Our goal is to achieve a multi-scale understanding of the molecular organization of living systems. How do monomeric building blocks form macromolecules? and how do macromolecules give rise to organelles and cells? To address these questions, we develop tools to probe, manipulate, and re-program how cells communicate with one another. We then leverage these tools to gain insight into natural biology and to overcome roadblocks in the development of cell therapies and tissue engineered systems.
Synthetic Mechanobiology: here we are engineering and investigating how cells send and interpret mechanical cues.
Viral Proteases & their Inhibitors as Biology Tools: we continue to develop new tools to control biological activities with increased spatiotemporal, molecular, and cellular resolution.
High Resolution Imaging of the Molecular Organization of Cells: an integrative goal of the lab is to bridge the “resolution gap” which has traditionally divided researchers along macromolecular and tissue/organismal level scales. To facilitate this goal, we are developing imaging tools to visualize molecular interactions along nanometer to micron-level dimensions.
Control of mRNA Localization and Translation in Cells: we developing new molecular tools to track and manipulate specified mRNAs in cells and organisms.
I. Synthetic Mechanobiology
Cells can sense and interpret mechanical stimuli from their environments and neighbors, but the ability to engineer customized mechanosensing capabilities has long been a challenge for synthetic and mechanobiology. To address this challenge, we are developing tension-tunable receptors that can be used to program relationships between extracellular and inter-cellular forces and gene expression changes.
We have generated tunable mechanoreceptors by stabilizing a force-sensitive component of Notch/SynNotch receptors known as the “negative regulatory region” (NRR), which must be mechanically unfolded for signaling to ensue. To permit the tuning of receptor sensitivities, we first stabilized the NRR by fusing it with an inhibitory anti-NRR antibody fragment, producing a strengthened NRR (sNRR) that required ten times more tension for signaling activity. This requirement was then lowered by altering binding interactions within the domain’s “mechanoactive” site using a structure-guided mutagenesis approach. This strategy allowed us to populate the sensitivity gap between NRR and sNRR domains, resulting in 14 mechanoreceptor sequences with tensile sensitivities that span the biologically relevant picoNewton (pN) range. When expressed by cells, these proteins could be used to activate gene expression responses following stimulation with extracellular and intercellular tensional cues.
Cell based therapeutics are difficult to develop because of the limited ways to control their activities following infusion into the body. To address this challenge, my lab has pioneered novel “chemogenetic” methods to regulate synthetic mammalian cell functions using orthogonal small molecules:
II. Viral Proteases and their Inhibitors as Synthetic Biology Tools
In Tague, Dotson, Tunney, et. al. (2018) we developed a method in which the NS3 cis-protease from hepatitis C virus (HCV) can be used as a ligand-inducible connection to control the function and localization of engineered proteins in mammalian cells. To demonstrate the versatility of this approach, we designed drug-sensitive transcription factors and transmembrane signaling proteins, the activities of which can be tightly and reversibly controlled using clinically tested antiviral protease inhibitors. In addition, we have recently devised schemes which biorthogonal ligation chemistry is exploited to control cell signaling and gene expression in mammalian cells. Collectively, this work represents important contributions to the synthetic biology toolkit by providing ways to control cell based therapeutics using safe and orthogonal small molecules.
An integrative goal of the lab is to achieve a “multi-scale” understanding of cell-cell communication, with the ultimate ambition of bridging the “resolution gap” which has traditionally divided researchers along macromolecular and tissue/organismal level scales. To facilitate this goal, we are developed imaging tools that can be used to visualize molecular interactions along nanometer to micron-level scales.
III. High Resolution Imaging of the Molecular Organization of Cells
We are designing tools for tagging and tracking biomolecules and sub cellular structures using correlative light and electron microscopy (CLEM), expansion microscopy (ExM), and other complementary techniques.
mRNA localization is an essential regulator of countless multicellular phenomena (including epithelial polarization, synaptic plasticity, and stem cell differentiation), yet the functions and regulatory mechanisms of localized mRNAs remain poorly understood. A primary bottleneck in the understanding of these systems is the limited ways in which these transcripts can be analyzed and controlled in living cells. To overcome these limitations, we developing new molecular tools to track and control specified mRNA sequences in cells and organisms.
IV. Imaging, Manipulation, and Control of mRNA Localization and Translation
A long-standing ambition of the lab is to understand localized mRNA translation in the context of cell-cell signaling and synaptic plasticity. It is known that cells and neurons possess localized mRNAs which are trafficked to and translated within discrete subcellular compartments and spatial regulation in this manner is thought to define local proteome compositions during memory formation.