David Thai

Bio: David Thai is an academic researcher. The author has contributed to research in topics: TEV protease & Organelle assembly. The author has an hindex of 1, co-authored 1 publications receiving 18 citations.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments

Abstract: We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Designer membraneless organelles sequester native factors for control of cell behavior

Abstract: Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

Aptamer-based optical manipulation of protein subcellular localization in cells.

Abstract: Protein-dominant cellular processes cannot be fully decoded without precise manipulation of their activity and localization in living cells. Advances in optogenetics have allowed spatiotemporal control over cellular proteins with molecular specificity; however, these methods require recombinant expression of fusion proteins, possibly leading to conflicting results. Instead of modifying proteins of interest, in this work, we focus on design of a tunable recognition unit and develop an aptamer-based near-infrared (NIR) light-responsive nanoplatform for manipulating the subcellular localization of specific proteins in their native states. Our results demonstrate that this nanoplatform allows photocontrol over the cytoplasmic-nuclear shuttling behavior of the target RelA protein (a member of the NF-κβ family), enabling regulation of RelA-related signaling pathways. With a modular design, this aptamer-based nanoplatform can be readily extended for the manipulation of different proteins (e.g., lysozyme and p53), holding great potential to develop a variety of label-free protein photoregulation strategies for studying complex biological events. Optogenetic manipulation of protein localisation in cells involves the creation of fusions that can influence activity. Here the authors develop a near-infrared light-responsive aptamer-based system to regulate the nuclear-cytoplasmic shuttling of NF-κB subunit RelA.

HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells.

Abstract: Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension.

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer

Abstract: Many dynamic biological processes are regulated by protein–protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein–protein interactions.

Fabricating an intelligent cell-like nano-prodrug via hierarchical self-assembly based on the DNA skeleton for suppressing lung metastasis of breast cancer.

Abstract: Cancer metastasis is a major cause of high mortality in breast cancer. Despite the progress achieved in nanomaterial-based treatments, the cure rate remains unsatisfactory, owing to their poor biocompatibility and non-specific recognition. Inspired by the cell-mimetic strategy, in this work, we fabricated an intelligent cell-like nano-prodrug (Dox-MPK@MDL) for lung metastasis of breast cancer. Specifically, a DNA tetrahedron dendrimer was selected to act as a rigid internal cytoskeleton, and then sequentially coated with a liposome and macrophage membrane to form cell-like Dox-MPK@MDL via hierarchical self-assembly. Here, it should be noted that pH-sensitive Dox-MPK prodrugs were synthesized and inserted into the DNA-based cytoskeleton (the Dox group is an intercalator of double stranded DNA) in advance for the next anti-metastatic therapy. Our results show that Dox-MPK@MDL specifically targeted the sites of lung metastasis via the biomimetic metastasis-homing effects and intelligently triggered Dox release at the metastatic cancer cells, thereby leading to the significant inhibition of lung metastasis. All these features help to enhance the anti-metastatic therapy efficiency of Dox while maximally reducing side-effects.