Collaborative Research: Tools 4 Cells: Developing Next Generation Methods for Studying Cytoskeletal Factors in the Cell Nucleus

近年来，基因在细胞核中的定位对于开启和关闭它们很重要，这一点已经变得很清楚。然而，人们对基因定位是如何调控的知之甚少。该项目的目标是了解肌动蛋白，一种在组织细胞质中起核心作用的蛋白质，如何参与基因组的组织和基因表达的控制。这需要新的工具，精确扰动肌动蛋白只在细胞核中，以避免任何混淆的影响，由于其细胞质部分的中断。这些工具包括光激活的核肌动蛋白丝的稳定剂和选择性降解核肌动蛋白的系统。 总之，这些工具将能够对核肌动蛋白进行精细控制，而不会影响其在细胞其他部分的功能。 该项目的更广泛影响包括其内在价值，因为所有有核细胞都可能含有肌动蛋白，并且开发的工具将传播给该领域的其他研究人员，预计将广泛影响我们对控制基因组定位和基因使用的基本机制的理解。此外，UIC是一个少数服务机构，芝加哥大学参与了许多努力，从STEM学科代表性不足的群体中招收学生。该项目将保持这一传统，并通过让来自这些群体的学生参与科学，为加强STEM人才队伍做出贡献。基于肌动蛋白的细胞骨架因子，对于定义细胞形状至关重要，也存在于细胞核中，在那里它们与转录和染色质组织有关。揭示这些影响背后的机制是复杂的挑战，具体针对细胞骨架因子的核池的动态扰动。现有技术引入了伪影，并且在快速时间尺度上不可操纵。在这个项目中，将开发新的和更锋利的工具。这些措施包括适应LILAC，光活化探针，跟踪和/或稳定肌动蛋白丝在细胞核中，而不扰动细胞质肌动蛋白。反之亦然，为了能够精确地控制核肌动蛋白降解，将生长素诱导的降解决定子（AID）标签与内源性β-肌动蛋白融合，并与强核定位的TIR 1泛素连接酶组合。总之，这些损失（降解决定子耗尽）和获得功能（LILAC）核肌动蛋白诱导的操作在克隆细胞系将使肌动蛋白动力学对核过程的直接影响的发现。这些工具将被广泛的特点和用于研究肌动蛋白在核组织中的作用。高分辨率染色质构象捕获（Micro-C）将用于揭示核肌动蛋白动力学对精细尺度染色质重组的影响。校准的ChIP-seq和PRO-seq将产生对染色质状态的相关变化和对新生转录的功能影响的互补见解。调整核肌动蛋白动力学在纳米尺度上对Pol II聚类的直接影响将在活细胞和固定细胞中使用定量超分辨率显微镜进行研究。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查进行评估，被认为值得支持的搜索.

In recent years it has become clear that the positioning of genes in the cell nucleus is important for turning them on and off. However, little is known about how gene positioning is regulated. The goal of this project is to understand how actin, a protein that plays a central role in organizing the cytoplasm, is also involved in the organization of the genome and the control of gene expression. This requires novel tools for the precise perturbation of actin only in the cell nucleus to avoid any confounding effects due to disruptions of its cytoplasmic fraction. These tools include a light-activatable stabilizer of nuclear actin filaments and a system to selectively degrade nuclear actin. Together, these tools will enable fine control over nuclear actin without impacting its functions in other parts of the cell. The Broader Impacts of this project include its intrinsic merit as all nucleated cells likely contain actin and the developed tools will be disseminated to other researchers in the field and are expected to broadly impact our understanding of basic mechanisms that control genome positioning and gene usage. Further, UIC is a minority serving institution and the University of Chicago participates in numerous efforts to recruit students from groups underrepresented in STEM disciplines. This project will maintain this tradition and contribute to strengthening the STEM workforce by heavily involving students from these groups in science.Actin-based cytoskeletal factors, essential for defining cell shape, are also present in the nucleus, where they have been linked to transcription and chromatin organization. Uncovering the mechanisms behind these effects is complicated by the challenges of specifically targeting the nuclear pool of cytoskeletal factors for dynamic perturbation. Existing techniques introduce artifacts and are not manipulable on rapid timescales. In this project, new and sharper tools will be developed. These include an adaptation of LILAC, a photoactivated probe, to track and/or stabilize actin filaments in the nucleus, without perturbations to cytoplasmic actin. Vice versa, to enable precise temporal control of nuclear actin degradation, an auxin inducible degron (AID) tag will be fused to endogenous beta-actin and combined with a strongly nuclear-localized TIR1 ubiquitin ligase. Together, these loss- (degron depletion) and gain-of-function (LILAC) nuclear actin inducible manipulations in clonal cell lines will enable the discovery of direct effects of actin dynamics on nuclear processes. These tools will be broadly characterized and used to investigate the role of actin in nuclear organization. High-resolution chromatin conformation capture (Micro-C) will be used to reveal the impact of nuclear actin dynamics on fine-scale chromatin reorganization. Calibrated ChIP-seq and PRO-seq will generate complementary insights into associated changes in chromatin state and functional impacts on nascent transcription. Direct effects of tuning nuclear actin dynamics on Pol II clustering at the nanometer scale will be investigated using quantitative superresolution microscopy in live and fixed cells.The project is co-funded by the Genetic Mechanisms program in the Division of Molecular and Cellular BiosciencesThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.