Jared Toettcher - US grants

Abstract Harnessing optogenetics to diagnose and therapeutically rewire cancer cell signaling In recent years, advances in microscopy, sequencing, and proteomics have had a dramatic impact on our ability to probe the inner workings of cells and tissues. These tools have revealed that cells are remarkably complex information processing devices: external inputs activate many intracellular networks with complex dynamics. Yet despite the progress in measuring the cell's outputs, we have lacked analogous control over the inputs we can deliver to probe and perturb these complex networks. By replacing chemical stimuli with light, optogenetic tools can be used to apply and remove inputs with high spatial and temporal precision. Here, I propose to bring this precise input control to bear on two long-standing problems in cancer biology, a field where it has so far had limited impact. First, I will develop an approach termed ?optogenetic profiling?, which aims to directly measure how growth signaling is altered in tumor cells by measuring cellular responses to a rich set of input stimuli. Rooted in engineering, this approach is akin to probing an electronic circuit with a different signals to characterize its function, and can be highly informative even when the exact wiring diagram is unknown. This approach may offer a key to interpreting genomic data, allowing us to group cell lines with different mutations into shared functional classes. It could also have a large impact on treatment: identifying which pathway is deregulated may immediately suggest which targeted pathway inhibitors will be effective. Second, I will explore how light-induced protein aggregation can be used to therapeutically rewire cancer cell signaling. Tumor cells rely on signaling changes that both amplify proliferation and suppress apoptosis, but current therapies are typically limited to inhibiting pro-growth signaling. Here, I will test whether light-induced co-clustering of signaling proteins can perform two other therapeutic functions: amplifying apoptotic signaling or diverting growth inputs to cell-death outputs. The studies proposed will not only uncover fundamental principles in cell signaling but could usher in new approaches for cancer diagnosis (by functionally profiling signaling pathway responses) and treatment (by engineering gain-of-fuction therapies based on signaling enzyme clustering).

Summary One of the grand challenges of modern biology is to understand how gene activity is controlled in space and time, in the context of native chromosomes and in individual living cells. The goal of this proposal is to tackle exactly this challenge: we will develop new approaches to measure and manipulate long-range chromosomal interactions and quantify their effects on gene expression, in real-time and in living cells and tissues. By quantitatively mapping the relationship between transcription factor assembly (e.g. formation of biomolecular condensates), chromosome organization and transcription kinetics, our study will define how gene expression is controlled at unprecedented resolution. Transcriptional regulation forms the basis of cellular differentiation during organismal development, and its defects underlie a variety of disease states, from developmental disorders to cancer. Yet current methods are limited: traditional live-imaging lacks the spatial resolution to accurately define chromosome organization at the scale of individual genes, while bulk assays using fixed material are ill-suited for studying temporal dynamics. In addition, membrane-less nuclear condensates, which form through liquid-liquid phase separation, are thought to play key but as-yet-undefined roles in regulating transcription. To address these challenges, we will develop new imaging methods to measure chromosomal distances in living cells and build optogenetic tools to assemble/disassemble chromosome loops and nuclear condensates. We will deploy these tools to examine regulatory interactions at genomic scales characteristic of enhancer? promoter interactions in flies and mammals (from tens to hundreds of kilobases), and study their implications in the context of cell fate specification in the developing Drosophila embryo. The resulting technologies will be applied to analogous transcriptional loci in mouse embryonic stem cells and organoids derived from these cells. Together, the proposed studies will help reveal how robust mechanisms of cell type specification emerge from stochastic processes such as transcriptional bursts, fluctuations in the size and stability of biomolecular condensates, and dynamic instability of chromatin architecture. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately allow us to take control of gene activity by re-engineering the transcriptional programs underlying developmental and disease processes.

Cells can be coaxed into forming organ-like structures outside of the body. These “organoids” would help scientists study organ development, function, and disease. Organoids often fail to form in a reproducible manner, currently limiting their utility. This project will invent new approaches to reliably build organoids that mimic the lung. The investigators will track and control organoid formation using light and mechanical forces. The project will introduce high school and college students from underrepresented communities to scientific research. The project will also share its approaches by building an international symposium.

The minimal functional unit of the lung is the alveolus, which is comprised of alveolar epithelial type I (AT1) cells interspersed with type II (AT2) cells, surrounded by a meshwork of myofibroblasts that helps maintain 3D structure. The ability to reproducibly generate organoids that mimic the alveoli of the lung would have immense promise for studies aimed at understanding tissue function, the fundamental processes of respiratory infection, and the biomechanics of tissue structures during health and disease. Unfortunately, current protocols to generate alveolar organoids fail to reproduce native tissue structure. This RECODE project will uncover the rules necessary to differentiate alveolar progenitor cells into precise ratios of AT1:AT2 cells, and the contractile signaling that permits myofibroblasts to fold the epithelium into an alveolus. This transformational goal will be accomplished via a highly innovative combination of expertise from quantitative developmental biology, mechanobiology, biomaterials, computational modeling, and synthetic biology, which will be used to identify the spatiotemporal dynamics that governs alveolar differentiation and morphogenesis. The proposed research is divided into three main conceptual advances. Aim 1 focuses on using real-time fluorescent reporters, mathematical modeling, and optogenetics approaches to define the biochemical signaling dynamics necessary for specifying bipotent progenitors into AT2 cells. Aim 2 takes advantage of synthetic materials, real-time reporters, and optogenetics to uncover the mechanical signaling necessary for specifying bipotent progenitors into AT1 cells. Aim 3 combines computational modeling, 3D printing, and optogenetics to uncover and reproduce the patterns of contraction used by myofibroblasts to fold the epithelium into the alveolus. Altogether, this work will identify the design rules required to construct organoids that reproducibly differentiate into tissue structures that mimic alveoli within the lung.

This RECODE award is co-funded by the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences, the Developmental Systems Cluster in the Division of Integrative Organismal Systems, and the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems.

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.