Molecular mechanisms in the mammalian cell nucleus

Abstract/Project Summary My research group strives to gain detailed information about cellular nanoscale structures, dynamics, and molecular mechanisms by designing and applying innovative and versatile single-molecule imaging tools, labeling schemes, and analysis algorithms. The goal of our research is to improve our understanding of cellular function and pathogenesis by answering biophysical questions related to normal function, laminopathies, ciliopathies, cancers, and other diseases. One of our primary research interests and long-term goals is understanding the mammalian cell nucleus, including its related and anchored proteins, its chromatin organization and dynamics, and how they relate to gene regulation in healthy cells and during disease progression. In this proposal, we will build upon our expertise in fluorescence single-molecule tracking and super- resolution imaging, engineered point spread functions (PSFs) for 3D detection of single molecules with tens of nanometer precision, light sheet illumination for reduced out-of-focus background fluorescence, photobleaching, and photodamage during whole live-cell imaging, our recently developed dCas9-based nanobody array labeling approaches for long-term tracking of chromatin dynamics with excellent spatiotemporal resolution, and our recently developed light sheet-compatible microfluidic system for precise control of the extracellular environment. We will implement and expand these innovations to address a range of biophysical questions related to molecular mechanisms behind cellular function in healthy cells and during disease progression, including how the dynamics of chromatin organization affect gene expression and what the molecular mechanisms are that underlie the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS). To achieve our goals, we will pursue the following main thrusts: (1) develop and implement approaches for labeling and live-cell 3D tracking of specific chromosomal loci pairs, such as enhancer-promoter (EP) pairs, and gene expression during adjustable extracellular conditions. These measurements will yield novel data with unprecedented spatiotemporal resolution on enhancer and promoter dynamics, EP separation, contact frequency, and contact duration, and establish how these parameters relate to transcriptional output. This will enable us to uncover fundamental physical mechanisms of gene regulation and will set the stage for future research on interaction kinetics of various regulatory elements implicated in chromatin reorganization and gene regulation. (2) Define the molecular interactions of the mutated protein progerin in HGPS and map how its nanoscale spatial distribution and interactions with other proteins and with chromatin are affected by treatment with available and novel therapies. This thrust will be of great significance for human health by evaluating the molecular mechanisms of pathogenesis and treatment strategies for HGPS and the results may have wide-ranging medical implications also for other laminopathies. Our versatile methods are generalizable and enable future research on molecular mechanisms in a wide range of biological systems and diseases, including other laminopathies.