Exocytosis-endocytosis coupling at presynaptic terminals

突触前末端的胞吐作用-内吞作用耦合

基本信息

批准号：
9673997
负责人：
Xuelin Lou
金额：
$ 23.1万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2020-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9673997
关键词：
Actins Action Potentials Address Affect Animal Genetics Animal Model Area Auditory Brain Brain Diseases Brain Stem Cell physiology Cells Cellular biology Cerebellar Mossy Fibers Chemical Synapse Clathrin Communication Coupling Data Disease Dynamin Dynamin I Electric Capacitance Electron Microscopy Endocytosis Endocytosis Pathway Equilibrium Exocytosis Failure Fire - disasters Frequencies Functional disorder Future Genetic Genetic Models Glutamates Goals Guanosine Triphosphate Phosphohydrolases Health Image Kinetics Knock-out Knockout Mice Knowledge Laboratories Learning Link Measurement Measures Mediating Membrane Membrane Proteins Modeling Molecular Monitor Moods Morphology Natural regeneration Nerve Neurons Neurosciences Noise PHluorin Pharmacology Physiological Play Presynaptic Terminals Process Property Proteins Recycling Reporting Research Resolution Role Running Sampling Signal Transduction Surface Synapses Synaptic Vesicles Techniques Temperature Testing Time Vesicle Work base biophysical techniques experimental study granule cell improved in vivo inhibitor/antagonist insight mental state mouse model presynaptic response statistics temporal measurement time use tool

项目摘要

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.

项目摘要这项工作的长期目标是阐明胞吞作用 - 细胞增多症偶联的基本机制在中央神经末端。许多类型的突触常规传输高频动作电位通过活性区域的高速囊泡融合。融合的突触囊泡及其相关蛋白必须是通过内吞作用检索。除了再生新的突触囊泡以备将来使用，这对于平衡神经末端的表面积并保持完整的超微结构。尽管数十年广泛的研究，化学突触中内吞作用的机制尚未完全解决，尤其是在生理温度。有力的证据表明，内吞作用的不同模式发生在对不同的突触活性的反应和内吞作用比囊泡融合慢了几个数量级。但是，最近的形态学研究提出了一种超快内吞作用，仅发生在生理上温度并取代其他形式的内吞作用。这是一个有吸引力的模型，因为它有效在高速囊泡融合过程中最小化神经末端表面积的不平衡。另一方面，该模型建立在固定突触的静态图像的统计上，需要足够的功能数据在生理温度下测试和表征内吞作用。内吞作用的完全改变进入新的，无网蛋白独立的内吞作用模式的途径也提出了许多有趣的新问题。 Dynamin 1是一个大的GTPase，是在突触时网格蛋白介质内吞作用所必需的，但其在其他中的作用内吞作用的形式，例如散装内吞作用和超快内吞作用，较清晰和有争议。在这个提案，我们将通过突触前终端的电容记录来解决这些问题生理温度。时间溶解电容测量（CM）具有很高的时间分辨率，并且灵敏度，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性内吞作用分辨率CM在生理温度下。我们将使用Hold的花萼，快速谷氨酸能中央突触在听觉脑干中。我们将使用新策略在CM录音过程中克服几个技术限制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，将受到监测。其次，我们将使用Dynyin-1 有条件的敲除小鼠作为有价值的遗传模型；它在生理温度的内吞作用将根据各种突触活动进行研究。这应该为Dynamin 1提供重要的见解1 在体内功能。该项目将进一步提高该领域，并最近解决几个关键问题由于该领域的快速进步而提高。它将提高我们对动力学和分子的知识在类似于体内的疾病下，中央突触处的胞吐 - 胞胞细胞增多症的机制，我们期望对神经元和神经科学的细胞生物学产生广泛的影响。