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.

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