Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14

IF 3.2 3区生物学 Q3 CELL BIOLOGY Cell and Tissue Research Pub Date : 2024-02-26 DOI:10.1007/s00441-024-03875-w

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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.

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通过 CRISPR/Cas9 介导的内源性 DNJ-14 标记分析半胱氨酸串联蛋白/DNAJC5 在优雅子中的组织分布情况

摘要半胱氨酸串联蛋白（CSP）是分子伴侣 DnaJ/Hsp40 家族的成员。CSP富集于神经元，主要定位于突触小泡。苍蝇、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，但大部分注意力还是集中在 CSP 的神经元功能上。在这里，我们利用基因组编辑技术荧光标记秀丽隐杆线虫的 CSP 同源物 dnj-14，以确定哪些组织优先表达 CSP，从而可能导致观察到的突变体表型。用wrmScarlet替换dnj-14会导致强烈的趋化缺陷，这与其他dnj-14无效突变体一样。与此相反，在框架内插入报告因子以创建 DNJ-14-wrmScarlet 融合蛋白对趋化性没有影响，这表明 C 端标记不会损害 DNJ-14 的功能。WrmScarlet荧光在报告株的肠道、头/咽、精巢和外阴/子宫中最明显，表明DNJ-14优先在这些组织中表达。将 DNJ-14-wrmScarlet 株系与 GFP 标记株系杂交，证实了肠道和咽部的表达，但只观察到与神经元 GFP 的部分重叠。肠道中的 DNJ-14-wrmScarlet 荧光在饥饿时增加，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（精巢和外阴/子宫）中的富集与哺乳动物睾丸特异性表达的CSPβ和CSPγ异构体相似。此外，CSPα信使 RNA 在人类近端消化道中高度表达，这表明 CSP 在胃肠道系统中可能具有保守但被忽视的功能。

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来源期刊

Cell and Tissue Research 生物-细胞生物学

CiteScore

7.00

自引率

2.80%

发文量

142

审稿时长

1 months

期刊介绍： The journal publishes regular articles and reviews in the areas of molecular, cell, and supracellular biology. In particular, the journal intends to provide a forum for publishing data that analyze the supracellular, integrative actions of gene products and their impact on the formation of tissue structure and function. Submission of papers with an emphasis on structure-function relationships as revealed by recombinant molecular technologies is especially encouraged. Areas of research with a long-standing tradition of publishing in Cell & Tissue Research include: - neurobiology - neuroendocrinology - endocrinology - reproductive biology - skeletal and immune systems - development - stem cells - muscle biology.