SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the γ-tubulin–mediated addition of centriolar microtubules

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Figure S1. GFP fusions with SAS-4 and SAS-6 can functionally substitute for the corresponding endogenous proteins. Because no mutant alleles in sas-4 or sas-6 are currently available, we used an RNAi-based approach to confirm functionality of GFP fusions with SAS-4 and SAS-6. Strains were generated expressing each GFP fusion from an integrated transgene rendered insensitive to targeting by dsRNAs that deplete protein expressed from the endogenous locus. (A and B) RNAi-resistant (RR) transgenes encoding GFP:SAS-4 and GFP:SAS-6 were generated by changing the nucleotide sequence of the regions in red without altering the encoded amino acid sequence or codon bias. Quantitative immunoblotting for SAS-4 (A) or SAS-6 (B) confirmed that the GFP fusions were expressed at levels comparable to those of the endogenous proteins and that dsRNAs corresponding to the regions altered in the RR transgenes selectively depleted the endogenous proteins. For each blot, an extract prepared from wild-type N2 worms (left lane), serial dilutions of extract prepared from worms expressing the RR transgene (center lanes), and extract prepared from worms expressing the RR transgene injected with the dsRNA (right lane) were analyzed; numbers indicate the percentage of amount loaded in 100% lanes. Blots were reprobed for α-tubulin as a loading control (bottom blot panels). Asterisks in B denote contaminant bands. (C and D) Importantly, expression of GFP:SAS-4 (C) or GFP:SAS-6 (D) from the RNAi-resistant transgenes but not from transgenes in which the endogenous sequence was unaltered restored centriole duplication and rescued the embryonic lethality that results from depletion of the endogenous proteins. DIC and fluorescence images are shown for embryos in the second mitotic division together with a schematic interpretation of the phenotype and embryo viability measurements (n = 8–14 worms and 140–1,044 embryos per condition). Embryos from animals carrying the unaltered transgene (left) fail centriole duplication and have monopolar spindles in each daughter cell at the two-cell stage. Expression of GFP:SAS-4 or GFP:SAS-6 from the RR transgenes (SAS-4, right; and SAS-6, middle) rescues spindle bipolarity and embryonic viability. Viability of embryos derived from adults carrying the gfp::sas-6^RR transgene is <100% even without dsRNA treatment, likely because of integration of this transgene into an essential locus (the strain is an obligate heterozygote for the transgene insertion). This may also explain why only 50% of the embryos laid by dsRNA-treated worms carrying the gfp::sas-6^RR transgene are viable, because 25% of the offspring carry no transgene. Brood sizes were similar for dsRNA-treated worms carrying unaltered and RNAi-resistant transgenes (not depicted). Times (min:s) in image panels are relative to nuclear envelope breakdown of the first embryonic division. Bars, 10 μm.

Materials and methods. Strains containing integrated transgenes directing the expression of GFP:SAS-4 or GFP:SAS-6 that are insensitive to dsRNA targeting the endogenous message (OD111 and OD115 for sas-4 and sas-6, respectively) were generated by resequencing ∼500 bp at the 5′ end of sas-4 (ATG to MscI site) and ∼300 bp at the 3′ end of sas-6 (NdeI site to stop codon) by shuffling alternative codons within the sequence (thereby maintaining codon bias) and replacing introns with synthetic introns optimized for C. elegans expression (provided by A. Fire, Stanford University, Stanford, CA). Strains were maintained at 25°C. Immunoblotting of sas-4 and sas-6(RNAi) and wild-type control worms was performed as described previously (Hannak, E., M. Kirkham, A.A. Hyman, and K. Oegema. 2001. J. Cell Biol. 155:1109–1116). SAS-4 and SAS-6 were detected using antibodies raised against residues 1–120 and 401–492 of SAS-4 and SAS-6, respectively. Polyclonal antibodies were raised and affinity purified as described previously (Oegema, K., A. Desai, S. Rybina, M. Kirkham, and A.A. Hyman. 2001. J. Cell Biol. 153:1209–1226). Blots were reprobed with DM1α (Sigma-Aldrich) as a loading control.