Strategy for Compositional Analysis of the Hair Cell Mechanotransduction Complex Using TIRF Microscopy.
Strategy for Compositional Analysis of the Hair Cell Mechanotransduction Complex Using TIRF Microscopy.
复制标题
使用 TIRF 显微镜对毛细胞力转导复合物进行成分分析的策略。
DOI：
10.1017/s1431927619007062
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Gouaux,Eric
中科院分区：
文献类型：
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作者：
Clark,Sarah;Elferich,Johannes;Gai,Jingpeng;Goehring,April;Mitra,Jaba;Ha,Taekjip;Gouaux,Eric
位于内耳毛束尖端的机械传导机制负责我们对运动和声音的感觉。每个束由约100个静纤毛组成，呈阶梯状排列，通过尖端连接、由原钙粘蛋白15（PCDH 15）和钙粘蛋白-23（CDH 23）组成的细胞外蛋白丝连接[1]。由声音和流体运动引起的发束的偏转导致位于较低尖端-链接插入部位的机械转导通道（MET）复合体的打开，从而产生电信号。除了尖端连接蛋白外，MET复合物还有三种推定的组分：脂肪瘤HMGIC融合伴侣样5蛋白LHFPL 5（也称为TMHS）[2，3]、跨膜内耳蛋白TMIE和跨膜样通道蛋白TMC 1/2 [4，5]，它们可能是复合物的孔形成亚基[6]。据估计，每个静纤毛只有一个MET复合体，这意味着每个小鼠耳蜗大约有1阿托摩尔复合体[6]。这种低丰度的物质不能通过常规的分析方法检测，如ELISA或western blot。在这里，我们提出了一种策略，以克服这一限制，并分析从小鼠耳蜗MET复合物：单分子下拉（SiMPull）。SiMPull结合了传统的下拉分析与单分子全内反射（TIRF）显微镜，以检查细胞或组织提取物中的蛋白质复合物[7]。固定在盖玻片上的抗体用于从溶液中捕获MET复合物，另一种与荧光团连接的抗体或抗体片段（Fab）使复合物可视化（图1A）。这种方法能够研究单个分子，从而允许分析低丰度复合物的组成和化学计量。
Mechanotransduction machinery located at the tips of hair bundles in the inner ear are responsible for our sensations of movement and sound. Each bundle is composed of~ 100 stereocilia organized in a staircase array that are connected by tip-links, extracellular protein filaments composed of protocadherin 15 (PCDH15) and cadherin-23 (CDH23)[1]. Deflection of the hair bundle by sound and fluid movement leads to opening of the mechanotransduction channel (MET) complex located at the lower tip-link insertion site, resulting in an electrical signal. There are three putative components of the MET complex aside from the tip-link proteins: the lipoma HMGIC fusion partner-like 5 protein LHFPL5 (also known as TMHS)[2, 3], the transmembrane inner ear protein TMIE, and the transmembrane-like channel proteins TMC1/2 [4, 5], which are the likely pore-forming subunits of the complex [6]. It is estimated that there is only one MET complex per stereocilia, which means there is approximately 1 attomole of complex per mouse cochlea [6]. This low abundance of material cannot be detected by conventional methods of analysis, such as ELISA or western blot.Here we present a strategy to overcome this limitation and analyze the MET complex from mouse cochlea: single molecule pulldown (SiMPull). SiMPull combines a conventional pull-down assay with single molecule total internal reflection (TIRF) microscopy to examine protein complexes from cell or tissue extracts [7]. An antibody immobilized on a coverslip is used to capture the MET complex from solution, and another antibody or antibody fragment (Fab) linked to a fluorophore enables visualization of the complex (Figure 1A). This approach enables the study of individual molecules, thus allowing analysis of the composition and stoichiometry of a low abundance complex.