Fig. 2

Loss of TRiC Subunits Causes Lack of Functional TRiC at Sarcomeric Z-disks

(A) cct3^sa1761 harbors an essential splice site mutation in cct3 (red arrow).

(B) RT-PCR using oligonucleotides targeting regions marked by green arrows in (A) generated a 334-bp amplicon with WT embryos and a single 415-bp amplicon with cct3^sa1761 homozygotes. Amplicons were identified by sequencing.

(C) Western blot analysis using antibodies against human CCT3 revealed epitope loss in cct3^sa1761 homozygotes (GAPDH served as a loading control).

(D) CRISPR/Cas9-mediated deletion of 14 bp from exon 5 of cct4 causes a frameshift evoking premature stop codons in cct4⁻¹⁴.

(E) Western blot analysis using antibodies against CCT4 revealed epitope loss in cct4⁻¹⁴ homozygotes (GAPDH served as a loading control).

(F) At 3 dpf, the birefringence was significantly reduced in cct3^sa1761 and cct4⁻¹⁴ mutants compared with siblings. Data are mean ± SEM; ^∗∗∗∗p < 0.0001 by Student’s t test; n = 3.

(G) GFP fluorescence of Tg(cry:mCherry,-600unc:cct3GFP) localized to sarcomeric Z-disks as identified by co-localization with mCherryCaaX-positive t-tubules introduced by Tg(acta1:mCherryCaaX) in siblings and cct3^sa1761 homozygotes (arrows) (n = 5 per genotype).

(H) Expression of the Cct3-GFP fusion protein ameliorated the birefringence of cct3^sa1761 homozygotes with high significance. Data are mean ± SEM; ^∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey’s post hoc test; n = 3.

(I) The striated pattern obtained with antibodies against Cct4 in siblings was severely compromised in cct3^sa1761 (n = 5 per genotype).

(J) Compared with siblings, localization of Cct3-GFP to Z-disks is severely compromised in cct4⁻¹⁴ homozygotes transgenic for Tg(cry:mCherry,-600unc:cct3GFP) and Tg(acta1:mCherryCaaX) (n = 4 per genotype).

(K) Immunohistochemistry detected the localization of Cct5 at actinin-positive Z-disks in siblings but not in cct4⁻¹⁴ homozygotes (n = 3 per genotype).

(L) In cct5^tf212b homozygotes and siblings that harbor Tg(cry:mCherry,-600unc:cct3GFP) and Tg(acta1:mCherryCaaX), GFP-tagged Cct3 localized to Z-disks (arrows) (n = 3 per genotype).

(M) Representative TEM micrographs of cct3^sa1761 present a reduced amount of myofibrils (n = 3 per genotype). Importantly, although the sarcomeres appear organized, electron-dense rods were detected at Z-disks in cct3^sa1761 (red arrowheads). White arrowheads point to t-tubules at Z-disks. Boxed areas are shown at higher magnification.

See also Figures S4, S5, and S7.