Detecting intracellular signalling in behaving axonal growth cones.

检测轴突生长锥行为的细胞内信号传导。

• 批准号: RGPIN-2016-06516
• 负责人: Kania, Artur
• 金额: $ 2.4万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616360
• 关键词: Detecting; intracellular; signalling; behaving; axonal

Neuronal circuits develop with great precision and specificity, starting with the highly accurate guidance of neuronal axons. Over the past two decades much work has been directed towards the identification of axonal guidance cues and their receptors, but how their signalling coordinates the dynamics of growth cone behaviour remains poorly understood. Part of the reason for this is the low spatial and temporal resolution of our understanding of growth cone intracellular signalling pathways. To resolve this, we propose to use a specific class of spinal motor neurons (LMCs), obtained from embryonic chicken explants and a recently-developed mouse embryonic stem cell line that can be easily induced to form spinal motor neurons. Our in vitro analysis indicates that LMCs respond to ephrin-B signals that activate the EphB tyrosine kinase pathway. Recent protein engineering efforts also generated a number of fluorescent indicators of intracellular signalling, such as receptor phosphorylation, signal relay protein conformation changes, or small GTPase activation and cytoskeleton remodelling. However, very few of such indicators have been used in the context of live growth cones, and essentially none in molecularly-defined neurons. We propose to use fluorescent indicators of cellular signalling events to study the live dynamics of axon guidance signalling in spinal motor neurons, in order to link axon guidance receptor signal relay with growth cone dynamics at high spatial and temporal resolution, through the following Aims: Aim 1 Assess EphB signalling dynamics in LMC neurons following treatments with ephrin-B family of proteins. These experiments will first characterise the general response properties of a recently developed EphB sensor, and will be later combined with whole growth cone imaging in order to relate biochemical level events with growth cone morphology changes. Aim 2 Assess cytoskeletal response dynamics in LMCs following ephrin-B treatment. Here, we will take advantage of small GTPase activation biosensors, which will be first characterised in the context of ephrin-B stimulation, and subsequently imaged in the context of whole growth cone dynamics. Aim 3 Link EphB and cytoskeletal signalling events in live growth cones. This will be done by simultaneously imaging EphB signalling, small GTPase activation and cytoskeletal rearrangement.

神经元电路的发展具有极高的精确度和特异度，从神经轴突的高度精确引导开始。在过去的二十年里，许多工作都集中在轴突引导信号及其受体的识别上，但它们的信号如何协调生长锥行为的动态仍然知之甚少。部分原因是我们对生长激素细胞内信号通路的理解在空间和时间上的分辨率很低。 为了解决这一问题，我们建议使用一类特定的脊髓运动神经元(LMC)，从鸡胚胎外植体和最近开发的小鼠胚胎干细胞系中获得，这种干细胞可以很容易地诱导形成脊髓运动神经元。我们的体外分析表明，LMCs对Ephin-B信号做出反应，激活EphB酪氨酸激酶途径。最近的蛋白质工程工作也产生了许多细胞内信号的荧光指示物，如受体磷酸化、信号转导蛋白构象变化，或小GTP酶激活和细胞骨架重构。然而，很少有这样的指标被用于活的生长锥体的背景下，基本上没有用于分子定义的神经元。我们建议使用细胞信号事件的荧光指示剂来研究脊髓运动神经元中轴突引导信号的实时动力学，以便在高空间和时间分辨率下将轴突引导受体信号传递与生长锥动力学联系起来，通过以下目的： 目标1 评估EphB蛋白家族蛋白治疗后LMC神经元中的EphB信号动力学。这些实验将首先描述最近开发的EphB传感器的一般响应特性，然后将与整个生长锥成像相结合，以便将生化水平事件与生长锥形态变化联系起来。 目标2 评估肾上腺素-B治疗后肺小管上皮细胞的细胞骨架反应动力学。在这里，我们将利用小的GTP酶激活生物传感器，它将首先在eaffin-B刺激的背景下表征，然后在整个生长锥动态的背景下成像。 目标3 在活的生长锥体中将EphB和细胞骨架信号事件联系起来。这将通过同时成像EphB信号、小GTP酶激活和细胞骨架重排来完成。