Maeda Reo, Pacentine Itallia V, Erickson Timothy, Nicolson Teresa
Oregon Hearing Research Center and the Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239.
Oregon Hearing Research Center and the Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239
J Neurosci. 2017 Mar 22;37(12):3231-3245. doi: 10.1523/JNEUROSCI.2216-16.2017. Epub 2017 Feb 20.
Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. The significance of these protein-protein interaction domains of PCDH15 in hair cells has not been determined. Here, we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) in -null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior and hair cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype and that the CD1- or CD3-specific regions were dispensable for hair bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain was deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for the function of Pcdh15a in zebrafish hair cells. Tip links transmit force to mechanotransduction channels at the tip of hair bundles in sensory hair cells. One component of tip links is Protocadherin 15 (PCDH15). Here, we demonstrate that, when transgenically expressed, either zebrafish Pcdh15a-cytodomain 1 (CD1) or Pcdh15a-CD3 can rescue the phenotype of a -null mutant. Even when lacking the specific regions for CD1 or CD3, truncated Pcdh15a that contains the so-called common region (CR) at the cytoplasmic/membrane interface still has the ability to rescue similar to full-length Pcdh15a. In contrast, Pcdh15a lacking the entire cytoplasmic domain is not functional. These results demonstrate that the CR plays a key role in the mechanotransduction complex in hair cells.
原钙黏蛋白15（PCDH15）作为顶连接的一个组成部分，是感觉毛细胞机械转导所必需的。PCDH15的异构体在其胞质结构域（CD1、CD2和CD3）上存在差异，但共享细胞外和跨膜（TMD）结构域，以及一个被称为共同区域（CR）的细胞内结构域。在异源表达系统中，PCDH15的TMD和CR均已被证明能与机械转导复合物的成员相互作用。PCDH15的这些蛋白质-蛋白质相互作用结构域在毛细胞中的重要性尚未确定。在这里，我们通过评估Pcdh15a转基因介导的听觉/前庭行为、毛细胞形态和活性的拯救情况，研究了斑马鱼Pcdh15a的两种异构体（CD1和CD3）在基因敲除突变体中的定位和功能。我们发现单独任何一种异构体都能够拯救Pcdh15a基因敲除的表型，并且CD1或CD3特异性区域对于毛束完整性以及用FM4-64标记毛细胞（用作机械转导的替代指标）而言是可有可无的。当CR或TMD结构域被删除时，突变蛋白定位于静纤毛尖端，但无法拯救FM4-64标记。破坏这两个结构域导致Pcdh15a完全无法定位于毛束。我们的研究结果表明，TMD和胞质CR结构域是斑马鱼毛细胞中Pcdh15a发挥功能所必需的。顶连接将力传递给感觉毛细胞毛束尖端的机械转导通道。顶连接的一个组成部分是原钙黏蛋白15（PCDH15）。在这里，我们证明，当转基因表达时，斑马鱼Pcdh15a胞质结构域1（CD1）或Pcdh
