Showing 1 - 7 of 7 results
Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry.
Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells.
Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering.
The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular “plug and play” design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads (‘PHD beads’), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of ∼109 protein molecules per bead (Ø 20 μm). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a ∼50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis.
Improved Photocleavable Proteins with Faster and More Efficient Dissociation.
The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.
SPLIT: Stable Protein Coacervation using a Light Induced Transition.
Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membrane-less organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membrane-less organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.
Hydrogels With Tunable Mechanical Properties Based on Photocleavable Proteins.
Hydrogels with photo-responsive mechanical properties have found broad biomedical applications, including delivering bioactive molecules, cell culture, biosensing, and tissue engineering. Here, using a photocleavable protein, PhoCl, as the crosslinker we engineer two types of poly(ethylene glycol) hydrogels whose mechanical stability can be weakened or strengthened, respectively, upon visible light illumination. In the photo weakening hydrogels, photocleavage leads to rupture of the protein crosslinkers, and decrease of the mechanical properties of the hydrogels. In contrast, in the photo strengthening hydrogels, by properly choosing the crosslinking positions, photocleavage does not rupture the crosslinking sites but exposes additional cryptical reactive cysteine residues. When reacting with extra maleimide groups in the hydrogel network, the mechanical properties of the hydrogels can be enhanced upon light illumination. Our study indicates that photocleavable proteins could provide more designing possibilities than the small-molecule counterparts. A proof-of-principle demonstration of spatially controlling the mechanical properties of hydrogels was also provided.
Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.
Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.
Genetically Encoded Photocleavable Linkers for Patterned Protein Release from Biomaterials.
Given the critical role that proteins play in almost all biological processes, there is great interest in controlling their presentation within and release from biomaterials. Despite such outstanding enthusiasm, previously developed strategies in this regard result in ill-defined and heterogeneous populations with substantially decreased activity, precluding their successful application to fragile species including growth factors. Here, we introduce a modular and scalable method for creating monodisperse, genetically encoded chimeras that enable bioactive proteins to be immobilized within and subsequently photoreleased from polymeric hydrogels. Building upon recent developments in chemoenzymatic reactions, bioorthogonal chemistry, and optogenetics, we tether fluorescent proteins, model enzymes, and growth factors site-specifically to gel biomaterials through a photocleavable protein (PhoCl) that undergoes irreversible backbone photoscission upon exposure to cytocompatible visible light (λ ≈ 400 nm) in a dose-dependent manner. Mask-based and laser-scanning lithographic strategies using commonly available light sources are employed to spatiotemporally pattern protein release from hydrogels while retaining their full activity. The photopatterned epidermal growth factor presentation is exploited to promote anisotropic cellular proliferation in 3D. We expect these methods to be broadly useful for applications in diagnostics, drug delivery, and regenerative medicine.