SPATIAL DYNAMICS OF PROTEIN TURNOVER IN COCHLEA

毛细胞静纤毛在听觉中起着重要作用。 毛细胞静纤毛的主要蛋白质成分是肌动蛋白，组织成定向的细丝，其中一些附着在富含肌动蛋白的角质板上。 毛细胞肌动蛋白丝的变化在噪声性和药物性听力损失中都很常见。 尽管肌动蛋白对毛细胞的正常结构和功能至关重要，而且含有肌动蛋白的结构的破坏与毛细胞损伤有着明确的联系，但人们对毛细胞中肌动蛋白周转的动力学，甚至是一般的蛋白质周转的动力学知之甚少。在高风险/高影响赠款的任期内使用多重成像质谱的新方法，我们已经成像和测量的百分比营业额的蛋白质使用非放射性标记物，15 N亮氨酸，在各种耳蜗位置：静纤毛，覆膜，Reissner膜螺旋神经节和颞骨。 质量图像的分辨率很容易达到50 nm。 结合15 N/14 N或13 C/12 C同位素比值测量的高精度，它提供了沿着静纤毛或其他耳蜗结构映射蛋白质（肌动蛋白）周转的可能性。我们使用的多重成像质谱仪（MIMS）已捐赠给哈佛，并安装在我们的实验室。 本研究拟利用MIMS技术研究雏鸡耳蜗蛋白质的亚细胞定位和周转率。 将在正常条件下、耳毒性损伤后和再生期间对雏鸡进行研究。 将特别注意静纤毛和覆膜。 我们将寻找蛋白质周转和DNA复制之间的关联，特别是在再生过程中。多重成像质谱的使用可能会开辟一个全新的研究领域，药物诱导的细胞损伤的影响。 此外，重同位素标记与多重成像质谱法的结合可能会在听觉和前庭研究的其他领域中找到广泛的用途。

The hair-cell stereocilia play a major role in hearing. The main protein component of hair-cell stereocilia is actin, organized in oriented filaments, some of them attached to the actin-rich cuticular plate. Changes in hair-cell actin filaments are common both in noise-induced and in drug-induced hearing loss. Despite the central importance of actin to the proper structure and function of hair cells, and the clear association of disruptions of actin-containing structures with hair-cell damage, little is known about the dynamics of actin turnover, or even of protein turnover in general, in hair cells. Using the new methodology of Multiple Imaging Mass Spectrometry during the tenure of a High Risk/High Impact grant, we have imaged and measured the percentage turnover of protein using a non radioactive marker, 15N leucine, in a variety of cochlear location: stereocilia, tectorial membrane, Reissner membrane spiral ganglion and temporal bone. The resolution of the mass images is easily 50 nm. Together with the high precision of the 15N/ 14N or 13C/12C isotopic ratios measurements, it provides the possibility to map the turnover of protein (actin) along stereocilia or other cochlear structures. The Multiple Imaging Mass Spectrometer (MIMS) that we used has been donated to Harvard and is installed in our laboratory. We propose to use the MIMS to studying the subcellular location and turnover rate of protein in the cochlea of chicks. Studies will be performed on chicks in normal conditions, after ototoxic insult, and during regeneration. Special attention will be given to the stereocilia and to the tectorial membrane. We will look for association between protein turnover and DNA replication, particularly during regeneration. The use of Multiple Imaging Mass Spectrometry may open up a whole new area of investigation into the cellular effects of drug- induced injury. Furthermore, the combination of heavy isotope labeling with Multiple Imaging Mass Spectrometry may find wide use in other areas of auditory and vestibular research.