Showing 1 - 8 of 8 results
Optical Sensors and Actuators for Probing Proximity-Dependent Biotinylation in Living Cells.
Proximity-dependent biotinylation techniques have been gaining wide applications in the systematic analysis of protein-protein interactions (PPIs) on a proteome-wide scale in living cells. The engineered biotin ligase TurboID is among the most widely adopted given its enhanced biotinylation efficiency, but it faces the background biotinylation complication that might confound proteomic data interpretation. To address this issue, we report herein a set of split TurboID variants that can be reversibly assembled by using light (designated "OptoID"), which enable optogenetic control of biotinylation based proximity labeling in living cells. OptoID could be further coupled with an engineered monomeric streptavidin that permits real-time monitoring of biotinylation with high temporal precision. These optical actuators and sensors will likely find broad applications in precise proximity proteomics and rapid detection of biotinylation in living cells.
Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety.
Chimeric antigen receptor (CAR) T cell-based immunotherapy, approved by the US Food and Drug Administration, has shown curative potential in patients with haematological malignancies. However, owing to the lack of control over the location and duration of the anti-tumour immune response, CAR T cell therapy still faces safety challenges arising from cytokine release syndrome and on-target, off-tumour toxicity. Herein, we present the design of light-switchable CAR (designated LiCAR) T cells that allow real-time phototunable activation of therapeutic T cells to precisely induce tumour cell killing. When coupled with imaging-guided, surgically removable upconversion nanoplates that have enhanced near-infrared-to-blue upconversion luminescence as miniature deep-tissue photon transducers, LiCAR T cells enable both spatial and temporal control over T cell-mediated anti-tumour therapeutic activity in vivo with greatly mitigated side effects. Our nano-optogenetic immunomodulation platform not only provides a unique approach to interrogate CAR-mediated anti-tumour immunity, but also sets the stage for developing precision medicine to deliver personalized anticancer therapy.
Optogenetic Control of Non-Apoptotic Cell Death.
Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.
Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling.
Genetically encoded photoswitches have enabled spatial and temporal control of cellular events to achieve tailored functions in living cells, but their applications to probe the structure-function relations of signaling proteins are still underexplored. We illustrate herein the incorporation of various blue light-responsive photoreceptors into modular domains of the stromal interaction molecule 1 (STIM1) to manipulate protein activity and faithfully recapitulate STIM1-mediated signaling events. Capitalizing on these optogenetic tools, we identify the molecular determinants required to mediate protein oligomerization, intramolecular conformational switch, and protein-target interactions. In parallel, we have applied these synthetic devices to enable light-inducible gating of calcium channels, conformational switch, dynamic protein-microtubule interactions and assembly of membrane contact sites in a reversible manner. Our optogenetic engineering approach can be broadly applied to aid the mechanistic dissection of cell signaling, as well as non-invasive interrogation of physiological processes with high precision.
A molecular toolbox for interrogation of membrane contact sites.
Membrane contact sites (MCSs) are specialized subcellular compartments formed by closely apposed membranes from two organelles. The intermembrane gap is separated by a distance ranging from 10 to 35 nm. MCSs are typically maintained through dynamic protein-protein and protein-lipid interactions. These intermembrane contact sites constitute important intracellular signalling hotspots to mediate a plethora of cellular processes, including calcium homeostasis, lipid metabolism, membrane biogenesis and organelle remodelling. In recent years, a series of genetically encoded probes and chemogenetic or optogenetic actuators have been invented to aid the visualization and interrogation of MCSs in both fixed and living cells. These molecular tools have greatly accelerated the pace of mechanistic dissection of membrane contact sites at the molecular level. In this review, we present an overview on the latest progress in this endeavour, and provide a general guide to the selection of methods and molecular tools for probing interorganellar membrane contact sites.
CRAC channel-based optogenetics.
Store-operated Ca²+ entry (SOCE) constitutes a major Ca2+ influx pathway in mammals to regulate a myriad of physiological processes, including muscle contraction, synaptic transmission, gene expression, and metabolism. In non-excitable cells, the Ca²+ release-activated Ca²+ (CRAC) channel, composed of ORAI and stromal interaction molecules (STIM), constitutes a prototypical example of SOCE to mediate Ca2+ entry at specialized membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and the plasma membrane (PM). The key steps of SOCE activation include the oligomerization of the luminal domain of the ER-resident Ca2+ sensor STIM1 upon Ca²+ store depletion, subsequent signal propagation toward the cytoplasmic domain to trigger a conformational switch and overcome the intramolecular autoinhibition, and ultimate exposure of the minimal ORAI-activating domain to directly engage and gate ORAI channels in the plasma membrane. This exquisitely coordinated cellular event is also facilitated by the C-terminal polybasic domain of STIM1, which physically associates with negatively charged phosphoinositides embedded in the inner leaflet of the PM to enable efficient translocation of STIM1 into ER-PM MCSs. Here, we present recent progress in recapitulating STIM1-mediated SOCE activation by engineering CRAC channels with optogenetic approaches. These STIM1-based optogenetic tools make it possible to not only mechanistically recapture the key molecular steps of SOCE activation, but also remotely and reversibly control Ca²+-dependent cellular processes, inter-organellar tethering at MCSs, and transcriptional reprogramming when combined with CRISPR/Cas9-based genome-editing tools.
Optical control of membrane tethering and interorganellar communication at nanoscales.
Endoplasmic reticulum (ER) forms an extensive intracellular membranous network in eukaryotes that dynamically connects and communicates with diverse subcellular compartments such as plasma membrane (PM) through membrane contact sites (MCSs), with the inter-membrane gaps separated by a distance of 10-40 nm. Phosphoinositides (PI) constitute an important class of cell membrane phospholipids shared by many MCSs to regulate a myriad of cellular events, including membrane trafficking, calcium homeostasis and lipid metabolism. By installing photosensitivity into a series of engineered PI-binding domains with minimal sizes, we have created an optogenetic toolkit (designated as 'OptoPB') to enable rapid and reversible control of protein translocation and inter-membrane tethering at MCSs. These genetically-encoded, single-component tools can be used as scaffolds for grafting lipid-binding domains to dissect molecular determinants that govern protein-lipid interactions in living cells. Furthermore, we have demonstrated the use of OptoPB as a versatile fusion tag to photomanipulate protein translocation toward PM for reprogramming of PI metabolism. When tethered to the ER membrane with the insertion of flexible spacers, OptoPB can be applied to reversibly photo-tune the gap distances at nanometer scales between the two organellar membranes at MCSs, and to gauge the distance requirement for the free diffusion of protein complexes into MCSs. Our modular optical tools will find broad applications in non-invasive and remote control of protein subcellular localization and interorganellar contact sites that are critical for cell signaling.
Near-infrared photoactivatable control of Ca(2+) signaling and optogenetic immunomodulation.
The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca(2+) oscillations and Ca(2+)-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca(2+)-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca(2+)-modulated activities with tailored function.