Phenotypes of zebrafish larvae knocked down with different concentrations of <i>ca10a</i> and <i>ca10b</i> antisense morpholinos.

<p>Phenotypes of zebrafish larvae knocked down with different concentrations of <i>ca10a</i> and <i>ca10b</i> antisense morpholinos.</p

Inactivation of <i>ca10a</i> and <i>ca10b</i> Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish

<div><p>Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the <i>CA11</i> gene, but instead possess two co-orthologs of <i>CA10</i>. We analyzed the expression pattern of zebrafish <i>ca10a</i> and <i>ca10b</i> genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish <i>ca10a</i> and <i>ca10b</i> reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish <i>ca10a</i> and <i>ca10b </i>genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of <i>ca10a</i> and <i>ca10b</i> showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the <i>ca10a</i> and <i>ca10b</i> morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of <i>ca10a</i> and <i>ca10b</i> expression in zebrafish larvae leads to a movement disorder.</p></div

Structural basis of vesicle formation at the inner nuclear membrane

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM

Silencing of <i>ca10a</i> and <i>ca10b</i> in zebrafish larvae.

<p><b>A)</b> Schematic presentation of matured <i>ca10a</i> mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). <b>B)</b> Schematic structure of unprocessed mRNA for <i>ca10a</i> with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. <b>C)</b> Schematic depiction of unprocessed mRNA for <i>ca10b</i> and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). <b>D)</b> Gel electrophoresis showing RT-PCR analysis of <i>ca10a</i> morphant mRNA injected with MO2. <b>E)</b> RT-PCR gel image of <i>ca10b</i> morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in <b>D</b> and Lane 3 and 4 in <b>E)</b> compared with wild type mRNAs of <i>ca10a</i> and <i>ca10b</i> in wild type fish (Lane 3 in <b>D</b> and lane 2 <b>E</b>). <b>F)</b> The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both <i>ca10a</i> and <i>ca10b</i>, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.</p