Control of actin filament networks by Arp2/3 complex and its regulators

Arp2/3 复合物及其调节剂对肌动蛋白丝网络的控制

• 批准号: 10163397
• 负责人: Bradley J Nolen
• 金额: $ 25万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10163397
• 关键词: 20 year old; Actins; Administrative Supplement; Automatic Data Processing; Cells; Color; Complex; Data; Data Collection; Equipment; Fluorescence Microscopy; Funding; Goals; Image; Label; Laboratories; Lasers; Lighting; Microfilaments; Microscope; Noise; Protein Family; Proteins; Research; Signal Transduction; Work; experience; experimental study; fluorophore; genetic regulatory protein; improved; instrument; parent grant; prevent; single molecule

Project abstract Here we request funding to purchase of a Leica TIRF microscope with an iLas2 circular TIRF laser launch. This instrument will be a critical piece of equipment in my laboratory, and will be the primary data generator for at least half of the projects ongoing in my lab. In R35GM136319, the parent grant for this supplement, my laboratory proposes to determine how WASP and WDS family proteins activate Arp2/3 complex to nucleate actin filaments. Determining the mechanism of these important regulatory proteins and how they work together to assemble functional actin networks in cell depends on our ability to visualize their activities using single molecule total internal reflection fluorescence microscopy (smTIRF). While our lab is currently equipped with a TIRF microscope, our ability to carry out multi-wavelength smTIRF experiments has been limited by our ability to select the optimal TIRF angle for each fluorophore during data collection. This has prevented us from carrying out three color smTIRF experiments in which actin and two actin regulatory proteins are labeled, each with a different fluorophore. It has also complicated our efforts to visualize with single molecule sensitivity proteins labeled with fluorescent proteins (e.g. mNeonGreen), limiting the kinds of experiments that we can do. Our current TIRF microscope also suffers from uneven illumination issues that prevent us from using the entire field of view of TIRF images and make automated data processing more challenging. Therefore, we request funds to purchase a new TIRF microscope that will eliminate these issues, thereby greatly enhancing our ability to carry out multiwavelength smTIRF experiments. We believe this instrument, a Leica TIRF microscope with a iLas2 circular TIRF illuminator, will greatly enhance our ability to accomplish the specific goals in the parent grant and will increase the impact of our research on the field.

项目摘要 在此，我们请求资金购买配备 iLas2 圆形 TIRF 激光器的徕卡 TIRF 显微镜 发射。该仪器将是我实验室的关键设备，并将成为主要数据 我的实验室中至少一半正在进行的项目的生成器。在 R35GM136319 中，家长为此授予的补助金 补充，我的实验室建议确定 WASP 和 WDS 家族蛋白如何激活 Arp2/3 与肌动蛋白丝成核的复合体。确定这些重要调节蛋白的机制 它们如何协同工作以在细胞中组装功能性肌动蛋白网络取决于我们可视化的能力 他们的活动使用单分子全内反射荧光显微镜（smTIRF）。虽然我们的 实验室目前配备了TIRF显微镜，我们有能力进行多波长smTIRF 实验受到我们为每个荧光团选择最佳 TIRF 角度的能力的限制。 数据收集。这使得我们无法进行三色 smTIRF 实验，其中肌动蛋白 两种肌动蛋白调节蛋白被标记，每种蛋白都有不同的荧光团。也让我们的心情变得复杂 努力用荧光蛋白标记的单分子敏感性蛋白质进行可视化（例如 mNeonGreen），限制了我们可以做的实验种类。我们当前的 TIRF 显微镜也受到影响 照明不均匀问题使我们无法使用 TIRF 图像的整个视场，并且 使自动化数据处理更具挑战性。因此，我们请求资金购买新的 TIRF 显微镜将消除这些问题，从而大大提高我们的执行能力 多波长 smTIRF 实验。我们相信这款仪器是带有 iLas2 的 Leica TIRF 显微镜 圆形 TIRF 照明器，将大大增强我们完成父资助中特定目标的能力 并将增加我们研究对该领域的影响。