Curated Optogenetic Publication Database

Abstract: Inflammasomes are multiprotein platforms which control caspase-1 activation, leading to the processing of proinflammatory cytokines into mature and active cytokines IL-1β and IL-18, and to pyroptosis through the cleavage of gasdermin-D (GSDMD). Inflammasomes assemble upon activation of specific cytosolic pattern recognition receptors (PRRs) by damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). They converge to the nucleation of apoptosis-associated speck-like containing a caspase activation and recruitment domain (ASC) to form hetero-oligomers with caspase-1. Studying inflammasome encoding activities remains challenging because PAMPs and DAMPs are sensed by a large diversity of cytosolic and membranous PRRs. To bypass the different signals required to activate the inflammasome, we designed an optogenetic approach to temporally and quantitatively manipulate ASC assembly (i.e. in a PAMP- or DAMP-independent manner). We reveal that controlling light-sensitive oligomerization of ASC is sufficient to recapitulate the classical features of inflammasomes within minutes, and enabled us to decipher the complexity of volume regulation and pore opening during pyroptosis. Overall, this approach offers interesting perspective to decipher PRR signaling pathways in the field of innate immunity.

Abstract: Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control both passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

Abstract: Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein-protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.

Abstract: Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the ‘survival of motor neuron protein’ (SMN) is required for the formation of three different membraneless organelles (MLOs), we hypothesized that at least one region of SMN employs a unifying mechanism of condensation. Unexpectedly, we show here that SMN’s globular tudor domain was sufficient for dimerization-induced condensation in vivo, while its two intrinsically disordered regions (IDRs) were not. The condensate-forming property of the SMN tudor domain required binding to its ligand, dimethylarginine (DMA), and was shared by at least seven additional tudor domains in six different proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs – gems and Cajal bodies – were separate or overlapping. These findings show that the combination of a tudor domain bound to its DMA ligand – DMA-tudor – represents a versatile yet specific interaction module that regulates MLO assembly and defines their composition.

Abstract: Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Abstract: Previous studies with various Src family kinase biosensors showed that the nuclear kinase activities are much suppressed compared to those in the cytosol, suggesting that these kinases are regulated differently in the nucleus and in the cytosol. In this study, using Fyn as an example, we first engineered a Fyn biosensor with a light-inducible nuclear localization signal (LINuS) to demonstrate that the Fyn kinase activity is significantly lower in the nucleus than in the cytosol. To understand how different equilibrium states between Fyn and the corresponding phosphatases are maintained in the cytosol and nucleus, we further engineered a Fyn kinase domain with LINuS. The results revealed that the Fyn kinase can be actively transported into the nucleus upon light activation and upregulate the biosensor signals in the nucleus. Our results suggest that there is limited transport or diffusion of Fyn kinase between the cytosol and nucleus in the cells, which is important for the maintenance of different equilibrium states of Fyn in situ.

Abstract: Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.

Abstract: The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.

Abstract: We developed VIEW-MOD (Versatile Illumination Engine with a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.

Abstract: Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.

Abstract: The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

Abstract: Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms.