SPATIAL DYNAMICS OF PROTEIN TURNOVER IN COCHLEA

毛细胞立体纤毛在听力中起着重要作用。毛细胞立体纤毛的主要蛋白质成分是肌动蛋白，排列成取向的细丝，其中一些附着在富含肌动蛋白的角质板上。毛细胞肌动蛋白细丝的变化在噪音引起的和药物引起的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明显相关，但人们对毛细胞中肌动蛋白周转的动态，甚至一般的蛋白质周转的动态知之甚少。在高风险/高影响拨款期间，我们使用多重成像质谱仪的新方法，使用非放射性标记物15N亮氨酸成像并测量了不同耳蜗处的蛋白质周转百分比：立体纤毛、覆盖膜、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。再加上15N/14N或13C/12C同位素比值测量的高精度，它提供了沿着立体纤毛或其他耳蜗结构绘制蛋白质(肌动蛋白)周转的可能性。我们使用的多重成像质谱仪(MIMS)已经捐赠给哈佛大学，并安装在我们的实验室中。我们建议使用MIMS来研究雏鸡耳蜗亚细胞位置和蛋白质的周转率。研究将在正常条件下、耳毒性侮辱后和再生期间进行。将特别注意立体纤毛和覆盖膜。我们将寻找蛋白质周转和DNA复制之间的联系，特别是在再生过程中。多重成像质谱仪的使用可能会为药物损伤的细胞效应研究开辟一个全新的领域。此外，重同位素标记与多重成像质谱学的结合可能会在听觉和前庭研究的其他领域找到广泛的用途。

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.