Hair Cell Polarization and Sensory Bundle Development

项目总结/摘要 这一建议解决了mInsc-LGN-Gαi蛋白复合物在静纤毛中的未表征作用 延伸，并将LGN-Gαi定义为差异静纤毛所需的Myo 15的长期受欢迎的货物 在毛细胞发育期间跨行生长。在缺乏LGN或Gαi的情况下， 发育不良，形成一个不成熟的外观束，只保留了一个浅楼梯模式。有趣的是， 在缺乏Myo 15、Whirlin和Eps 8的情况下，已经报道了类似的缺陷，这三种蛋白质富集在 所有静纤毛的顶端数量与它们的高度成比例。我们在这里表明，LGN和Gαi具有相同的 尖端定位，但仅限于第一静纤毛行。引人注目的是，LGN和Gαi未能在 没有Myo 15。相比之下，Myo 15-Whrn-Eps 8在所有发育不良的行中以类似的低量存在， LGN或Gαi突变体，表明LGN-Gαi通过指定 第一排通过交叉突变体和使用遗传学，蛋白质免疫定位和蛋白质结合 生物化学，我们建议解决扩展的Myo 15复合物的功能，现在包括LGN-Gαi，其中 所有的蛋白质显然都具有独立的、互补的功能来形成纤维束。 我们先前表明mInsc-LGN-Gαi首先占据并产生“裸露区”，即毛发的平坦区域。 细胞顶端，早期微绒毛排除勾勒出形成束的V形或半圆形边缘。 基于亚细胞区室之间LGN-Gαi蛋白量动态和平衡的证据， 我们建议，LGN-Gαi限制在第一行可以通过它们在第二行的先前富集来指示。 邻近的裸区。LGN-Gαi促进第一行Myo 15-Whrn-Eps 8表达量增加， 进入最高的排，进而通过倾斜的末端连杆影响较短排的高度，如前所述。到 测试的新想法，楼梯模式是建立回收平面极性信息，我们提出 特定阶段的功能丧失和获得方法，包括使用耳蜗的探索性实验 外植体，旨在阐明和操纵LGN-Gαi的功能在亚细胞水平。 毛束的阶梯状结构对听力至关重要，并被认为有助于 对声音刺激的方向敏感性，但在分子水平上仍无法解释。通过发现新的 Myo 15复合体的成员，并阐明了如何在各行中创造生长的不对称性，这项工作 显著提高了我们对DFNB 3（MYO 15），DFNB 31/USH 2D（WHRN）， DFNB 102（EPS 8）和DFNB 82/Chudley-McCullough综合征（LGN/GPSM 2）。

PROJECT SUMMARY/ABSTRACT This proposal addresses the uncharacterized role of the mInsc-LGN-Gαi protein complex in stereocilia elongation, and defines LGN-Gαi as a long sought-after cargo of Myo15 required for differential stereocilia growth across rows during hair cell development. In the absence of LGN or Gαi, postnatal stereocilia are stunted and form an immature-looking bundle that only retains a shallow staircase pattern. Interestingly, very similar defects have been reported in the absence of Myo15, Whirlin and Eps8, three proteins enriched at the tips of all stereocilia in amounts proportional to their height. We show here that LGN and Gαi share the same tip localization, but are restricted to the first stereocilia row. Strikingly, LGN and Gαi fail to traffic to tips in absence of Myo15. In contrast, Myo15-Whrn-Eps8 are found in similar low amounts across all stunted rows in LGN or Gαi mutants, suggesting that LGN-Gαi instruct differential stereocilia identity across rows by specifying the first row. By intercrossing mutants and using genetics, protein immunolocalization and protein binding biochemistry, we propose to solve the function of an extended Myo15 complex now including LGN-Gαi where all proteins apparently have independent, complementary functions to shape the bundle. We previously showed that mInsc-LGN-Gαi first occupy and generate the 'bare zone', the flat region of the hair cell apex where early microvilli exclusion outlines the V-shaped or semi-circular edge of the forming bundle. Based on evidence for dynamic and balanced LGN-Gαi protein amounts between subcellular compartments, we propose that restriction of LGN-Gαi to the first row could be instructed by their prior enrichment at the adjacent bare zone. LGN-Gαi would promote higher amounts of Myo15-Whrn-Eps8 at the first row, its growth into the tallest row in turn influencing the height of shorter rows via oblique tip-links, as proposed previously. To test the novel idea that the staircase pattern is established by recycling planar polarity information, we propose stage-specific loss- and gain-of-function approaches, including exploratory experiments using cochlear explants aimed at elucidating and manipulating LGN-Gαi function at the subcellular level. The staircase-like architecture of the hair bundle is essential for hearing and considered instrumental for direction-sensitivity to sound stimuli, but remains largely unexplained at the molecular level. By uncovering new members of the Myo15 complex and clarifying how asymmetry of growth is created across rows, this work notably improves our understanding of hereditary hearing loss in DFNB3 (MYO15), DFNB31/USH2D (WHRN), DFNB102 (EPS8) and DFNB82/Chudley-McCullough syndrome (LGN/GPSM2).