Curated Optogenetic Publication Database

Abstract: Necroptosis is a programmed lytic cell death involving active cytokine production and plasma membrane rupture through distinct signaling cascades. However, it remains challenging to delineate this inflammatory cell death pathway at specific signaling nodes with spatiotemporal accuracy. To address this challenge, we developed an optogenetic system, termed Light-activatable Receptor-Interacting Protein Kinase 3 or La-RIPK3, to enable ligand-free, optical induction of RIPK3 oligomerization. La-RIPK3 activation dissects RIPK3-centric lytic cell death through the induction of RIPK3-containing necrosome, which mediates cytokine production and plasma membrane rupture. Bulk RNA-Seq analysis reveals that RIPK3 oligomerization results in partially overlapped gene expression compared to pharmacological induction of necroptosis. Additionally, La-RIPK3 activates separated groups of genes regulated by RIPK3 kinase-dependent and -independent processes. Using patterned light stimulation delivered by a spatial light modulator, we demonstrate precise spatiotemporal control of necroptosis in La-RIPK3-transduced HT-29 cells. Optogenetic control of proinflammatory lytic cell death could lead to the development of innovative experimental strategies to finetune the immune landscape for disease intervention.

Abstract: Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell-cell communication precisely during necroptosis. Receptor-interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non-canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3-mediated necroptosis. Opsin-free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light-sensitive protein-protein interaction to modulate cell signaling. Compared to chemical-based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand-free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3-mediated necroptosis in colorectal HT-29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system Support Protocol: Quantification of the titer of lentivirus Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT-29 cells Basic Protocol 3: Optimization of optogenetic stimulation conditions Basic Protocol 4: Time-stamped live-cell imaging of HT-29 lytic cell death Basic Protocol 5: Quantification of HT-29 lytic cell death.

Abstract: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.

Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.

Abstract: Membrane fusion during synaptic transmission mediates the trafficking of chemical signals and neuronal communication. The fast kinetics of membrane fusion on the order of millisecond is precisely regulated by the assembly of SNAREs and accessory proteins. It is believed that the formation of the SNARE complex is a key step during membrane fusion. Little is known, however, about the molecular machinery that mediates the formation of a large pre-fusion complex, including multiple SNAREs and accessory proteins. Syntaxin, a transmembrane protein on the plasma membrane, has been observed to undergo oligomerization to form clusters. Whether this clustering plays a critical role in membrane fusion is poorly understood in live cells. Optogenetics is an emerging biotechnology armed with the capacity to precisely modulate protein-protein interaction in time and space. Here, we propose an experimental scheme that combines optogenetics with single-vesicle membrane fusion, aiming to gain a better understanding of the molecular mechanism by which the syntaxin cluster regulates membrane fusion. We envision that newly developed optogenetic tools could facilitate the mechanistic understanding of synaptic transmission in live cells and animals.

Abstract: Ligand-independent activation of receptor tyrosine kinases (RTKs) allows for dissecting out the receptor-specific signaling outcomes from the pleiotropic effects of the ligands. In this regard, RTK intracellular domains (ICD) are of interest due to their ability to recapitulate signaling activity in a ligand-independent manner when fused to chemical and optical dimerizing domains. A common strategy for synthetic activation of RTKs involves membrane tethering of dimerizer-RTK ICD fusions. Depending on the intrinsic signaling capacity, however, this approach could entail undesirable baseline signaling activity in the absence of stimulus, thereby diminishing the system's sensitivity. Here, we observed toxicity in early Xenopus laevis embryos when using such a conventional optogenetic design for the fibroblast growth factor receptor (FGFR). To surpass this challenge, we developed a cytoplasm-to-membrane translocation approach, where FGFR ICD is recruited from the cytoplasm to the plasma membrane by light, followed by its subsequent activation via homo-association. This strategy results in the optical activation of FGFR with low background activity and high sensitivity, which allows for the light-mediated formation of ectopic tail-like structure in developing Xenopus laevis embryos. We further generalized this strategy by developing optogenetic platforms to control three neurotrophic tropomyosin receptor kinases, TrkA, TrkB, and TrkC. We envision that these ligand-independent optogenetic RTKs will provide useful toolsets for the delineation of signaling sub-circuits in developing vertebrate embryos.