ANALYSIS OF CALI EXPERIMENTS WITH VIRTUAL CELL

虚拟细胞 CALI 实验分析

• 批准号: 8362508
• 负责人: Kenneth A Jacobson
• 金额: $ 1.05万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362508
• 关键词: Actins; Cell Line; Cell physiology; Cells; Chimeric Proteins; Complement; Dendritic Cells; Filament; Funding; Generations; Grant; Growth; Label; Lasers; Light; Mediating; Microfilaments; Modeling; National Center for Research Resources; Phenotype; Physiological; Principal Investigator; Proteins; Reagent; Research; Research Infrastructure; Resources; Role; Source; Structure; System; Techniques; United States National Institutes of Health; chromophore; cost; knock-down; loss of function; research study; spatiotemporal; virtual

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Chromophore-assisted laser inactivation (CALI) is a light-mediated technique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. We developed a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrated that CALI of EGFP-CapZbeta increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We showed that these effects are completely dependent upon knocking down the endogenous protein. To fully interpret these results and also guard against over-interpretation, a quantitative analysis, which build on the Virtual Cell dendritic actin nucleation model, will be developed.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 发色团辅助激光灭活（Cali）是一种光介导的技术，可提供精确的时空控制蛋白质失活，从而更好地了解蛋白质在细胞功能中的作用。 EGFP已被有效用作Cali发色团，其对靶蛋白的共通胶附着避免了使用外源添加的标记试剂。 EGFP-CALI的潜在缺点是，Cali表型可以被内源性的，未标记的蛋白质遮盖，这种蛋白质不容易被轻灭。在用功能性EGFP融合蛋白救出的缺陷细胞中进行EGFP-CALI实验可以实现更完整的功能丧失。我们开发了一种改良的慢病毒系统，用于快速有效地产生与生理水平的EGFP融合蛋白相辅相成的敲低细胞系。我们证明了EGFP-Capzbeta的Cali增加了未覆盖的肌动蛋白丝，从而增强了细丝生长并形成了许多突出结构。我们表明这些作用完全取决于击倒内源性蛋白。为了充分解释这些结果并防止过度解释，将开发基于虚拟细胞树突状肌动蛋白成核模型的定量分析。