Molecular mechanisms of Synaptotagmin 1 mediated synaptic vesicle fusion

这个项目的目标，其中福尔斯属于生物科学和生物技术的优先领域，是获得新的基本见解突触递质释放的分子机制。该项目的特点是通过整合光遗传学、膜片钳电生理学和荧光成像来开发新的实验工具（与Scientific合作）。突触囊泡融合的时间由囊泡Ca 2+传感器synaptotagmin（不同突触中的Syt 1、2和9）精确控制，但其作用机制仍然是个谜。我们建议测试的假设，形成的Ca 2+敏感的Syt 1寡聚环起着重要的作用，诱发囊泡释放的同步。我们将利用我们的合作者J. Rothman（耶鲁大学）的最新发现，他产生了一种Syt 1突变体（F349 A），在生化测定中对Syt 1寡聚化产生显性负效应。如果Syt 1寡聚环是必不可少的同步发射器释放然后过度表达的F349 A突变体在突触前末梢应该desscreenise囊泡fusion.Testing这一假设需要测量诱发囊泡释放亚毫秒精度。这在培养的随机连接的神经元中很难实现，因此需要使用有组织的脑组织。我们将使用转基因小鼠，其中EYFP标记的通道视紫红质-2（ChR 2）在CaMKII启动子的控制下在锥体神经元中表达（殖民地在我们实验室建立）。我们将携带WT或F349 A Syt 1与mCherry融合的慢病毒构建体注射到体内小鼠新皮质中。几周后，我们将制备急性脑切片，并使用黄色和红色荧光来鉴定表达ChR 2的细胞，这些细胞也表达重组Syt 1构建体。用477 nm激光刺激识别的单个神经元，并从突触后细胞记录光学诱发的突触后电流（EPSC）。通过这种方式，我们将比较F349 A和WT Syt 1构建体对快速诱发递质释放的影响，并确定Syt 1寡聚化是否是囊泡融合同步所必需的。这些实验提出了几个技术挑战，这些挑战完全在我们的工业合作伙伴的专业知识范围内。首先，我们需要将光刺激限制在单个突触前神经元，避免在相邻神经元及其纤维中产生尖峰。为了解决这个问题，学生将开发新的软件功能，在最近上市的科学激光应用刺激和解锁（LASU）系统中实现时空刺激模式的精确控制。第二，我们需要确保EPSC是通过单个神经元的尖峰产生的。神经元中的尖峰的成像共表达遗传编码的Ca 2+指示剂（例如GCaMP 6）潜在地提供了对光刺激的特异性的强有力的控制。因此，我们将沿着Syt 1构建体共注射GCaMP 6病毒构建体。这里的挑战是ChR 2和GCaMP 6的激发光谱之间的重叠。为了克服这一点，我们将集成EM-CCD相机和基于LED的落射荧光成像系统与LASU。EM-CCD的使用将允许我们将GCaMP 6激发光的强度降低到ChR 2激活阈值以下。作为一种应急措施，我们还将使用成对的全细胞膜片钳记录。

The goal of this project, which falls into the Bioscience and Biotechnology priority area, is to obtain new fundamental insights into the molecular mechanism of synaptic transmitter release. This project features the development of new experimental tools (with Scientifica) via integration of optogenetics, patch-clamp electrophysiology and fluorescence imaging.The timing of synaptic vesicle fusion is precisely controlled by vesicular Ca2+ sensor synaptotagmin (Syt1, 2, and 9 in different synapses), yet the mechanism of its action remains enigmatic. We propose to test the hypothesis that formation of Ca2+-sensitive Syt1 oligomeric rings plays a major role in synchronisation of evoked vesicular release. We will capitalise on recent findings of our collaborator J. Rothman (Yale) who produced a Syt1 mutant (F349A) that exerts a dominant-negative effect on Syt1 oligomersation in biochemical assays. If Syt1 oligomeric rings are essential for synchronisation of transmitter release then overexpression of the F349A mutant in presynaptic terminals should desynchronise vesicle fusion.Testing this hypothesis requires measurements of evoked vesicle release with a submillisecond precision. This is difficult to achieve in randomly connected neurons in culture and thus requires the use of organised brain tissue. We will use transgenic mice where EYFP tagged channelrhodopsin-2 (ChR2) is expressed in pyramidal neurons under the control of CaMKII promoter (the colony is established in our lab). We will inject lentiviral constructs carrying either WT or F349A Syt1 fused with mCherry into mouse neocortex in vivo. Several weeks later we will prepare acute brain slices and use yellow and red fluorescence to identify ChR2 expressing cells which also express recombinant Syt1 constructs. Identified individual neurons will be stimulated with 477 nm laser and optically evoked post-synaptic currents (EPSCs) will be recorded from a post-synaptic cell. In this way, we will compare the effects of F349A and WT Syt1 constructs on fast evoked transmitter release and will establish whether Syt1 oligomerisation is required for synchronisation of vesicle fusion.These experiments pose several technical challenges that are squarely within the expertise of our industrial partner. First, we need to restrict photostimulation to a single presynaptic neuron avoiding spike generation in the neighbouring neurons and their fibres. To address this, the student will develop new software capabilities that will implement precise control of the spatio-temporal stimulation patters in the recently marketed Scientifica Laser Applied Stimulation and Uncaging (LASU) system.Second, we need to ensure that EPSCs are generated by spiking of a single neuron. Imaging of spikes in neurons co-expressing a genetically encoded Ca2+ indicator (e.g. GCaMP6) potentially provides a powerful control for the specificity of photostimulation. Therefore, along with Syt1 constructs we will co-inject GCaMP6 viral constructs. The challenge here is the overlap between the excitation spectra of ChR2 and GCaMP6. To overcome this we will integrate an EM-CCD camera and an LED-based epifluorescence imaging system with the LASU. The use of EM-CCD will allow us to reduce the intensity of GCaMP6 excitation light below the ChR2 activation threshold.The use of the above approach should allow parallel stimulation of Syt1 expressing neurons. As a contingency we will also use paired whole-cell patch-clamp recordings.