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

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. This project will also contribute to strengthening the STEM workforce by heavily involving students 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 Biosciences This 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.