Exocytosis-endocytosis coupling at presynaptic terminals

项目摘要 本工作的长期目标是阐明胞吐-胞吞耦合的基本机制 在中枢神经末梢。许多类型的突触常规地传递高频动作电位 通过活性区的高速囊泡融合。融合的突触囊泡及其相关蛋白质必须 通过内吞作用回收。除了再生新的突触囊泡供将来使用外， 平衡神经末梢的表面积并保持完整的超微结构。尽管经过数十年的 尽管进行了广泛的研究，但化学突触的内吞作用机制尚未完全阐明，特别是在 生理温度强有力的证据表明，不同模式的内吞发生在 细胞对不同的突触活动有不同的反应，内吞作用比囊泡融合慢几个数量级。 然而，最近的形态学研究提出了一种超快的内吞作用，仅发生在生理性的细胞内。 温度和取代其他形式的内吞作用。这是一个有吸引力的模型，因为它有效地 在高速率囊泡融合期间使神经末梢的表面积的不平衡最小化。另一方面，在一项研究中， 该模型建立在固定突触静态图像的统计基础上，需要足够的功能数据 以测试和表征生理温度下的内吞作用。内吞作用的完全改变 新的、网格蛋白独立的内吞作用模式也提出了许多有趣的新问题。 发动蛋白1是一种大的GTdR，其是突触处网格蛋白介导的内吞作用所必需的，但其在其他神经元中的作用， 内吞作用的形式如大量内吞作用和超快内吞作用不太清楚和有争议。在这 建议，我们将解决这些问题的电容记录从突触前终端在 生理温度时间分辨电容测量（Cm）具有高的时间分辨率， 敏感性，因此是一种合适的方法。首先，我们将通过高时间来表征突触内吞作用- 在生理温度下的分辨率Cm。我们将使用Held的花萼，一个快速的突触， 在听觉脑干中。我们将克服几个技术限制在厘米记录使用新的战略 并提取在生理温度下可能存在的任何快速内吞作用。不同的突触活动， 包括自发的单囊泡内吞作用。其次，我们将使用发动蛋白-1 条件性基因敲除小鼠作为一种有价值的遗传模型;其在生理温度下的内吞特性 将被研究对各种突触活动的反应。这将为发动蛋白1提供重要的见解 在体内发挥作用。该项目将进一步推动该领域的发展，并解决最近的几个关键问题 在这一领域的快速发展。这将促进我们在动力学和分子方面的知识 在类似于体内的条件下，我们研究了中央突触的胞吐-胞吞耦合机制， 预计对神经元细胞生物学和神经科学产生广泛影响。

PROJECT SUMMARY The long-term goal of this work is to elucidate the fundamental mechanism of exocytosis-endocytosis coupling at the central nerve terminals. Many types of synapses routinely transmit high-frequency action potentials through high-rate vesicle fusion at active zones. Fused synaptic vesicles and their associated proteins must be retrieved by endocytosis. In addition to regenerating new synaptic vesicles for future use, it is critical for balancing the surface area of the nerve terminals and maintaining intact ultrastructures. Despite decades of extensive research, the mechanism of endocytosis at chemical synapses is not fully addressed, particularly at physiological temperature. Strong evidence suggests that different modes of endocytosis take place in response to different synaptic activity, and endocytosis is a few orders of magnitude slower than vesicle fusion. However, recent morphological studies propose an ultrafast endocytosis that only occurs at a physiological temperature and replaces other forms of endocytosis. This is an attractive model because it efficiently minimizes the imbalance of surface area of nerve terminals during high-rate vesicle fusion. On the other hand, this model is built on the statistics of static images of fixed synapses, and sufficient functional data are required to test and characterize endocytosis at physiological temperature. The complete change of endocytosis pathways into a new, clathrin-independent endocytosis mode also raises many interesting new questions. Dynamin 1 is a large GTPase that is required for clathrin-mediate endocytosis at synapses, but its role in other forms of endocytosis such as bulk endocytosis and ultrafast endocytosis is less clear and controversial. In this proposal, we will address these questions by capacitance recordings from presynaptic terminals at physiological temperature. The time-resolve capacitance measurement (Cm) has high temporal resolution and sensitivity and thus is a suitable approach. First, we will characterize synaptic endocytosis by high time- resolution Cm at physiological temperature. We will use the calyx of Held, a fast glutamatergic central synapse in the auditory brainstem. We will overcome several technical limits during Cm recordings using new strategies and extract any fast endocytosis that may be present at physiological temperature. Different synaptic activities, including spontaneous single vesicle endocytosis, will be monitored. Secondly, we will use dynamin-1 conditional knockout mice as a valuable genetic model; its endocytosis properties at physiological temperate will be studied in response to various synaptic activities. This should provide significant insight into dynamin 1 function in vivo. This project will advance the field a step further and address several key questions recently raised by the rapid progress in this field. It will advance our knowledge on the kinetics and molecular mechanism of exocytosis-endocytosis coupling at central synapses under a condition similar to in vivo, and we expect a broad impact on cell biology of neurons and neuroscience.