C. elegans strains

Strains used in this study and their genotypes are listed in Table S1 and were maintained on nematode growth medium (NGM) plates seeded with OP50 E. coli at 20°C using standard techniques (Stiernagle, 2006). Re-encoded nmy-2 transgene fused to mCherry in strain GCP22 was generated by Mos1-mediated single-copy transgene insertion (MosSCI) onto chromosome II, tTi5605 (Frøkjaer-Jensen et al., 2008; Frøkjær-Jensen et al., 2012). The nmy-2 promoter region (5.2 kb), ORF and 3′UTR (1.3 kb) were cloned as overlapping fragments from genomic DNA. A region of ∼400 bp in exon 12 was re-encoded so that transgenic nmy-2, and not endogenous nmy-2, could be specifically depleted by RNAi. mCherry was cloned from pDC122 and all fragments were assembled with pCFJ151 backbone through Gibson assembly (Gibson et al., 2009). Single-copy transgene insertions were generated by injecting a mixture of target plasmid pAC71, plasmid with transposase pCFJ601 and plasmids carrying the selection markers pCFJ90, pCFJ104 and pGH8 into the strain EG6429, as described previously (Frøkjaer-Jensen et al., 2008; Frøkjær-Jensen et al., 2012). Transgene integrations were confirmed by PCR of regions spanning each side of the insertion, by sequencing of the entire genomic DNA locus and by fluorescence microscopy.

To generate point mutants in nmy-2 and unc-54, the endogenous loci of nmy-2 and unc-54 were modified using CRISPR/Cas9-mediated genome editing. Two or three single guide RNAs (sgRNAs) were cloned into the pDD162 vector (Dickinson et al., 2013) and injected together with a single-stranded repair template carrying the modified sequence of interest flanked by 35-50 bp homology regions (IDT ultramer) into gonads of young adult N2 hermaphrodites. All sgRNAs and repair templates used are listed in Table S2. To facilitate the identification of successfully injected animals, injection mixes contained a sgRNA and a repair template to generate the R92C mutation in the dpy-10 gene, which causes a dominant roller phenotype (Arribere et al., 2014; Levy et al., 1993). All point mutations were verified by DNA sequencing. To remove potential off-target mutations, mutants were subjected to six rounds of outcrossing with N2 animals. nmy-2(S251A) and nmy-2(R252A) homozygous animals were sterile. To propagate these animals, two approaches were used. In the first approach, nmy-2(S251A) and nmy-2(R252A) animals were balanced with hT2 (from strain JK2739) to generate strains GCP629 and GCP513, respectively. Heterozygous mutant animals were fertile and easily identifiable, and homozygous animals were sterile (their gonads are shown in Fig. 2A). In the second approach, we were able to keep the nmy-2(S251A) and nmy-2(R252A) animals in homozygosity after crossing the strains GCP629 and GCP513 with GCP22 to generate the strains GCP618 and GCP592, respectively, which contained mutated endogenous nmy-2 on chromosome I and transgenic nmy-2 (re-encoded for RNAi resistance and fused to mCherry but otherwise wild type) on chromosome II. The expression of transgene-encoded NMY-2::mCherry kept nmy-2(S251A) and nmy-2(R252A) animals fertile and led to the production of viable embryos.

RNA interference

RNAi was performed by feeding hermaphrodites with bacteria that express dsRNA. DNA fragments of target genes were cloned into the L4440 vector, which was transformed into HT115 E. coli. Primers in Table S3 were used to amplify nmy-2 fragments from N2 cDNA (nmy-2_RNA#1) or from the synthesized re-encoded region (nmy-2_RNA#2). These fragments were cloned using the EcoRV restriction site in L4440. For RNAi of myo-3 or unc-54, the corresponding L4440 vectors were obtained from the Ahringer library (Source Bioscience) and sequenced to confirm the gene target. Expression of dsRNAs in transformed HT115 was induced with 1 mM IPTG and the bacteria were fed to hermaphrodites as described below.

As protein levels in newly fertilized embryos gradually decrease with increasing duration of RNAi treatment of the mother (Kirkham et al., 2003; Velarde et al., 2007), different RNAi conditions were used to deplete NMY-2 depending on the desired level of depletion. RNAi treatment conditions were different depending on the use of nmy-2_RNA#1, nmy-2_RNA#2 or nmy-2_RNA#3:

In experiments where specific depletion of transgenic NMY-2::mCherry^sen was desired [labeled as nmy-2::mCherry^sen(RNAi) in the figures], nmy-2_RNA#2 was used in GCP22, GCP618 or GCP592 L4 hermaphrodites at 20°C. Mild depletions (22-26 h of RNAi treatment) were used in Figs 4A,B,D,E, 6A, and 7A-D. Under these conditions, most embryos were able to complete cytokinesis. Penetrant depletions (36-38 h of RNAi treatment) were used in Figs 4B,C and 7E. In these conditions all one-cell embryos failed cytokinesis. More-penetrant depletions (38-48 h of RNAi treatment) were used in Fig. 3D. Under these conditions hermaphrodites presented non-compartmentalized gonads and stopped producing embryos, and embryos laid at earlier time points were not viable.

In experiments whose purpose was to deplete endogenous NMY-2 to an extent that did not preclude cytokinesis [labeled as nmy-2(RNAi) in the figures], nmy-2_RNA#1 was used in L4 hermaphrodites at 20°C (Fig. 6, Fig. S3). Mild depletions (26-28 h of RNAi treatment) were used in GCP21, GCP401 and GCP420 strains in Fig. 5F,H and Fig. S5H. Under these conditions most nmy-2(S250A) and all nmy-2(R718C) embryos were able to complete cytokinesis albeit more slowly than controls. Partial depletions (30-32 h of RNAi treatment) were used in strains GCP21 (Fig. 6B), GCP179 (Fig. S3A-C) and GCP22 (Fig. S3D-G). Under these conditions most embryos were able to complete cytokinesis albeit more slowly than controls. In Fig. S3D-G, both endogenous and transgenic NMY-2::mCherry were simultaneously depleted using nmy-2_RNA#3.

Depletion of MYO-3 was accomplished by incubating L1 hermaphrodites of strains GCP565 and GCP619 (Fig. 1D), GCP523 (Fig. S4A) and GCP524 (Fig. S5B) for 72 h at 20°C. Depletion of MYO-3, UNC-54 or simultaneous depletion of MYO-3 and UNC-54 in Fig. 1D,F,G, Figs S4A and S5B were performed by feeding L1 hermaphrodites of strain N2 for 72 h at 20°C. For double RNAi of myo-3 and unc-54, bacteria expressing each of the constructs were mixed 1:1 before seeding RNAi plates (Fig. 1F).

Live imaging

Gravid hermaphrodites were dissected and one-cell embryos were mounted in a drop of M9 (86 mM NaCl, 42 mM Na₂HPO₄, 22 mM KH₂PO₄ and 1 mM MgSO₄) on 2% agarose pads overlaid with a coverslip. Live imaging of cytokinesis was performed at 20°C. Images were acquired on a spinning disk confocal system (Andor Revolution XD Confocal System; Andor Technology) with a confocal scanner unit (CSU-X1; Yokogawa Electric Corporation) mounted on an inverted microscope (Ti-E, Nikon) equipped with a 60×1.42 oil-immersion Plan-Apochromat objective, and solid-state lasers of 405 nm (60 mW), 488 nm (50 mW) and 561 nm (50 mW). For image acquisition, an electron multiplication back-thinned charge-coupled device camera (iXon; Andor Technology) was used. Acquisition parameters, shutters and focus were controlled using Andor iQ3 software. For central plane imaging in one-cell embryos, 6×1 μm z stacks were collected in the 488 nm and 561 nm channels every 10 s in embryos of GCP22, GCP592, GCP618, GCP21, GCP401, GCP420 and GCP179 strains. For cortical imaging in one-cell embryos, 7×0.5 μm z stacks were collected in the 488 nm and 561 nm channels every 5 s in embryos of GCP22, GCP592 and GCP618 strains. For imaging GCP401 and GCP420 embryos, the same protocol was applied using the 488-nm channel.

Embryonic viability and egg laying assay

In Figs S4E and S5F, L4 hermaphrodites of strains GCP21, GCP401 or GCP420 were placed on NGM plates and singled out onto fresh plates after 40 h at 20°C. Adult gravid hermaphrodites were allowed to lay eggs for 8 h. In Fig. 3D, GCP22, GCP618 or GCP592 L4 hermaphrodites were placed on plates with bacteria expressing nmy-2_RNA#2 for 40 h at 20°C. Adult hermaphrodites were singled out onto fresh RNAi plates and left to lay eggs for 8 h. In all cases, animals were removed after the egg laying interval and embryos were left to hatch at 20°C for 24 h before counting. The number of hatched and unhatched (dead) embryos was counted and the fraction of viable embryos calculated by dividing the number of hatched embryos by the total number of progeny. To measure egg-laying rates in Fig. 1E, Figs S4B and S5C, L4 hermaphrodites of strains N2, GCP523, GCP524, GCP565 or GCP619 were placed on NGM plates and singled out onto fresh plates after 40 h at 20°C. Adult hermaphrodites were allowed to lay eggs for 8 h. The egg laying rate was calculated by dividing the total number of eggs on the plate by the number of hours during which eggs were laid.

Worm protein extracts and immunoblotting

For protein sample preparation in Fig. 3C and Fig. S4D, 60 L4 hermaphrodites of strains N2, GCP22, GCP592 GCP618 or GCP401 were grown on NGM plates for 46 h at 20°C and pelleted at 750 g. Animals were washed three times in M9 medium supplemented with 0.1% Triton X-100. Lysis was performed by resuspending the pellet in SDS-PAGE sample buffer [250 mM Tris (pH 6.8), 30% (v/v) glycerol, 8% (w/v) SDS, 200 mM DTT and 0.04% (w/v) bromophenol blue] with one-third the volume of quartz sand (Sigma). Tubes were subject to three 5 -min cycles of alternating boiling at 95°C and vortexing, after which the quartz sand was pelleted and the supernatant recovered. Protein samples were resolved by SDS-PAGE in a 7.5% gel and transferred to 0.2-μm nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris, 140 mM NaCl, and 0.1% Tween, pH 7.6) and probed at 4°C overnight with the following primary antibodies: anti-NMY-2 (1:10,000, home-made rabbit polyclonal antibody against residues 945-1368) and anti-α-tubulin (1:5000, mouse monoclonal DM1-α, T6199, Sigma). Membranes were washed three times with TBS-T and incubated for 1 h at room temperature with the following HRP-conjugated secondary antibodies: goat anti-rabbit (1:5000, 111-035-144, Jackson ImmunoResearch) and goat anti-mouse (1:5000, 115-035-003, Jackson ImmunoResearch). Membranes were washed three times with TBST and proteins were visualized by chemiluminescence using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and a ChemiDoc XRS+ System with Image Lab Software (Bio-Rad).

Protein sequence alignments

Myosin protein sequences for alignments shown in Fig. 1B,C, Figs S1 and S5A were obtained from the Uniprot database with the following accession numbers: P08799|MYS2_DICDI; G5EBY3|G5EBY3_CAEEL; Q20641|Q20641_CAEELQ99323|MYSN_DROME; F1QC64|F1QC64_DANRE; F8W3L6|F8W3L6_DANRE; F1R3G4|F1R3G4_DANRE; 93522|O93522_XENLA; Q04834|Q04834_XENLA; Q8VDD5|MYH9_MOUSE; Q61879|MYH10_MOUSE; Q6URW6|MYH14_MOUSE; P35579|MYH9_HUMAN; P35580|MYH10_HUMAN; Q7Z406|MYH14_HUMAN; and P02566|MYO4_CAEEL. Sequences were aligned using Jalview software (Waterhouse et al., 2009) and the muscle algorithm with default parameters (Edgar, 2004).

Protein expression and purification

Primers used for cloning of expression constructs are listed in Table S4, and plasmid information is provided in Table S5. DNA coding for wild-type NMY-2 S1 (amino acids 1-874), NMY-2 HMM (amino acids 1-1364), full-length MLC-4, full-length MLC-5 and full-length LET-502 was amplified from C. elegans cDNA and cloned into the pACEbac1 expression vector using Gibson assembly (Gibson et al., 2009). NMY-2 constructs were tagged N-terminally with 6×His followed by a flexible linker (GlySerGlySerGly). LET-502(1-469), MLC-4 and MLC-5 were tagged N-terminally with StrepTagII followed by a flexible linker (GlySerGlySerGly). To obtain the vectors for expression of NMY-2 point mutants (S250A, S251A and R252A), the pACEbac1 vector containing wild-type nmy-2 cDNA was used as a template for site-directed mutagenesis. To obtain the pACEbac1 vector for expression of LET-502(1-469), the pACEbac1 containing full-length let-502 cDNA was amplified using back-to-back primers to delete the coding sequence downstream of amino acid 469. The resulting vector was re-ligated in a single reaction combining PNK kinase for PCR product phosphorylation (NEB), DpnI for template removal by digestion (Thermo-Fisher Scientific) and DNA T4 ligase (NEB). Plasmids were transformed in DH5-alpha or TOP10 competent bacteria and all constructs were verified by Sanger DNA sequencing.

Bacmid recombination was performed in DH10EMBacY bacteria (Geneva Biotech) and Sf21 cells were transfected with each of the bacmids using X-tremeGene HP DNA Transfection Reagent (Roche). Virus production was performed as previously described (Bieniossek et al., 2008). For large-scale purification of NMY-2 S1 fragments or NMY-2 HMM fragments bound to MLC-4 and MLC-5 (referred to as NMY-2_S1 and NMY-2_HMM, respectively), corresponding baculoviruses were used to co-infect 500 ml cultures of Sf21 cells (0.8×10⁶ cells/ml, SFM4 medium; Hyclone). Cells were harvested by centrifugation at 3000 g for 5 min. Pellets were resuspended in lysis buffer [15 mM MOPS, 300 mM NaCl, 15 mM MgCl₂, 0.1% Tween 20, 1 mM EDTA, 3 mM NaN₃, 1 mM DTT (pH 7.3)] supplemented with EDTA-free Complete Protease Inhibitor Cocktail (Roche), sonicated and incubated for 20 min with 1 mM ATP to detach myosin from actin. Lysates were cleared by centrifugation at 34,000 g for 40 min. NMY-2_S1 was purified by batch affinity chromatography using Strep-Tactin Sepharose (IBA). Beads were washed with wash buffer [10 mM MOPS, 500 mM NaCl, 5 mM MgCl₂, 0.1% Tween 20, 1 mM EDTA, 3 mM NaN₃, 1 mM DTT (pH 7.3)] supplemented with 1 mM ATP in the first wash and eluted on a gravity column with elution buffer [10 mM MOPS, 500 mM NaCl, 2.5 mM desthiobiotin, 3 mM NaN₃, 1 mM DTT (pH 7.3)]. NMY-2_S1 was further purified by size-exclusion chromatography using a Superose 6 increase 10/300 column (GE HealthCare) pre-equilibrated with 10 mM MOPS, 500 mM NaCl and 1 mM EDTA (pH 7.3). Fractions containing NMY-2_S1 were pooled and concentrated in 50 MWCO Amicon Ultra-15 Centrifugal Filter Units (Merck-Millipore). NMY-2_HMM was purified by a tandem Strep-Tactin-6× His-tag affinity chromatography approach. The purification in Strep-Tactin Sepharose was similar to that used for NMY-2_S1, except that Tween-20 was excluded from the lysis and wash buffers, and 10 mM imidazole was added to the lysis buffer. Eluates from Strep-Tactin affinity chromatography were incubated with Ni-NTA beads (Thermo Fisher Scientific), washed with wash buffer 2 [10 mM MOPS, 500 mM NaCl, 25 mM imidazole, 3 mM NaN₃, 1 mM DTT (pH 7.3)] and eluted in elution buffer 2 [10 mM MOPS, 500 mM NaCl, 250 mM imidazole, 3 mM NaN₃, 1 mM DTT (pH 7.3)]. The eluate was concentrated using 50 MWCO Amicon Ultra-15 Centrifugal Filter Units (Merck-Millipore) and dialyzed overnight in low-salt buffer [10 mM MOPS, 25 mM KCl, 2 mM MgCl₂, 1 mM EDTA (pH 7.3)]. After dialysis, the samples were centrifuged at 2000 g for 5 min and supernatants were retained. For both NMY-2_S1 and NMY-2_HMM, glycerol and DTT were added to a final concentration of 10% (vol/vol) and 1 mM, respectively. Aliquots were flash-frozen in liquid nitrogen and stored at −80°C.

For LET-502(1-469) expression, baculoviruses were used to infect large-scale Sf21 cultures and protein was extracted from cells following similar procedures as described above but using the same buffer for lysis and washes [50 mM Tris, 150 mM NaCl, 3 mM NaN₃ 10% glycerol, 1 mM DTT (pH 7.3)] supplemented with EDTA-free Complete Protease Inhibitor Cocktail (Roche). LET-502(1-469) was purified by batch affinity chromatography using Strep-Tactin Sepharose (IBA). Beads were washed with lysis/wash buffers and eluted on a gravity column with elution buffer 3 [50 mM Tris, 150 mM NaCl, 3 mM NaN₃ 10% glycerol, 2.5 mM desthiobiotin (pH 7.3)]. Eluates were concentrated in a 10 MWCO Amicon Ultra-15 Centrifugal Filter Units (Merck-Millipore) and dialyzed overnight in dialysis buffer 2 [50 mM Tris, 150 mM NaCl, 3 mM NaN₃, 10% glycerol (pH 7.3)].

F-actin co-sedimentation assay

G-actin was from lyophilized human platelets (APHL99, Cytoskeleton) or rabbit muscle. Rabbit muscle G-actin was extracted from muscle acetone powder (Sigma, M6890) as described previously (Sellers, 1998) and lyophilized by freeze-drying after adding 2 mg of sucrose per mg of G-actin. Lyophilized G-actin was resuspended in water at 10 mg/ml and diluted to 1 mg/ml in G-actin buffer with ATP [5 mM Tris, 0.2 mM CaCl₂, 0.2 mM ATP (pH 8)]. Polymerization was induced by addition of 10× F-actin buffer [1× F-actin buffer: 5 mM Tris, 0.2 mM CaCl₂, 50 mM KCl, 2 mM MgCl₂, 1 mM ATP (pH 8)]. F-actin used in each round of co-sedimentation assays was from the same batch and source. Wild-type or mutant NMY-2_S1 was dialyzed overnight in F-actin buffer without ATP and supplemented with 1 mM DTT. All samples were pre-cleared by centrifugation at 150,000 g for 1 h in an Optima XP centrifuge with a TLA-100 rotor (Beckman-Coulter) prior to the assay. For the co-sedimentation assay, an equal amount (∼2 µM) of NMY-2_S1 was incubated with F-actin stock (final concentrations were 14.7 µM F-actin and 0.7 mM ATP) or a similar volume of F-actin buffer (negative control) for 30 min at room temperature. A similar approach was used for the estimation of the dissociation constant (Kd): 0.5 µM NMY-2_S1 was incubated with different amounts of actin as indicated in the plots in Figs 2E and 5C. Samples were centrifuged at 150,000 g for 1.5 h at room temperature. Supernatants (SIs) were removed and SDS-PAGE sample buffer was added to 1× final concentration. Pellets (PI) were resuspended in 30 µl of water and mixed by pipetting every 2 min over a period of 10 min on ice. SDS-PAGE sample buffer was added to 1× final concentration. To determine the ability of NMY-2_S1 to detach from F-actin, pellets prepared as above were resuspended in F-actin buffer supplemented with 50 mM ATP (without F-actin) over a period of 10 min on ice. After 30 min of incubation at room temperature, samples were centrifuged at 150,000 g for 1.5 h at room temperature. Supernatant and pellet fractions (SII, PII) were collected and prepared for loading in gels as described above. After addition of SDS-PAGE sample buffer, samples were incubated for 4 min at 95°C to denature proteins and run on 20-4% TGX gradient precast gels (Bio-Rad) in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Gels were stained with Blue Safe Coomassie (NZYtech) according to manufacturer's instructions (Figs 2C,F,H, 5A, Figs S2 and S5D). To verify the proteins bands corresponding to NMY-2 S1 fragments, immunoblotting with the mouse His-H8 antibody against the 6xHis tag (1:2500, 05-949 Merck Millipore) and a goat anti-mouse HRP-conjugated secondary antibody (1:5000, Jackson ImmunoResearch) was performed as described in the immunoblotting section.

In vitro phosphorylation assay

In vitro phosphorylation of MLC-4 was performed according to Haldeman et al. (2014). NMY-2_HMM (20 µg) were mixed with 0.5 µM of LET-502(1-469) in phosphorylation buffer [20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 50 mM NaCl, 1 mM CaCl₂]. The phosphorylation reaction was initiated by adding 2 mM ATP to the mix. The mix was incubated for 1 h at 24°C. The samples were precipitated with three volumes of acetone at −20°C and centrifuged at 15,000 g for 5 min. Acetone was removed and protein pellets were resuspended in urea sample buffer [8 M urea, 33 mM Tris-glycine (pH 8.6), 0.17 mM EDTA, 10 mM DTT added immediately before use] to a final concentration of ∼3.3 µg/µl. The entire sample was loaded without prior heating and separated in 4-20% TGX gradient 10-well precast gels (Bio-Rad) with Tris-glycine running buffer. Gels were stained with Blue Safe Coomassie stain (NZYtech) according to the manufacturer's instructions (Fig. 2H).

In vitro motility assay

In vitro motility assays were performed according to Sellers (1998). Actin was polymerized from lyophilized G-actin as described for co-sedimentation assays and a 2 µM stock was labeled by incubating F-actin overnight with rhodamine-phalloidin (1 unit of phalloidin for 100 µl of F-actin; Molecular Probes, Thermo Fisher Scientific) in labelling buffer [10 mM MOPS (pH 7.3), 0.1 mM EGTA, 3 mM NaN₃]. Motility chambers consisted of a microscopy slide taped to a 18×18 cm No. 1.5H coverslip (Marienfeld, Germany) previously coated with 1% nitrocellulose (2% collodion solution for microscopy, diluted to 1% with amyl acetate; Sigma-Aldrich). Two pieces of double-sided tape (Tesa) were used to form a ∼7 mm channel inside the chamber. NMY-2_HMM at 1 µg/µl in a high-salt solution [20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 500 mM NaCl] were flowed into the chamber. BSA solution [20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 500 mM NaCl, 1% BSA] was flowed to block the sites of the coverslip that did not bind NMY-2_HMM. Low-salt buffer [20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 50 mM NaCl] was used to remove unbound BSA. To block dead myosin heads and induce phosphorylation of MLC-4, the chamber was incubated with a solution containing unlabeled actin, LET-502(1-469) and ATP [20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 50 mM NaCl, 1 mM CaCl₂, 0.5 µM LET-502 (1-469), 5 µM unlabeled actin, 1 mM ATP] for four minutes. After washing, labeled F-actin solution [20 nM rhodamine-phalloidin F-actin, 1 mM DTT, 20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 50 mM NaCl] was flowed into the chamber. Unbound labeled F-actin was removed with low-salt buffer and assay buffer added [20 mM MOPS (pH 7.3), 0.1 mM EGTA, 5 mM MgCl₂, 25 mM KCl, 50 mM DTT, 1 mM ATP and 0.7% methylcellulose solution]. Chambers were imaged at 21°C every 2 s for 300 s total, using the microscope setup described above and the perfect focus system (Nikon) to maintain focus overtime.

F-actin and DNA labelling of body wall muscles and gonads

Phalloidin labelling of muscle actin shown in Fig. 1G and Fig. S4C was carried out in N2 adult animals depleted of UNC-54 or in adult hermaphrodites of strain GCP523, GCP565 and GCP619. Animals were collected and washed twice in M9 buffer, fixed with 4% paraformaldehyde (20% aqueous solution, Electron Microscopy Sciences), diluted in 1x cytoskeleton buffer (10 mM MES-KOH pH 6.1, 138 mM KCl, 3 mM MgCl₂, 2 mM EGTA) containing 0.32 M sucrose (Cramer and Mitchison, 1993) for 15 min, permeabilized with acetone at −20°C for 5 min, washed with PBS containing 0.5% Triton X-100 and 30 mM glycine (PBS-TG) for 10 min, and stained with 1:40 Oregon Green-phalloidin (Molecular Probes, Thermo Fisher Scientific) in PBS-TG for 30 min. After three 10 min washes with PBS-TG, animals were mounted with ProLong Antifade containing DAPI (Molecular Probes, Thermo Fisher Scientific). Labelling of gonads shown in Fig. 3A was carried out as described above in animals of strain N2, GCP513 and GCP623, but only the DAPI staining of the gonads is shown.

In Fig. 4A, DNA labelling of gonads was carried out in living adult hermaphrodites of strain GCP22, GCP592 and GCP618 depleted of NMY-2::mCherry^sen. Animals were transferred to a drop of water containing OP50 bacteria (1:10 dilution of a saturated culture) and 2 mg/ml Hoechst 33342 (Thermo Fisher Scientific) to label DNA and incubated for 1.5 h. After a recovery period of 30-60 min in NGM plates seeded with OP50, animals were anesthetized with levamisole (1 mg/ml, Sigma) and mounted on 2% agarose pads for imaging.

Image analysis, quantification and statistics

All microscopy image processing and measurements were carried out using Fiji (ImageJ; National Institutes of Health) (Schindelin et al., 2012) and Matlab (MathWorks). Z-stacks taken on the cell cortex were projected using the maximum intensity projection tool. Images within each figure panel are scaled equally. The equatorial region of the central plane was selected to create the kymographs shown in Figs 4C,D, 5F, Fig. S3C, using the Make Montage tool. Selected regions of 4 s time lapse movies (presented in Movie 1 for wild type, S251A and R252A NMY-2_HMM) were used to create 300 s time projections presented in Figs 2I and 5D by using the Temporal Color Code tool with the fire color scale.

Coomassie-stained SDS-PAGE gels were digitized in a GS-800 Calibrated Imaging Densitometer (Bio-Rad) and relative band intensity quantified in Image-Lab 5.2.1 (Bio-Rad). For each myosin mutant, the ability of NMY-2_S1 to co-sediment with F-actin was quantified by dividing the intensity of the band corresponding to the NMY-2 S1 fragment in the pellet by the sum of intensities of the NMY-2 S1 fragment bands in the supernatant and pellet. Graph plotting, linear regressions and statistical analyses were performed with Prism 7 or 8 (GraphPad Prism Software). Dissociation constant (Kd) estimations in Figs 2E and 5C were performed by fitting a ‘one-site specific binding’ model using a least squares nonlinear regression (GraphPad Prism Software). Error bars represent the 95% CI of the mean, except in Figs 2E and 5C where they represent the s.d., and in Fig. 7D where they represent the s.e.m. Statistical significance tests were performed using a Student t-test or one-way ANOVA with Bonferroni correction for multiple comparisons as indicated in figure legends.

Measurement of cytokinesis, ring assembly and furrow initiation time intervals, and ring constriction rate

Measurement of cytokinesis, ring assembly and furrow initiation time intervals, as well as ring constriction rate were performed in one-cell embryos of the following strains: GCP22, GCP592 and GCP618 (Fig. 4E); GCP21 and GCP401 (Fig. 5G-H); GCP21 and GCP420 (Fig. S5G-H); and GCP179 (Fig. S3B). This analysis only included embryos that completed ring constriction. Cytokinesis time was the interval between anaphase onset (myosin or Lifeact signal are cytoplasmic but absent from chromosomes, allowing the identification of the moment when the metaphase plate devoid of signal transitions into two separated DNA masses at anaphase) and the time when the contractile ring reached a diameter of ∼5 µm. Ring assembly time was the time interval between anaphase onset and the establishment of a shallow deformation in the equatorial region (time point when the equator shows first sign of deformation). Furrow initiation time corresponded to the time interval between the establishment of the shallow deformation and the time when the plasma membranes of the nascent daughter cells became juxtaposed to one another (back-to-back membrane configuration). During ring constriction, the distance between the two sides of the ring was measured in the z-plane where this distance was widest at each time point and plotted against time. Ring constriction rate was the slope of the linear region between ∼70% and 30% ingression. All intervals and ring constriction rate were determined based on imaging of the embryo central plane.

Fluorescence intensity measurements

To quantify the levels of Lifeact::GFP at the tip of the cytokinetic furrow in one-cell embryos, the mean fluorescence intensity in a 10-pixel wide, 30-pixel long (1.8×5.3 µm) region drawn over the tip of the furrow at 50% ingression was determined and the mean camera background was subtracted (a 40×40 pixel region placed outside of the embryos; Fig. 6A). The average intensity at the furrow tip is presented as a percentage of the corresponding controls. In Fig. 6B, quantification of actin levels in the contractile ring of ABa cells was carried out in 4-cell embryos of strain GCP22 by measuring the mean LifeAct::GFP fluorescence intensity in a manually traced 0.7 µm line over the circumference of the ring. The mean fluorescence intensity in a circle drawn in the cytoplasmic region at each time point was subtracted. Before quantification, each time-lapse movie was corrected for fluorescence intensity decay using the ImageJ bleach correction tool and the simple ratio method. Data from multiple rings were pooled and plotted against ring perimeter. The mean of data points that fell in overlapping 5 µm intervals was calculated and plotted against the perimeter at the center of each interval.

In Fig. S3D, NMY2::mCherry^sen intensity was calculated by measuring the average fluorescence intensity of a 130×35 pixel region placed over the cortical equatorial band and the camera mean fluorescence background was subtracted (a 40×40 pixel region placed outside of the embryos).

Analysis of band width and bundle alignment at the equatorial cortex

Equatorial actin band width in Fig. 7C was the length of a line traced across the band, perpendicularly to the division plane in cortical projections at shallow deformation. Values were normalized to embryo length along the anterior-posterior axis measured in the central plane, and averaged for each condition. Deviation from vertical alignment (°) over time in Fig. 7D was measured using the Directionality plugin for ImageJ. A region of 30×70 pixels corresponding to the furrow region was selected for each movie. The average directionality (angle α, in degrees), was calculated for every frame of each movie using the local gradient orientation method. Deviation from vertical alignment (in degrees) was calculated for each angle α, by subtracting the value of this angle from 90°. Absolute numbers were plotted.

Liquid thrashing assay

Hermaphrodites of strains N2, GCP523, GCP524, GCP565 or GCP619 were synchronized at the L1 stage by alkaline bleach treatment of adult gravid hermaphrodites (0.8% bleach, 250 mM NaOH; Stiernagle, 2006) to extract embryos, which were left to hatch overnight in M9 medium. L1 hermaphrodites were plated and grown at 20°C. Young adult hermaphrodites were transferred to an M9 droplet and left to acclimatize for 2 min after which images were acquired at ∼40 fps average in a SMZ 745T stereoscope (Nikon) mounted with a QIClic CCD camera (QImaging) and controlled by Micro-Manager software (Open Imaging). Body bends swimming frequencies in Fig. 1D,F and Figs S4A and S5B were automatically quantified using the wrMTrck plug-in using standard parameters (Nussbaum-Krammer et al., 2015) in ImageJ. Image background was removed by subtracting the average intensity projection of the stack and animals were segmented using Otsu intensity thresholding.