Abstract

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP’s neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα’s role in microautophagy. DNJ-14’s enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPβ and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.

中文翻译：

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通过 CRISPR/Cas9 介导的内源 DNJ-14 标记分析线虫中半胱氨酸串蛋白/DNAJC5 的组织分布

摘要

半胱氨酸串蛋白 (CSP) 是分子伴侣 DnaJ/Hsp40 家族的成员。CSP 在神经元中富集，主要定位于突触小泡。果蝇、蠕虫、小鼠和人类的 CSP 编码基因突变会导致神经元功能障碍、神经变性和寿命缩短。因此，尽管 CSP 也在非神经元细胞中表达，但大多数注意力都集中在 CSP 的神经元功能上。在这里，我们使用基因组编辑来荧光标记秀丽隐杆线虫 CSP 直系同源物dnj-14，以确定哪些组织优先表达 CSP，从而可能有助于观察到的突变表型。用 wrmScarlet替换dnj-14会导致强烈的趋化性缺陷，如其他dnj-14无效突变体所见。相比之下，在框内插入报告基因以创建 DNJ-14-wrmScarlet 融合蛋白对趋化性没有影响，表明 C 端标记不会损害 DNJ-14 功能。WrmScarlet 荧光在报告菌株的肠道、头/咽、受精囊和外阴/子宫中出现最明显，表明 DNJ-14 优先在这些组织中表达。将 DNJ-14-wrmScarlet 菌株与 GFP 标记菌株杂交证实了肠道和咽部表达，但仅观察到与神经元 GFP 的部分重叠。DNJ-14-wrm 肠道中的猩红色荧光因饥饿而增加，这可能与哺乳动物 CSPα 在微自噬中的作用有关。DNJ-14 在蠕虫生殖组织（受精囊和外阴/子宫）中的富集与哺乳动物中 CSPβ 和 CSPγ 亚型的睾丸特异性表达相似。此外，CSPα 信使 RNA 在人类近端消化道中高表达，表明 CSP 在胃肠系统内可能具有保守但被忽视的功能。