Fig 1.
Comparison of zebrafish vinculin isoforms.
(A) Phylogenetic analysis of the zebrafish vinculin genes with vinculin from other model systems based on the percentage identity on amino acid level. Percentage identity (in parenthesis) shown is compared to the human sequence. Used NCBI reference sequences: Drosophila (NP_476820.1), C. elegans (NP_501104.2), Human (NP_003364.1), Mouse (NP_033528.3), Chicken (NP_990772.1), Xenopus (NP_001090722.1). The protein sequences of the zebrafish vinculin isoforms were based on our own cDNA clones. (B) Schematic representation of vinculin protein structure. Binding partners of key regions are shown. (C-E) Multiple sequence alignment of vertebrate vinculin isoforms using ClustalOmega. Amino acid conservation is shown as bars underneath the alignment, with higher conservation shown as higher yellow bars, and lower conservation as lower darker yellow bars. Key regions are highlighted using bounded boxes. The alignment in (C) shows the first four helices of the D1 head domain, the alignment in (D) shows the loop domain, and the alignment in (E) shows the last three helices of the tail region.
Fig 2.
Zebrafish vinculin A and vinculin B localization.
Fixed α-catenin-depleted MDCK epithelial cells expressing either α-catenin-mCherry (top two rows, depicted in red) or α-cateninΔVBS-mCherry, which lacks the vinculin binding domain (bottom two rows, depicted in red). In addition, cells express zebrafish vinculinA-GFP or vinculinB-GFP (both depicted in green) and were stained for F-actin (blue). Asterisks mark Focal Adhesions, White arrows mark Focal Adherens Junctions and Yellow Arrowheads mark Linear Adherens Junctions.
Fig 3.
Generation of vinculin A-deficient zebrafish mutants using TALENs.
(A) Schematic representation of the endogenous vcla locus targeted by TALEN gene editing technology at the exon4-intron4 boundary. The TALEN arms flank a BclI restriction enzyme recognition site (highlighted in red) used for screening mutant alleles through restriction fragment length polymorphism (RFLP) analysis. (B) RFLP analysis of embryos injected with increasing dosages of vcla TALEN mRNA. Uncleaved bands represent efficient TALEN activity. (C) Summary of the range of mutations found at the vcla TALEN target locus in the F1 offspring of vcla TALEN-injected fish. Arrow denotes the mutation of the vcla mutant used in further experiments. (D) Sequence chromatograms from cDNA of wild-type vinculin (top) and of the vcla Δ8B mutant allele (bottom).
Fig 4.
Generation of vinculin B-deficient zebrafish using CRISPR-Cas.
(A) Schematic representation of the endogenous vclb locus showing the CRISPR guide RNA target site in exon 1. (B) Sequence chromatograms of genomic DNA from non-injected control (top) and vclb CRISPR-injected embryos. The arrowhead denotes the expected CRISPR cleavage site (3 nucleotides upstream of the PAM site). (C) Summary of mutations found in the F1 offspring of vclb CRISPR-injected fish of either a wild-type (top) or vcla mutant (bottom) background. Arrows denote the mutation of the vclb mutant used in further experiments. (D) Amino acid sequence of vclb in wild-type (top) and vclb mutant (bottom). Asterisk denotes the premature stop codon found in the vinculin B coding sequence of vclb mutants.
Fig 5.
Cardiac and skeletal muscle phenotypes of vinculin-null mutants.
(A) Western blot of lysates from the posterior half of WT, vcla-/- and vcla-/-vclb-/- embryos at 5 dpf, probed for vinculin and β-actin. (B) Some vinculin mutants show cardiac edema of which representative mild and severe cases are depicted. Wild type and vcla-/-vclb-/- mutants at 3 dpf (left) and 5 dpf (right). For corresponding images of the other genotypes described in (C), see supplemental S6 Fig. (C) Quantification of the presence of cardiac edemas in offspring from a vcla-/-vclb+/- incross. Classification as depicted in (B). Data was obtained from three independent experiments. WT embryos from an independent WT strain were analyzed as control from two independent experiments. Data is represented as mean ± s.e.m. A two-tailed paired student t-test was performed to compare the incidence of severe edemas between 3 dpf and 5 dpf within each genotype (see S7 Fig). (D) Immunostaining of actin in skeletal muscle of 5 dpf embryos. Images at the bottom are zoomed in parts of the upper images as indicated by the yellow squares (see S8 Fig for additional images and quantifications).