ANALYSIS OF CALI EXPERIMENTS WITH VIRTUAL CELL

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 发色团辅助激光失活(CALI)是一种光介导的技术，它提供了对蛋白质失活的精确时空控制，使人们能够更好地了解蛋白质在细胞功能中的作用。EGFP已经被有效地用作钙生色团，它与目标蛋白的共翻译结合避免了不得不使用外源添加的标记试剂。EGFP-CALI的一个潜在缺点是，CALI的表型可能被内源性的、不受光灭活影响的未标记蛋白所掩盖。在用功能性EGFP融合蛋白拯救的缺陷细胞中进行EGFP-CALI实验，可以实现更完全的功能丧失。我们开发了一种改进的慢病毒系统，用于快速高效地产生击倒细胞系，并补充生理水平的EGFP融合蛋白。我们证明了EGFP-CapZβ的CALI增加了未封顶的肌动蛋白细丝，导致了微丝生长和大量突起结构的形成。我们证明，这些效应完全依赖于击倒内源蛋白质。为了充分解释这些结果并防止过度解释，将开发建立在虚拟细胞树突状肌动蛋白成核模型基础上的定量分析。

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.