TRIM67 as a novel regulator of exocytosis in developing neurons

TRIM67 作为发育中神经元胞吐作用的新型调节剂

• 批准号: 9516753
• 负责人: Fabio Lee Urbina
• 金额: $ 3.52万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-15 至 2020-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9516753
• 关键词: Affect; Autistic Disorder; Axon; Brain; Cell membrane; Characteristics; Chemotactic Factors; Complex; Computer software; Cues; Cytoplasm; Data; Defect; Development; Diffuse; Diffusion; Embryo; Event; Exocytosis; Fluorescence Microscopy; Frequencies; Growth Cones; Image; Image Analysis; In Vitro; Knockout Mice; Manuscripts; Mediating; Membrane; Microfluidic Microchips; Microfluidics; Microscopic; Molecular; Morphogenesis; Morphology; Mus; NTN1 gene; Neurons; Phenotype; Play; Positioning Attribute; Preparation; Process; Regulation; Regulation of Exocytosis; Resolution; Role; Running; SNAP receptor; Schizophrenia; Secretory Vesicles; Synapses; System; Testing; Tetanus; Time; Titan; Transport Vesicles; Vesicle; Vesicle Transport Pathway; axon growth; axon guidance; experimental study; fluorescence imaging; improved; live cell imaging; mutant; nervous system disorder; neuron development; novel; prevent; receptor; response; software development; spatiotemporal; target SNARE proteins; trafficking; ubiquitin-protein ligase; vesicular SNARE proteins

PROJECT SUMMARY/ABSTRACT The expansion of the plasma membrane is a critical, continuous process in the developing neuron. The addition of membrane material primarily occurs through the secretory system, in which secretory vesicles are transported to and fuse with the plasma membrane. These vesicles undergo SNARE-complex mediated fusion, in which a v-SNARE on the vesicle and t-SNAREs on the plasma membrane interact and fuse the vesicular membrane and plasma membrane together, opening a fusion pore and expelling vesicular cargo. Presumably, membrane addition occurs primarily through the classic mechanism of exocytosis, full-vesicle fusion (FVF), in which the fusion pore dilates and the vesicle collapses into the plasma membrane. During kiss-and-run (KNR) fusion, however, the fusion pore closes and the vesicle remains intact. The high temporal and spatial resolution of imaging needed to capture these events has been a roadblock in differentiating between these two mechanisms of fusion. Using my newly developed image analysis software, which automatically detects and characterizes exocytic events, I have been able to discern between two modes of exocytosis in developing neurons, which are consistent with FVF and KNR. Furthermore, the E3 ubiquitin ligase, TRIM67, biases the mode of exocytosis away from KNR and toward FVF. Our lab has previously shown that the guidance cue netrin-1 increases the frequency of exocytosis in mouse cortical neurons in vitro. Netrin-1 and its receptor DCC also regulate axon guidance, with axons turning toward higher concentrations of netrin-1. The asymmetrical addition of plasma membrane is hypothesized to underlie this turning and guidance of growing axons, which is expected to primarily occur through FVF. Preliminary findings suggest that TRIM67 is necessary for a netrin-1- depedendent increase in the frequency of exocytosis. This proposal will test the hypotheses that TRIM67 biases the mode of fusion toward FVF and that TRIM67 is involved in axon turning in response to netrin-1 through its ability to promote FVF. Our first aim will be to confirm that the two modes of exocytosis revealed by the automated image analysis are genuine FVF and KNR events and to identify the domains of TRIM67 necessary for biasing exocytosis toward FVF and the domains necessary for the netrin-1 response. Our second aim will determine how a gradient of netrin affects the spatial occurrence of exocytosis in the growth cone, to identify whether this exocytic response is disrupted in Trim67-/- growth cones and to identify axon guidance defects in Trim67-/- neurons using a novel microfluidic chamber.

项目摘要/摘要 在发育中的神经元中，质膜的扩张是一个关键的、连续的过程。这个 膜物质的添加主要通过分泌系统发生，在分泌系统中，分泌小泡是 运输到质膜并与质膜融合。这些囊泡经历了圈套复合体介导的融合， 其中囊泡上的v型陷阱和质膜上的t型陷阱相互作用并融合囊泡。 膜和质膜结合在一起，打开融合孔，排出囊泡货物。据推测， 膜添加主要通过胞吐作用的经典机制，即全囊泡融合(FVF)，在 融合孔扩张，囊泡塌陷进入质膜。在接吻后逃跑(KNR)期间 然而，融合后，融合孔关闭，小泡保持完好。高时空分辨率 捕捉这些事件所需的成像一直是区分这两个事件的障碍 核聚变机制。使用我最新开发的图像分析软件，它可以自动检测和 胞吐事件的特征，我已经能够在发育过程中区分两种胞吐作用模式 神经元，与FVF和KNR一致。此外，E3泛素连接酶TRIM67使 胞吐方式远离KNR，朝向FVF。我们的实验室之前已经表明，引导信号 Netrin-1可增加体外培养的小鼠皮质神经元胞吐频率。Netrin-1及其受体DCC 也调节轴突引导，轴突转向更高浓度的netrin-1。不对称 质膜的添加被认为是这种生长轴突的转折和引导的基础，这是 预计主要通过FVF发生。初步研究表明，TRIM67是Netrin-1所必需的。 胞吐频率呈递增趋势。这项提议将检验TRIM67的假设 使融合模式偏向FVF，并且TRIM67参与响应netrin-1的轴突转向 通过其促进FVF的能力。我们的第一个目标将是确认下列两种胞吐作用模式 自动图像分析是真正的FVF和KNR事件，并识别TRIM67的结构域 使胞吐偏向FVF和Netrin-1反应所必需的结构域所必需的。我们的 第二个目标将确定网状蛋白的梯度如何影响生长过程中胞吐作用的空间发生。 锥体，以确定这种胞吐反应是否在Trim67-/-生长锥体中被破坏并识别轴突 使用新型微流体室的Trim67-/-神经元的引导缺陷。