Curated Optogenetic Publication Database

Abstract: Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function—dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

Abstract: The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.

Abstract: The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

Abstract: Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

Abstract: The regulation of synaptic transmission is crucial for plasticity, homeostasis and learning. Chemical synaptic transmission is thus modulated to accommodate different activity levels, which also enables homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cAMP signaling enhances cholinergic neuron output, and these neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish. Optogenetic stimulation by photoactivated adenylyl cyclase (bPAC) resulted in elevated locomotion as measured in behavioural assays. Furthermore, post-synaptic patch-clamp recordings revealed that in bPAC transgenics, the frequency of miniature excitatory postsynaptic currents (mEPSCs) was increased after photostimulation. These results suggested that cAMP-mediated activation of ZF motor neurons leads to increased fusion of SVs, consequently resulting in enhanced neuromuscular activity. We generated mutants lacking the neuropeptide processing enzyme carboxypeptidase E (cpe), and the most abundant neuropeptide precursor in motor neurons, tachykinin (tac1). Both mutants showed exaggerated locomotion after photostimulation. cpe mutants exhibit lower mEPSC frequency during photostimulation and less large-amplitude mEPSCs. In tac1 mutants mEPSC frequency was not affected but amplitudes were significantly smaller. Exaggerated locomotion in the mutants thus reflected upscaling of postsynaptic excitability. cpe and tac1 mutant muscles expressed more nicotinic acetylcholine receptors (nAChR) on their surface. Thus, neuropeptide signaling regulates synaptic transmitter output in zebrafish motor neurons, and muscle cells homeostatically regulate nAChR surface expression, compensating reduced presynaptic input. This mechanism may be widely conserved in the animal kingdom.

Abstract: T cells regulate adaptive immune responses through complex signaling pathways mediated by T cell receptor (TCR). The functional domains of the TCR are combined with specific antibodies for the development of chimeric antigen receptor (CAR) T cell therapy. In this review, we first overview current understanding on the T cell signaling pathways as well as traditional methods that have been widely used for the T cell study. These methods, however, are still limited to investigating dynamic molecular events with spatiotemporal resolutions. Therefore, genetically encoded biosensors and optogenetic tools have been developed to study dynamic T cell signaling pathways in live cells. We review these cutting-edge technologies that revealed dynamic and complex molecular mechanisms at each stage of T cell signaling pathways. They have been primarily applied to the study of dynamic molecular events in TCR signaling, and they will further aid in understanding the mechanisms of CAR activation and function. Therefore, genetically encoded biosensors and optogenetic tools offer powerful tools for enhancing our understanding of signaling mechanisms in T cells and CAR-T cells.

Abstract: Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses.

Abstract: Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical-genetic approaches to probe the role of biomolecular condensates in health and disease.

Abstract: The structural plasticity of synapses is crucial for regulating brain functions. However, currently available methods for studying synapse organization based on split fluorescent proteins (FPs) have been limited in assessing synaptic dynamics in vivo due to the irreversible binding of split FPs. Here, we develop 'SynapShot', a method for visualizing the structural dynamics of intact synapses by combining dimerization-dependent FPs (ddFPs) with engineered synaptic adhesion molecules. SynapShot allows real-time monitoring of reversible and bidirectional changes of synaptic contacts under physiological stimulation. The application of green and red ddFPs in SynapShot enables simultaneous visualization of two distinct populations of synapses. Notably, the red-shifted SynapShot is highly compatible with blue light-based optogenetic techniques, allowing for visualization of synaptic dynamics while precisely controlling specific signaling pathways. Furthermore, we demonstrate that SynapShot enables real-time monitoring of structural changes in synaptic contacts in the mouse brain during both primitive and higher-order behaviors.

Abstract: Cellular processes can be modulated by physical means, such as light, which offers advantages over chemically inducible systems with respect to spatiotemporal control. Here we introduce an optogenetic gene expression system for Lactococcus lactis that utilizes a split T7 RNA polymerase linked to two variants of the Vivid regulators. Depending on the chosen photoreceptor variant, either ‘Magnets’ or ‘enhanced Magnets’, this system can achieve either high protein expression levels or low basal activity in the absence of light, exhibiting a fold induction close to 30, rapid expression kinetics, and heightened light sensitivity. This system functions effectively in liquid cultures and within cells embedded in hydrogel matrices, highlighting its potential in the development of novel engineered living materials capable of responding to physical stimuli such as light. The optogenetic component of this system is highly customizable, allowing for the adjustment of expression patterns through modifications to the promoters and/or engineered T7 RNA polymerase variants. We anticipate that this system can be broadly adapted to other Gram-positive hosts with minimal modifications required.

Abstract: Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with different types of responses. Here, we design and demonstrate a light-activated contact-dependent communication tool for synthetic cells. We utilize a split bioluminescent protein to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. Our modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

Abstract: When animals are infected by a pathogen, peripheral sensors of infection signal to the brain to coordinate a set of adaptive behavioral changes known as sickness behaviors. While the pathways that signal from the periphery to the brain have been intensively studied in recent years, how central circuits are reconfigured to elicit sickness behaviors is not well understood. Here we find that neuromodulatory systems linked to stress and satiety are recruited upon infection to drive sickness behaviors in C. elegans. Upon chronic infection by the bacterium Pseudomonas aeruginosa PA14, C. elegans decrease their feeding behavior, then display reversible bouts of quiescence, and eventually die. The ALA neuron and its neuropeptides FLP-7, FLP-24, and NLP-8, which control stress-induced sleep in uninfected animals, promote the PA14-induced feeding reduction. However, the ALA neuropeptide FLP-13 instead acts to delay quiescence and death in infected animals. Cell-specific genetic perturbations show that the neurons that release FLP-13 to delay quiescence in infected animals are distinct from ALA. A brain-wide imaging screen reveals that infection-induced quiescence involves ASI and DAF-7/TGF-beta, which control satiety-induced quiescence in uninfected animals. Our results suggest that a common set of neuromodulators are recruited across different physiological states, acting from distinct neural sources and in distinct combinations to drive state-dependent behaviors.

Abstract: The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Abstract: The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.

Abstract: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system, and introduce a powerful new tool for bacterial optogenetics.

Abstract: Chimeric antigen receptor (CAR)-T cell immunotherapy emerges as an effective cancer treatment. However, significant safety concerns remain, such as cytokine release syndrome (CRS) and "on-target, off-tumor" cytotoxicity, due to a lack of precise control over conventional CAR-T cell activity. To address this issue, a nano-optogenetic approach has been developed to enable spatiotemporal control of CAR-T cell activity. This system is comprised of synthetic light-sensitive CAR-T cells and upconversion nanoparticles acting as an in situ nanotransducer, allowing near-infrared light to wirelessly control CAR-T cell immunotherapy.

Abstract: By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

Abstract: Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

Abstract: Avena Sativa phototropin 1 Light-oxygen-voltage 2 domain (AsLOV2) is the model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change is initiated by the unfolding of the N-terminal helix in the dark state followed by the unfolding of the C-terminal helix. The light state is defined by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, β-sheets have been identified as crucial components for mediating allosteric signal transmission between the two termini. In this study, we combined microsecond all-atm molecular dynamics simulations and Markov state modeling of conformational states to quantify molecular basis of mutation-induced allostery in the AsLOV2 protein. Through a combination of computational investigations, we determine that the Hβ and Iβ strands are the most critical structural elements involved in the allosteric mechanism. To elucidate the role of these β-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive simulation analysis. The results highlighted the role of two hydrogen bond Asn482-Leu453 and Gln479-Val520 in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. The comprehensive atomistic-level analysis of the conformational landscapes revealed the critical functional role of β-sheet segments in the transmission of the allosteric signal upon the photoactivation of the light state.

Abstract: Mural cells are critical components of the cerebral vasculature. They are categorized into three primary subsets: arteriole smooth muscle cells (aSMCs), pericytes (PCs) and venule smooth muscle cells (vSMCs). It is well known that aSMCs can directly regulate cerebral blood flow (CBF) with their own contraction and dilation mechanisms. On the other hand, the direct involvement of PCs or vSMCs in CBF regulation is controversial. This ambiguity is largely due to the lack of specifically manipulable tools to isolate their function. To address this issue, we employed a set-subtraction approach by using a combination of tTA-mediated gene induction and Cre-mediated gene excision. We developed transgenic mice expressing optical actuators, channelrhodopsin-2 (ChR2) and photoactivated adenylyl cyclase (PAC) in smooth muscle actin (SMA)-negative mural cells that lack the machinery for SMA-mediated vasoregulation. Using these mouse models, we assessed CBF alterations in response to optical stimulation using laser Doppler techniques. Our results showed that optical stimulation induced notable CBF changes in both models. This study provides evidence for the potential regulatory role of PCs and vSMCs in cerebral hemodynamics and introduces powerful tools to specifically manipulate these cell types in vascular neurobiology.

Abstract: Presynaptic forms of plasticity occur throughout the nervous system and play an important role in learning and memory but the underlying molecular mechanisms are insufficiently understood. Here we show that the small GTPase Rab3 is a key mediator of cyclic AMP (cAMP)-induced presynaptic plasticity in Drosophila. Pharmacological and optogenetic cAMP production triggered concentration-dependent alterations of synaptic transmission, including potentiation and depression of evoked neurotransmitter release, as well as strongly facilitated spontaneous release. These changes correlated with a nanoscopic rearrangement of the active zone protein Unc13A and required Rab3. To link these results to animal behaviour, we turned to the established role of cAMP signalling in memory formation and demonstrate that Rab3 is necessary for olfactory learning. As Rab3 is dispensable for basal synaptic transmission, these findings highlight a molecular pathway specifically dedicated to tuning neuronal communication and adaptive behaviour.

Abstract: The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive spit transcription factors in the budding yeast, Saccharomyces cerevisiae. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.

Abstract: Optogenetics is an attractive synthetic biology tool for controlling the metabolic flux distribution. Here, we demonstrated optogenetic flux ratio control of glycolytic pathways consisting of the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP), and Entner-Doudoroff (ED) pathways by illuminating multicolor lights using blue light-responsive EL222 and green/red light-responsive CcaSR in Escherichia coli. EL222 forms a dimer and binds to a particular DNA sequence under blue light; therefore, target gene expression can be reduced or induced by inserting a recognition sequence into its promoter regions. First, a flux ratio between the PP and ED pathways was controlled by blue light using EL222. After blocking the EMP pathway, the EL222-recognition sequence was inserted between the -35 and -10 regions of gnd to repress the PP flux and was also inserted upstream of the -35 region of edd to induce ED flux. After adjusting light intensity, the PP:ED flux ratios were 60:39% and 29:70% under dark and blue light conditions, respectively. Finally, a CcaSR-based pgi expression system was implemented to control the flux ratio between the EMP and PP + ED pathways by illuminating green/red light. The EMP:PP:ED flux ratios were 80:9:11%, 14:35:51%, and 33:5:62% under green, red, and red and blue light, respectively.

Abstract: Inhibition of Bruton's tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph, R.E., et al., 2020, https://doi.org/10.7554/eLife.60470 ). Here we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.

Abstract: We report an integrated optogenetic and bioelectronic platform for stable and long-term modulation and monitoring of cardiomyocyte function in vitro. Optogenetic inputs were achieved through expression of a photoactivatable adenylyl cyclase (bPAC), that when activated by blue light caused a dose-dependent and time-limited increase in autonomous cardiomyocyte beat rate. Bioelectronic readouts were achieved through an integrated planar multi-electrode array (MEA) that provided real-time readouts of electrophysiological activity from 32 spatially-distinct locations. Irradiation at 27 μW/mm2 resulted in a ca. 14% increase in beat rate within 20-25 minutes, which remained stable for at least 2 hours. The beating rate could be cycled through repeated “on” and “off” states, and its magnitude was a monotonic function of irradiation intensity. Our integrated platform opens new avenues in bioelectronic medicine, including closedloop feedback systems, with potential applications for cardiac regulation including arrhythmia diagnosis and intervention.