Cul3‐Klhl18 ubiquitin ligase modulates rod transducin translocation during light‐dark adaptation

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Abstract

<p id="d196005e210">Adaptation is a general feature of sensory systems. In rod photoreceptors, light‐dependent transducin translocation and Ca <sup>2+</sup> homeostasis are involved in light/dark adaptation and prevention of cell damage by light. However, the underlying regulatory mechanisms remain unclear. Here, we identify mammalian Cul3‐Klhl18 ubiquitin ligase as a transducin translocation modulator during light/dark adaptation. Under dark conditions, <i>Klhl18 <sup>−/−</sup> </i> mice exhibited decreased rod light responses and subcellular localization of the transducin α‐subunit (Tα), similar to that observed in light‐adapted <i>Klhl18 <sup>+/+</sup> </i> mice. Cul3‐Klhl18 promoted ubiquitination and degradation of Unc119, a rod Tα‐interacting protein. Unc119 overexpression phenocopied Tα mislocalization observed in <i>Klhl18 <sup>−/−</sup> </i> mice. Klhl18 weakly recognized casein kinase‐2‐phosphorylated Unc119 protein, which is dephosphorylated by Ca <sup>2+</sup>‐dependent phosphatase calcineurin. Calcineurin inhibition increased Unc119 expression and Tα mislocalization in rods. These results suggest that Cul3‐Klhl18 modulates rod Tα translocation during light/dark adaptation through Unc119 ubiquitination, which is affected by phosphorylation. Notably, inactivation of the Cul3‐Klhl18 ligase and calcineurin inhibitors <span style="fixed-case">FK</span>506 and cyclosporine A that are known immunosuppressant drugs repressed light‐induced photoreceptor damage, suggesting potential therapeutic targets. </p><p class="first" id="d196005e241">Ubiquitination‐dependent degradation of the transducin trafficking regulator Unc119 mediates adjustment of light sensitivity in rod photoreceptors. <div class="boxed-text panel" id="embj2018101409-blkfxd-0002"> <a class="named-anchor" id="embj2018101409-blkfxd-0002">  </a> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/6926b93c-c723-4d3c-b927-80ab4e026203/PubMedCentral/image/EMBJ-38-e101409-g015.jpg"/> </div> <div class="panel-content"/> </div> </p>

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Phosphate-binding tag, a new tool to visualize phosphorylated proteins.

Eiji Kinoshita, Emiko Kinoshita-Kikuta, Kei Takiyama … (2006)

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).

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Electroporation and RNA interference in the rodent retina in vivo and in vitro.

Takahiko Matsuda, Constance L. Cepko (2004)

The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.

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The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.

Mary Mattapallil, Eric F Wawrousek, Chi-Chao Chan … (2012)

We noted an unexpected inheritance pattern of lesions in several strains of gene-manipulated mice with ocular phenotypes. The lesions, which appeared at various stages of backcross to C57BL/6, bore resemblance to the rd8 retinal degeneration phenotype. We set out to examine the prevalence of this mutation in induced mutant mouse lines, vendor C57BL/6 mice and in widely used embryonic stem cells. Ocular lesions were evaluated by fundus examination and histopathology. Detection of the rd8 mutation at the genetic level was performed by PCR with appropriate primers. Data were confirmed by DNA sequencing in selected cases. Analysis of several induced mutant mouse lines with ocular disease phenotypes revealed that the disease was associated 100% with the presence of the rd8 mutation in the Crb1 gene rather than with the gene of interest. DNA analysis of C57BL/6 mice from common commercial vendors demonstrated the presence of the rd8 mutation in homozygous form in all C57BL/6N substrains, but not in the C57BL/6J substrain. A series of commercially available embryonic stem cells of C57BL/6N origin and C57BL/6N mouse lines used to generate ES cells also contained the rd8 mutation. Affected mice displayed ocular lesions typical of rd8, which were detectable by funduscopy and histopathology as early as 6 weeks of age. These findings identify the presence of the rd8 mutation in the C57BL/6N mouse substrain used widely to produce transgenic and knockout mice. The results have grave implications for the vision research community who develop mouse lines to study eye disease, as presence of rd8 can produce significant disease phenotypes unrelated to the gene or genes of interest. It is suggested that researchers screen for rd8 if their mouse lines were generated on the C57BL/6N background, bear resemblance to the rd8 phenotype, or are of indeterminate origin.