ANALYSIS OF CALI EXPERIMENTS WITH VIRTUAL CELL

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 发色团辅助激光失活（卡利）是一种光介导的技术，可提供蛋白质失活的精确时空控制，从而更好地了解蛋白质在细胞功能中的作用。EGFP已被有效地用作卡利发色团，并且其与靶蛋白的共翻译连接避免了必须使用外源添加的标记试剂。EGFP-CALI的一个潜在缺点是卡利表型可能被内源性未标记的蛋白质遮蔽，该蛋白质对光失活不敏感。在用功能性EGFP融合蛋白拯救的缺陷细胞中进行EGFP-CALI实验允许实现更完全的功能丧失。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融合蛋白互补的敲减细胞系。我们证明了EGFP-CapZ β的卡利增加了未加帽的肌动蛋白丝，导致增强的丝生长和许多膨胀结构的形成。我们发现这些作用完全依赖于敲低内源性蛋白。为了充分解释这些结果，也防止过度解释，定量分析，这是建立在虚拟细胞树突状肌动蛋白成核模型，将开发。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.