Development of endocytosis in cochlear inner hair cells

哺乳动物内毛细胞（IHC）不知疲倦地将机械声刺激转化为分级的受体电位和分级的递质释放，在很宽的动态范围内具有出色的时间精度。在听力开始之前，IHC经历一个发育程序，在此期间它们产生Ca 2+驱动的动作电位。听觉前和成熟IHC之间的递质释放在许多方面不同，例如i）触发的种类（Ca 2+动作电位与分级受体电位），ii）Ca 2+效率，iii）关键分子和iv）突触的形态。尽管在IHC中充分研究了囊泡释放的功能和成熟，但对于先前插入的囊泡膜从质膜的内吞作用，情况并非如此。初步的数据强烈表明，内吞作用经历了一个成熟的过程，平行的发育修饰的胞吐作用。在这个项目中，我的目的是研究内吞作用在早产儿相比，成熟的IHC。具体目标是：1）阐明内吞作用的动力学和Ca ~（2+）依赖性; 2）鉴定内吞机制的分子组成; 3）分析内吞蛋白的作用。将使用膜片钳技术测量胞吞作用作为膜电容变化。我们将通过转录分析在mRNA水平上鉴定内吞机制的组成部分，并通过免疫标记在蛋白水平上使用光学显微镜和透射电子显微镜。最后，我们将阐明内吞蛋白在IHC中的作用，通过阻断它们的功能，并通过膜电容记录和使用可固定的膜染料的膜摄取成像研究对内吞作用的影响。这里获得的知识对于理解IHC的最终成熟以及IHC如何不知疲倦地将声音信息传递到听觉通路至关重要。这项研究将进一步作为未来项目的基础，涉及突变小鼠模型的内吞蛋白，了解人类耳聋。

Mammalian inner hair cells (IHC) indefatigably transduce mechanical sound stimuli into graded receptor potentials and graded transmitter release with excellent temporal precision over a wide dynamic range. Before the onset of hearing, IHCs undergo a developmental program during which they generate Ca2+-driven action potentials. Transmitter release differs in many regards between pre-hearing and mature IHCs, such as i) the kind of trigger (Ca2+ action potentials vs. graded receptor potentials), ii) Ca2+ efficiency, iii) crucial molecules and iv) morphology of the synapse. Whereas function and maturation of vesicle release is well studied in IHCs, this is not the case for endocytosis of previously inserted vesicle membrane from the plasma membrane. Preliminary data strongly suggest that endocytosis undergoes a process of maturation that parallels the developmental modifications of exocytosis. In this project I aim to characterise endocytosis in pre-mature compared to mature IHCs. The specific aims are: 1) to characterise the kinetics and Ca2+ dependence of endocytosis; 2) to identify the molecular components of the endocytic machinery; 3) to analyse the role of endocytic proteins. Endocytosis will be measured as membrane capacitance changes using the patch clamp technique. We will identify components of the endocytic machinery on mRNA level by transcript analysis and on protein level by immunolabelling using light microscopy and transmission electron microscopy. Finally, we will elucidate the role of endocytic proteins in IHCs by blocking their function and studying the resulting effect on endocytosis via membrane capacitance recordings and imaging of membrane uptake using a fixable membrane dye. The knowledge gained here is essential to understand final maturation of the IHC and how IHCs indefatigably transfer sound information to the auditory pathway. This study will further serve as a basis for future projects involving mutant mouse models of endocytic proteins for understanding human deafness.