Profilin is a conserved protein that binds actin monomers in a 1:1 ratio, inhibiting spontaneous actin filament nucleation and monomer addition onto filament pointed-ends, but stimulating ADP/ATP exchange on actin, permitting monomer addition to filament barbed-ends, and enhancing formin-mediated actin polymerization (Kovar et al., 2003; Lu & Pollard, 2001; Moseley et al., 2004; Pollard et al., 2000; Pollard & Cooper, 1984; Pring et al., 1992; Romero et al., 2004). The nematode Caenorhabditis elegans encodes three profilin genes, pfn-1 , pfn-2 , and pfn-3 . Neither pfn-2 nor pfn-3 is essential, but both impact muscle development, as their simultaneous deletion ( pfn-2∆ pfn-3∆ ) results in modestly fewer but abnormally wide sarcomeres in striated body-wall muscle (BWM) (Kimmich et al., 2024; Polet et al., 2006), and their absence strongly exacerbates BWM filamentous actin (F-actin) defects in tropomodulin ( unc-94 ) mutants (Yamashiro et al., 2008). Additionally, worms lacking pfn-3 have misshapen and unstable dense bodies, structures that normally aid in anchoring thin filaments at sarcomere Z-lines in BWM (Kimmich et al., 2024). PFN-2 and PFN-3 proteins likely cooperate with the formin FHOD-1 , as worms lacking FHOD-1 , or expressing FHOD-1 missing its profilin-binding Formin Homology-1 domain, also have unstable dense bodies and fewer BWM sarcomeres (Kimmich et al., 2024; Mi-Mi et al., 2012).

Based on immunostain, PFN-3 was reported to localize to dot-like structures at the tips of dense bodies in BWM (Polet et al., 2006). With this anti- PFN-3 being no longer available (personal communications, S. Ono, Ph.D., Emory University), we set out to create a functional fluorescently-tagged PFN-3 . Amino (N)-terminal tagging of human profilin-1 with mApple and (SGLRSRAQAS) linker was shown to preserve a range of biochemical and cellular functions (Pimm et al., 2022). Thus, we set out to N-terminally tag PFN-3 with mScarlet and an identical linker. Our attempts to tag the endogenous pfn-3 locus using CRISPR-Cas9 failed, but we successfully introduced a single copy mScarlet:: pfn-3 fusion transgene ( mSc:: pfn-3 ) to a neutral locus on Chromosome II using the Mos-I transposon-mediated single copy insertion (MosSCI) technique (Frøkjær-Jensen et al., 2008). We included in this transgene 2023 bp pfn-3 upstream sequence plus the first two codons of pfn-3 upstream of mScarlet::linker , and the entire pfn-3 coding region downstream of the linker, including all pfn-3 introns and exons ( Fig. 1 A ). Two independent transgenic lines were isolated and validated by PCR (see Methods).

Western blot analysis of whole worm extracts using anti-mCherry verified expression of 41.2 kDa N-terminally-tagged PFN-3 (mSc:: PFN-3 ), compared to 26.4 kDa free mScarlet expressed in control strains ( Fig. 1 B ). This 14.8 kDa difference is close to 13.5 kDa predicted for PFN-3 . In live worms of both independent mSc:: PFN-3 lines, the fusion protein was visible in BWM, the anal depressor muscle (AD), and vulval muscles (VM) ( Fig. 1 C ), consistent with previous expression studies (Durham et al., 2021; Roux et al., 2023), as well as in pharyngeal muscles (PM), PM3 through PM8 ( Fig. 1 D ). Among the sarcomeres of BWM in live worms, mSc:: PFN-3 was arranged in striations as well as irregular patches between striations ( Fig. 1 E, BWM Sarcomere ), while in the cytosol overlying the sarcomeres, mSc:: PFN-3 appeared diffuse ( Fig. 1 E, BWM Cytosolic ). We did not observe dot-like mSc:: PFN-3 structures in live animals, but in fixed worms stained with fluorescent phalloidin to show F-actin, we sometimes observed mSc::PFN-3-positive dots associated with gaps in F-actin indicative of dense bodies ( Fig. 1 F, arrows ). We suggest dense body-associated mSc:: PFN-3 is likely present as was indicated by immunostain (Polet et al., 2006), but this is obscured in live animals by additional diffuse or loosely bound mSc:: PFN-3 that is lost during fixation and staining.

To compare PFN-3 and FHOD-1 localizations, we crossed mSc:: pfn-3 into a background expressing functional FHOD-1 bearing a carboxyl-terminal GFP tag ( FHOD-1 ::GFP) (Mi-Mi et al., 2012). FHOD-1 ::GFP normally occupies bright puncta and patchy striations in BWM (Mi-Mi et al., 2012; Sundaramurthy et al., 2020). In live animals, FHOD-1 ::GFP extensively overlapped with mSc:: PFN-3 , but, to within our limits of detection, there were also some areas of non-overlap ( Fig. 1 G ). This supports the possibility that PFN-3 and FHOD-1 interact within the sarcomeres to promote sarcomere growth and proper dense body morphogenesis, while areas of non-overlap may mark an inactive pool of FHOD-1 and/or FHOD-1 acting independently of PFN-3 .

To test whether mSc:: PFN-3 is functional, we crossed mSc:: pfn-3 into a pfn-2∆ pfn-3∆ background. In wild-type worms, dense bodies are semi-regular in shape and spacing along striations, while dense bodies in pfn-2∆ pfn-3∆ mutants appear irregular (Kimmich et al., 2024). This defect can be quantified by measuring the percent area occupied by the dense body marker a-actinin/ ATN-1 in a field of view, as dense body malformation increases the relative area occupied by ATN-1 (Kimmich et al., 2024). Based on analysis of ATN-1 immunostain, mSc:: PFN-3 expression restored normal dense body morphology to pfn-2∆ pfn-3 ∆ worms ( Fig. 1 H & I ). pfn-2∆ pfn-3∆ mutants have a very modest but statistically significant reduction in the width of BWM cells, which correlates with the presence of fewer sarcomeres per cell (Kimmich et al., 2024; Mi-Mi et al., 2012). When we measured the width of phalloidin-stained BWM cells, again we observed mSc:: PFN-3 rescued pfn-2∆ pfn-3 ∆ mutant defects ( Fig. 1 J ). Additionally, we observed no dense body or cell size defects in otherwise wild-type animals expressing mSc:: PFN-3 ( Fig. 1 H-J ), suggesting the mSc:: pfn-3 transgene itself does not cause muscle defects.

Our results show mSc:: PFN-3 is functional with regard to sarcomere development in BWM, making it a useful tool for studying the muscle-specific functions of this profilin family member.

Plasmids

Plasmid pKM7 with mSc:: pfn-3 was generated using In-Fusion cloning (Takara Bio USA, San Diego, CA) to combine: 2023 bp pfn-3 upstream sequence (PCR-amplified from N2 genomic DNA); worm codon-optimized mScarlet with 10-amino acid SGLRSRAQAS linker (PCR-amplified from LP896 genomic DNA); pfn-3 coding region including introns and exons (from N2 genomic DNA); and vector backbone (from pCFJ90). lim-6 is positioned almost immediately downstream of pfn-3 , with an opposite orientation. In the hopes of avoiding induction of RNA interference against lim-6 , we chose to not include pfn-3 downstream sequence. Instead, we utilized the unc-54 3'UTR already present in pCFJ90, as this 3'UTR will support expression in most, if not all, somatic tissues (Mello and Fire, 1995). MosSCI recombination template plasmid pMK14 was generated by In-Fusion cloning mSc:: pfn-3 (from pKM7) into pCFJ151. Plasmid pKM16 expressing free mScarlet was generated using In-Fusion cloning to replace mCherry in BamHI/EcoRI-linearized pCFJ104 with mScarlet . All plasmid sequences were verified by next generation whole-plasmid sequencing (Genewiz, South Plainfield, NJ).

Worm Strains and Growth Conditions

Worms were maintained on nematode growth medium agar with OP50-1 bacteria food at 20°C (Brenner, 1974). MosSCI (Frøkjær-Jensen et al., 2008) was performed by gonadal microinjection of EG4322 adults with pKM14 (50 ng/µl), transposase-expression vector pJL43.1 (50 ng/µl), and GFP-expressing pDP401 (5 ng/µl), pSDV30 (2.5 ng/µl) and pSDV31 (5 ng/µl). Progeny of injected animals were grown to starvation and then screened for normal motility (from unc-119 (+) in pKM14) and absence of GFP (which otherwise would indicate the presence of an extrachromosomal array). Insertion at the ttTi5065 locus was confirmed by PCR-based detection of upstream and downstream insertion junctions, and failed detection of unmodified ttTi5065 locus. mSc:: pfn-3 coding regions were sequenced from two independent isolates ( DWP359 , DWP360 ) to confirm absence of mutations. mSc:: pfn-3 was outcrossed from DWP359 into a clean N2 background before crossing into DWP3 with fhod-1 ::gfp (resultant DWP396 ), or into DWP374 with pfn-2 (∆) pfn-3 (∆) (resultant DWP391 ). For soluble mScarlet expression, N2 adults were gonadally microinjected with pKM16 (5 ng/µl), pSDV30 (2.5 ng/µl), pDP401 (10 ng/µl), and pRS315 (82.5 ng/µl), and two independent mScarlet- and GFP-positive transformants were isolated ( DWP371 , DWP372 ).

Western blot analysis

Worms were collected and lysed as described previously (Yingling & Pruyne, 2021). Sample loads were normalized based on whole-lane Coomassie brilliant blue stain of worm extracts after SDS-PAGE. Nitrocellulose blots of normalized extracts were probed with Living Colors mCherry Monoclonal Antibody (diluted 1:1000) and peroxidase-conjugated goat anti- mouse (diluted 1:3000), as described previously (Mi-Mi et al., 2012).

Microscopy

Worms were fixed and stained with Alexa Fluor 488-phalloidin (diluted 1:250) to detect F-actin, or with antibody MH35 (diluted 1:10,000) to detect ATN-1 and FITC-conjugated goat anti- mouse (diluted 1:500), as previously (Kimmich et al., 2024). Live worms were immobilized for microscopy in 3.5 µL of 0.1 µm-diameter polystyrene nanobead suspension (Polysciences, Warrington, PA) between a coverslip and 10% agarose pad, as previously (Kimmich et al., 2024). Fixed or live worms were imaged on an SP8 Laser Scanning Confocal Microscope (Leica, Wetzlar, Germany) using LAS X software (version 3.5.2, build 4758; Leica) and HCX plan apochromat 63x/NA 1.4 oil immersion lambda objective. Deconvolution was performed using Huygens Essential software (Huygens Compute Engine 18.10.0, Scientific Volume Imaging, Hilversum, Netherlands). Images were false colored and subject to linear brightness adjustment using Adobe Photoshop CS4 (Adobe, San Jose, CA). Widths of BWM cells in phalloidin-stained day-1 adults were measured for two to four cells in each of 20 animals, in each of three independent replicate experiments, as previously (Kimmich et al., 2024; Mi-Mi et al., 2012; Sundaramurthy et al., 2020). Quantification of ATN-1 immunostain area in day-1 adults was performed for one image with 4-6 striations for each of 10 animals, in each of three independent replicate experiments, as previously (Kimmich et al., 2024). Analyses were performed blinded to strain identity.

Statistical Analysis

Graphs were designed in Prism 10 (version 10.1.1 ; GraphPad Software, Boston, MA). Data were analyzed using one-way analysis of variation with a Tukey's multiple comparison post hoc.p < 0.05was considered statistically significant. Data are shown as mean ± SD, and as individual measurements.