Fig. 1

A Missense Mutation in cct5 Causes Muscle Impairment within cct5^tf212b

(A) At 3 dpf, cct5^tf212b mutants appear similar to their siblings under bright-field conditions (n = 6 per genotype).

(B) Representative images of 3-dpf-old cct5^tf212b mutants under polarized light show a reduction in birefringence (quantified in C).

(C) Quantification of birefringence followed by normalization to 3-dpf-old siblings revealed a highly significant reduction in cct5^tf212b homozygotes through 7 dpf. Data are mean ± SEM; ^????p < 0.0001 homozygotes versus siblings of the same stage by one-way ANOVA with Tukey?s post hoc test; n = 3.

(D) SSLP-based mapping linked the phenotype-causing mutation to cct5 on chromosome 24. SSLP positions are shown in centimorgans and million base pairs according to the zebrafish genome assembly. Scale bar, 1 Mbp.

(E) Sequencing identified a missense mutation in exon 9 of cct5 (G422V).

(F) G422 (pink) forms part of the ATP-binding pocket (surface representation, green) of human CCT5. The glycine carbon is only 3.3 Å away from the adenosine nitrogen (stick representation, blue).

(G) Labeling of the sarcolemma with mCherryCaaX and the myofibril with Lifeact-GFP confirmed a reduced amount of myofibrils within cct5^tf212b homozygotes (n = 4 per genotype).

(H) H&E-stained sagittal sections of 6-dpf-old larvae confirmed the reduced amount of myofibrils in cct5^tf212b; arrows mark enlarged regions of the sarcoplasm (n = 8 per genotype).

(I) Representative transmission electron micrographs depicting organized sarcomeres, marked by brackets, in 3-dpf siblings and cct5^tf212b mutants (n = 3 per genotype).

(J) Immunohistochemistry with antibodies against actin (red) and ?-Actinin (green) enabled quantification of the sarcomere width that was not altered in cct5^tf212b (n = 3 per genotype).

(K) At the 18-somite stage, regularly spaced actinin-positive Z-bodies indicate the presence of pre-myofibrils in siblings and cct5^tf212b homozygotes (n = 4 per genotype).

(L) Maximal force generation of cct5^tf212b were significantly impaired compared with siblings. Data are mean ± SEM; ^????p < 0.0001 by Student?s t test; n = 9.

(M) Pulse duration and active force relationships are similar in both groups, suggesting unchanged motor neuron excitation. Data are mean ± SEM; n = 10.

(N) The insertion mutants cct1^hi3564Tg, cct2^hi1269Tg, cct5^hi2972Tg, and cct8^mn30Gt all feature a highly significant reduction in birefringence. Data are mean ± SEM; ^???p < 0.001 and ^????p < 0.0001 by Student?s t test; n = 3.

See also Figures S1?S5.