Francis Shimon P, Krey Jocelyn F, Krystofiak Evan S, Cui Runjia, Nanda Sonali, Xu Wenhao, Kachar Bechara, Barr-Gillespie Peter G, Shin Jung-Bum
Departments of Neuroscience.
Oregon Hearing Research Center & Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, and.
J Neurosci. 2015 Feb 4;35(5):1999-2014. doi: 10.1523/JNEUROSCI.3449-14.2015.
Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.
已知的耳聋基因中约有三分之一编码位于毛束中的蛋白质，毛束是感觉毛细胞的机械感受细胞器。在先前的研究中，我们使用质谱对毛束的蛋白质组进行了表征，从而发现了新的毛束蛋白。其中一种蛋白质是含Xin-肌动蛋白结合重复序列2（XIRP2），这是一种肌动蛋白交联蛋白，先前报道其在横纹肌中特异性表达。由于其他肌动蛋白交联蛋白的突变会导致听力损失，我们研究了XIRP2在听力功能中的作用。在内耳中，XIRP2在毛细胞中特异性表达，与毛束中富含肌动蛋白的结构、下方的表皮板和周向肌动蛋白带共定位。使用肽质谱分析表明，毛束含有一种先前未表征的XIRP2剪接变体，这表明XIRP2在毛细胞中的作用与在心肌细胞中报道的作用有显著差异。为了确定XIRP2在听力中的作用，我们应用了成簇规律间隔短回文重复序列（CRISPR）/Cas9介导的基因组编辑技术，在小鼠Xirp2基因中诱导靶向突变，从而消除内耳中XIRP2蛋白的表达。对所得Xirp2基因敲除小鼠的听力进行功能分析，发现其存在高频听力损失，对毛细胞进行超微结构扫描电子显微镜分析表明，这些小鼠的静纤毛发生退化。因此，我们得出结论，XIRP2是毛细胞静纤毛长期维持所必需的，其功能障碍会导致小鼠听力损失。
