Involvement of zebrafish Na+,K+ ATPase in myocardial cell junction maintenance

Na+,K+ ATPase is an essential ion pump involved in regulating ionic concentrations within epithelial cells. The zebrafish heart and mind (had) mutation, which disrupts the α1B1 subunit of Na+,K+ ATPase, causes heart tube elongation defects and other developmental abnormalities that are reminiscent of several epithelial cell polarity mutants, including nagie oko (nok). We demonstrate genetic interactions between had and nok in maintaining Zonula occludens-1 (ZO-1)–positive junction belts within myocardial cells. Functional tests and pharmacological inhibition experiments demonstrate that Na+,K+ ATPase activity is positively regulated via an N-terminal phosphorylation site that is necessary for correct heart morphogenesis to occur, and that maintenance of ZO-1 junction belts requires ion pump activity. These findings suggest that the correct ionic balance of myocardial cells is essential for the maintenance of epithelial integrity during heart morphogenesis

In Vivo Conditions to Identify Prkci Phosphorylation Targets Using the Analog-Sensitive Kinase Method in Zebrafish

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease

Pathways to understanding the genomic aetiology of osteoarthritis

Osteoarthritis is a common, complex disease with no curative therapy. In this review, we summarize current knowledge on disease aetiopathogenesis and outline genetics and genomics approaches that are helping catalyse a much-needed improved understanding of the biological underpinning of disease development and progression

Involvement of zebrafish Na+,K+ ATPase in myocardial cell junction maintenance.

Na(+),K(+) ATPase is an essential ion pump involved in regulating ionic concentrations within epithelial cells. The zebrafish heart and mind (had) mutation, which disrupts the alpha1B1 subunit of Na(+),K(+) ATPase, causes heart tube elongation defects and other developmental abnormalities that are reminiscent of several epithelial cell polarity mutants, including nagie oko (nok). We demonstrate genetic interactions between had and nok in maintaining Zonula occludens-1 (ZO-1)-positive junction belts within myocardial cells. Functional tests and pharmacological inhibition experiments demonstrate that Na(+),K(+) ATPase activity is positively regulated via an N-terminal phosphorylation site that is necessary for correct heart morphogenesis to occur, and that maintenance of ZO-1 junction belts requires ion pump activity. These findings suggest that the correct ionic balance of myocardial cells is essential for the maintenance of epithelial integrity during heart morphogenesis

Mutant Prkci^I316A has normal <i>in vivo</i> biological activity.

<p>(<b>A</b>) Reconstruction of confocal Z-stack sections of embryonic hearts at 28–30 hpf. Transgenic Tg[<i>cmlc2:GFP</i>]^twu34 one-cell stage embryos were injected with <i>prkci</i> MO alone or together with mRNA encoding Prkci^WT or analog-sensitive mutant forms of Prkci. Whereas the wild-type heart elongates into a heart tube and towards the left during cardiac jogging, heart development arrests at the cone stage and the heart remains at the embryonic midline in <i>prkci</i> morphants. In functional rescue experiments, injection of <i>prkci</i>MO together with mRNA encoding HisMyc-Prkci^WT or Prkci^I316A rescues heart tube elongation. In comparison, the analog-sensitive mutant form Prkci^V300A fails to rescue heart tube formation in <i>prkci</i> morphants. Percentiles indicate the occurrence of the most common phenotype as depicted in the images and numbers show the total of embryos tested. White dotted line indicates the embryonic midline. L, left; R, right. (<b>B</b>) Membrane localization of endogenous Prkci detected with an anti-Prkci antibody and exogenous Prkci^WT or Prkci^I316A in zebrafish cardiomyocytes detected with an anti-Myc antibody. Images are confocal reconstructions of single Z-stack sections of embryonic hearts marked by the transgenic reporter Tg[<i>cmlc2:GFP</i>]<i>^twu34</i> at 28–30 hpf. Expression of exogenous HisMyc-Prkci^WT or HisMyc-Prkci^I316A in cardiomyocytes reveals that both recombinant proteins localize to the cell membrane.</p

Thiophosphorylation of substrate proteins by Prkci^I316 in the zebrafish embryo.

<p>(<b>A</b>) Schematic diagram of the <i>in vivo</i> labeling method for the selective labeling of Prkci^I316A substrates during zebrafish development. (<b>B</b>) <i>In vivo</i> thiophosphorylation in zebrafish embryos injected at the one-cell stage with 200 μM <i>N⁶</i>-benzyl-ATPγS (6-bn-ATPγS) and mRNA encoding either Prkci^WT or Prkci^I316A (AS). Western blot analysis with rabbit monoclonal anti-thiophosphoester (α-thioP) C51-8 antibody (Epitomics) of 80% epiboly (6–8hpf) samples alkylated with 2.5 mM PNBM reveals a selectively labeled protein in the Prkci^I316A (AS) sample (asterisk).</p

Design of the analog-sensitive Prkci.

<p>(<b>A</b>) A space-creating mutation (“gatekeeper mutation”) is introduced into the kinase ATP binding pocket which allows the analog-sensitive (AS) kinase mutant to accept a bulky ATP analog (A*TP) required for the chemical genetic identification of kinase substrates [modified after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040000#pone.0040000-Cravatt1" target="_blank">[36]</a>]. (<b>B</b>) Alignment of the primary sequence of the ATP binding pocket within the kinase domains of Prkci, v-Src and c-Raf. The residues in red correspond to the amino acids mutated in v-Src <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040000#pone.0040000-Liu1" target="_blank">[1]</a>, c-Raf-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040000#pone.0040000-Hindley1" target="_blank">[28]</a>, and Prkci to enlarge the ATP binding pocket.</p