ZFIN ID: ZDB-FIG-180511-35
Berger et al., 2018 - In Vivo Function of the Chaperonin TRiC in α-Actin Folding during Sarcomere Assembly. Cell Reports   22:313-322 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Anatomical Terms:
Stage: Protruding-mouth
PHENOTYPE:
Fish:
Observed In:
Stage: Protruding-mouth

Fig. 2

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

(A) cct3sa1761 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 cct3sa1761 homozygotes. Amplicons were identified by sequencing.

(C) Western blot analysis using antibodies against human CCT3 revealed epitope loss in cct3sa1761 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 cct3sa1761 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 cct3sa1761 homozygotes (arrows) (n = 5 per genotype).

(H) Expression of the Cct3-GFP fusion protein ameliorated the birefringence of cct3sa1761 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 cct3sa1761 (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 cct5tf212b 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 cct3sa1761 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 cct3sa1761 (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.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
cct3 Ab1-cct3 WT standard conditions Protruding-mouth whole organism WB
Ab1-cct3 cct3sa1761/sa1761 standard conditions Protruding-mouth Not Detected whole organism WB
cct4 Ab1-cct4 WT standard conditions Protruding-mouth skeletal muscle cell Z disc IHC
Ab1-cct4 Protruding-mouth whole organism WB
Ab1-cct4 cct3sa1761/sa1761 standard conditions Protruding-mouth skeletal muscle cell IHC
Ab1-cct4 Protruding-mouth skeletal muscle cell Z disc IHC
Ab1-cct4 cct4pc34/pc34 standard conditions Protruding-mouth Not Detected whole organism WB
EGFP cct3sa1761/sa1761 ; pc22Tg ; pc32Tg standard conditions Protruding-mouth skeletal muscle cell Z disc IFL
pc22Tg ; pc32Tg ; cct4pc34/pc34 standard conditions Protruding-mouth skeletal muscle cell IFL
Protruding-mouth Not Detected skeletal muscle cell Z disc IFL
pc32Tg ; pc22Tg standard conditions Protruding-mouth skeletal muscle cell Z disc IFL
pc32Tg ; pc22Tg ; cct5tf212b/tf212b standard conditions Protruding-mouth skeletal muscle cell Z disc IFL
mCherry cct3sa1761/sa1761 ; pc22Tg ; pc32Tg standard conditions Protruding-mouth skeletal muscle cell T-tubule IFL
pc22Tg ; pc32Tg ; cct4pc34/pc34 standard conditions Protruding-mouth skeletal muscle cell T-tubule IFL
pc32Tg ; pc22Tg standard conditions Protruding-mouth skeletal muscle cell T-tubule IFL
pc32Tg ; pc22Tg ; cct5tf212b/tf212b standard conditions Protruding-mouth skeletal muscle cell T-tubule IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-actn IHC WT standard conditions Protruding-mouth skeletal muscle cell Z disc
IHC cct4pc34/pc34 standard conditions Protruding-mouth skeletal muscle cell Z disc
Ab1-cct5 IHC WT standard conditions Protruding-mouth skeletal muscle cell Z disc
IHC cct4pc34/pc34 standard conditions Protruding-mouth skeletal muscle cell
IHC Protruding-mouth skeletal muscle cell Z disc
Phenotype Details
Fish Conditions Stage Phenotype
cct3sa1761/sa1761 standard conditions Protruding-mouth myotome skeletal muscle cell has fewer parts of type skeletal muscle cell skeletal muscle myofibril, abnormal
Protruding-mouth skeletal muscle cell cct4 expression spatial pattern, abnormal
Protruding-mouth skeletal muscle cell sarcomere organization decreased process quality, abnormal
Protruding-mouth skeletal muscle cell skeletal muscle myofibril decreased amount, abnormal
Protruding-mouth skeletal muscle cell Z disc cct4 expression decreased amount, abnormal
Protruding-mouth skeletal muscle cell Z disc malformed, abnormal
Protruding-mouth skeletal muscle skeletal myofibril assembly decreased occurrence, abnormal
Protruding-mouth whole organism cct3 expression absent, abnormal
cct3sa1761/sa1761 ; pc22Tg ; pc32Tg standard conditions Protruding-mouth myotome skeletal muscle cell has fewer parts of type skeletal muscle cell skeletal muscle myofibril, ameliorated
Protruding-mouth skeletal muscle cell skeletal muscle myofibril decreased amount, ameliorated
Protruding-mouth skeletal muscle skeletal myofibril assembly decreased occurrence, ameliorated
cct4pc34/pc34 standard conditions Protruding-mouth myotome skeletal muscle cell has fewer parts of type skeletal muscle cell skeletal muscle myofibril, abnormal
Protruding-mouth skeletal muscle cell Ab1-cct5 labeling spatial pattern, abnormal
Protruding-mouth skeletal muscle cell skeletal muscle myofibril decreased amount, abnormal
Protruding-mouth skeletal muscle cell Z disc Ab1-cct5 labeling decreased amount, abnormal
Protruding-mouth skeletal muscle skeletal myofibril assembly decreased occurrence, abnormal
Protruding-mouth whole organism cct4 expression absent, abnormal
pc22Tg ; pc32Tg ; cct4pc34/pc34 standard conditions Protruding-mouth skeletal muscle cell EGFP expression spatial pattern, abnormal
Protruding-mouth skeletal muscle cell Z disc EGFP expression absent, abnormal
pc32Tg ; pc22Tg standard conditions Protruding-mouth myotome skeletal muscle cell has normal numbers of parts of type skeletal muscle cell skeletal muscle myofibril, normal
Protruding-mouth skeletal muscle cell skeletal muscle myofibril amount, normal
Protruding-mouth skeletal muscle skeletal myofibril assembly occurrence, normal
Acknowledgments:
ZFIN wishes to thank the journal Cell Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Rep.