PMC

(A-B) Proteins that co-purified with MAP::SMCL-1 but not untagged control adult extracts, identified by tandem affinity purification and MudPIT mass spectrometry. Numbers represent average NSAF values from two replicas. Co-purified proteins with the highest NSAF values are shown in (A), values for other condensin subunits are shown in (B), and all other proteins are shown in . Condensin SMC subunits are highlighted.

Phylogenetic tree built from all available Caenorhabditis species with sequenced and well-assembled genomes, other selected nematode species, and other selected model organisms. “+” symbol denotes the presence of SMCL-1-like protein based on similarity in a BLAST search and the additional criteria of short length, imperfect signature motif, and a Walker B motif lacking the catalytic glutamate (see and ). “1” denotes orthologs detected using a high-confidence Ensemble-COMPARA method and “2” denotes orthologs detected using BLAST-neighbor-joining tree methods.

(A) Heat shock regimen for data in (B-E). Bolt represents the single heat-shock pulse given to young adult hermaphrodites from the wild-type or inducible hs:smcl-1(+) transgenic strain. (B-E) Adult hermaphrodite gut tissue of the indicated strain and treatment was stained with DAPI to image DNA (green in merge) and immuno-stained with antibody against CAPG-1(B-C), DPY-27 (D) and DPY-28 (E), (red in merges). Antibody against SMCL-1 was also included in (B and C), showing overexpression upon heat shock (blue in merge). The foci of staining created by condensin I^DC subunit association with the X chromosomes are lost when SMCL-1 is overexpressed. (C) A mosaic animal in which anti-SMCL-1 staining indicates that one cell lacks the transgene (left) and shows foci of anti-CAPG-1, while a neighboring cells has the smcl-1 overexpression transgene (right) and CAPG-1 staining is weak and not localized to foci (also see ). HS = heat shock.

(A) The three condensin complexes in C. elegans. Subunits used for purification in bold. (B) MAP-tagged condensin transgenes and strategy for tandem affinity purification and protein identification. DPY-27 and KLE-2 were tagged at the N-terminus; DPY-26 was tagged at the C-terminus. The MAP tag (yellow) includes mVenus for visualization, and the FLAG epitope and SBP for tandem affinity purification. (C-D) Identities of (C) condensin proteins and (D) non-condensin proteins that co-purified during tandem affinity purification from adult extracts with MAP-tagged DPY-27, DPY-26, or KLE-2 but not in untagged control, and were identified by MudPIT mass spectrometry. Relative protein abundance in the sample was estimated by calculating the number of spectra identified, normalizing it to protein length, and dividing by the total spectra in the sample. These normalized spectral abundance factor (NSAF) values are shown as the average from two replicas. The non-condensin proteins with the five highest NSAF values are shown in D; SMCL-1 is highlighted. All other mass spectrometry data in – Tables.

(A) PCR and Western blot analyses confirming genomic deletion of smcl-1 by a MosDEL strategy (see ). (B-D) smcl-1(0) worms were assayed for phenotypes associated with condensin loss-of-function. (B) Viable progeny count: The number of total viable progeny produced throughout the lifetime of a hermaphrodite was counted from 20 wild-type and 20 smcl-1(0) worms. (C) X nondisjunction: Hermaphrodite and male progeny of unmated wild-type or smcl-1(0) hermaphrodite worms were counted to assay for an increase in spontaneous production of males, which indicates mis-segregation of the X chromosome. ~6,600 progeny were counted from each strain in four trials. (D) Hermaphrodite vs. male ratio: Hermaphrodite and male progeny of 10 mated wild-type or smcl-1(0) hermaphrodite worms were counted to assay for hermaphrodite-specific lethality. (E-F) The effect of deleting smcl-1 in the dpy-28(y1) hypomorphic, temperature-sensitive (ts) strain was assayed. (E) Viable progeny count: The number of total viable progeny produced throughout the lifetime of the worm was counted from 33 hermaphrodite worms of each specified genotype raised at the semi-permissive temperature of 18°C. (F) Brood size: The number of embryos laid throughout the lifetime of the worm was counted from 33 hermaphrodite worms of each genotype raised at the semi-permissive temperature of 18°C. Non-parametric Mann-Whitney test was used for all statistical analyses. *** denotes p ≤ 0.001; ** denotes p ≤ 0.01; * denotes p ≤0.05; ns = not significant. Bars represent 95% confidence intervals.

(A) Adult hermaphrodites from wild-type (WT) and a strain carrying the map::smcl-1 transgene driven by endogenous smcl-1 5’ and 3’ elements. A section of the germline is shown, imaged by DIC to show structures and fluorescent microscopy to detect mVenus expression from the MAP tag. Arrowheads denote the first four oocytes. (B) A typical SMC protein folds back on itself at a hinge domain, bringing coil regions together and creating a “head domain” (yellow) from ATPase domains in the N- and C-termini. SMCL-1 lacks predicted coil and hinge domains, but has N- and C-terminal ATPase domains that may be capable of forming a head domain (purple). (C) SMC head domain and the ATPase cycle, showing binding of ATP (red circle), ATP-dependent engagement of heads from two SMC proteins, and disengagement upon ATP hydrolysis. (D) SMCL-1 amino acid sequence aligned to C. elegans condensin SMC proteins. Shown are regions surrounding three conserved motifs found in SMCs and related ATPases: the Walker A motif, ABC transporter signature motif, and Walker B motifs, and their consensus sequences. SMCL-1 shares a conserved Walker A motif, but differs from consensus signature motif and Walker B motif at residues shown in red. Asterisk denotes catalytic amino acid required for ATP hydrolysis. x = any amino acid and h = hydrophobic amino acid.

(A) The conditional overexpression transgene. The smcl-1 genomic sequence is driven by the hsp-16.48 promoter and contains the unc-54 3’UTR. The transgene was co-injected with unc-119(+) as the positive selection marker into unc-119(ed9) mutant worms. Worms containing multicopy transgene arrays with both the overexpression transgene and positive selection marker are designated hs:smcl-1(+). (B) Western blot of standard lysate showing increase in SMCL-1 protein level after heat shock in hs:smcl-1(+) worms, compared to an actin control. (C) Heat shock regimen for data in (E). Bolt symbols represent heat-shock pulses administered from late embryo to early L2 larval stage. (D) Cartoon explaining segregation of the extrachromosomal transgene array. Parents that are hs:smcl-1(+) give rise to embryos without the overexpression array indicated hs:smcl-1(-), and with the overexpression array indicated hs:smcl-1(+) and shaded gray; upon hatching, the progeny are identified by their uncoordinated and non-uncoordinated movements, respectively. Mosaics are also produced (not depicted). (E) Viability of progeny from hs:smcl-1(+) or wild-type (WT) parent worms after no heat shock (-, bottom) or after heat-shock pulses (+, bottom). For hs:smcl-1(+) parents, y-axis represents percent of embryos that develop into uncoordinated hs:smcl-1(-) adults without the transgene (white bars) or into non-uncoordinated hs:smcl-1(+) adults with the transgene (gray bars). Note no transgene-expressing progeny survive heat shock-induced smcl-1 overexpression. For wild-type parents, y-axis represents percent of embryos that develop to adults (striped bars). (F) Heat shock regimen for data in (H). Bolt symbol represents a single 2-hour heat-shock pulse administered at early L1 larval stage to monitor effects of smcl-1 overexpression on nuclear divisions in the gut that occur at late L1 larval stage. (G) Adult gut cells with DNA stained by DAPI and imaged by fluorescence microscopy. Connected chromatin between nuclei (right) indicates a prior chromosome segregation defect. (H) Chromosome segregation defect in the gut. L1 progeny of transgenic hermaphrodites were subjected to heat shock (+, bottom) or no heat shock (-, bottom) Some L1s were uncoordinated hs:smcl-1(-) L1 larvae without the transgene (white bars); others were non-uncoordinated hs:smcl-1(+) with the smcl-1 overexpression transgene (gray bars). The number of connected nuclei per worm, which indicates defective chromosome segregation, was scored in >40 worms of each genotype. PD7271 is a control strain that contains an unrelated extrachromosomal array. HS = heat shock. ** denotes p ≤ 0.01; * denotes p ≤0.05; ns = not significant by a non-parametric Mann-Whitney test. Bars represent 95% confidence intervals.