Fascin 2b Is a Component of Stereocilia that Lengthens Actin-Based Protrusions

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a Kd≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.静纤毛（Stereocilia）是富含肌动蛋白的突起，可介导内耳的机械转导过程。为鉴定能够调控静纤毛细胞骨架的蛋白质，研究人员对斑马鱼（zebrafish）毛细胞的转录组进行了细致分析。其中一个极具潜力的候选蛋白为丝束蛋白2b（fascin 2b），这是一种已在视网膜感光细胞中被发现的丝状肌动蛋白捆绑蛋白。通过对斑马鱼毛细胞开展免疫标记实验，以及使用表达与绿色荧光蛋白（green fluorescent protein）融合的丝束蛋白2b的转基因斑马鱼，研究人员证实丝束蛋白2b可特异性定位于静纤毛。在体外实验中，将丝状肌动蛋白与携带丝束蛋白2b的重组融合蛋白共同孵育以测定二者的解离常数，最终测得Kd≈0.37 μM。电子显微镜观察结果显示，丝束蛋白2b与肌动蛋白丝形成的复合物可在体外组装成平行排列的肌动蛋白束。研究人员进一步发现，在COS-7细胞中表达丝束蛋白2b或另一种肌动蛋白捆绑蛋白埃斯平（espin），均可诱导细胞形成长丝状伪足（filopodia）。共表达实验表明，这两种蛋白可通过形成超长突起产生协同效应。通过在COS-7细胞及转基因毛细胞中使用模拟磷酸化或非磷酸化状态的磷酸化突变体丝束蛋白2b蛋白，研究人员证实，长丝状伪足的形成以及丝束蛋白2b向静纤毛的定位均依赖于丝氨酸38位点。在毛细胞中过表达野生型丝束蛋白2b，可使静纤毛长度较对照组显著增加。上述研究结果表明，丝束蛋白2b在静纤毛的形态塑造过程中发挥关键作用。