Japan) with a CFI Plan Fluor ELWD 40x (NA 0.60) objective. Images were captured by a Hamamatsu ORCA Flash 4.0 LT sCMOS camera (Hamamatsu Photonics, Shizuoka, Japan) and processed by NIS-Elements (Nikon Instruments) and Adobe Photoshop CS3.

Insertion of a STOP-IN cassette of 43 bp (Wang et al., 2018) to lev-11 E7c by CRISPR/Cas9-mediated genome editing was performed by SunyBiotech Co., Ltd (Fu Jian Province, China) to generate PHX4266 lev-11(syb4266), which was outcrossed with N2 wild-type three times to generate ON387 lev-11(syb4266). Genotyping was done by single-worm PCR using forward and reverse primers (GAA​GAG​GAG​TTG​CGC​GTC​GTT​GG and ACC​TCC​TTC​TGG​AGC​TTC​TGG​AC) that amplify a genomic fragment [851 bp for wild-type or 894 bp for lev-11(syb4266)] containing E7c, followed by digestion with Nhe I and agarose electrophoresis. Staining of worms with ATTO594-phalloidin (Millipore Sigma) was performed as described (Ono, 2001; 2022). Worm motility was determined by counting swinging motions of worms in M9 buffer as described (Epstein and Thomson, 1974). Brood size was determined by isolating single L4 worms in individual plates and counting number of progeny until no live worms were produced. The data were tested by Student’s t-test using SigmaPlot.