Curated Optogenetic Publication Database

Abstract: Cells and tissue integrity is constantly challenged by the necessity to adapt and respond to mechanical loads. Among the cellular components, the nucleus possesses mechano-sensing and mechanotransduction capabilities, yet the molecular mechanisms involved remain poorly defined. We postulated that the mechanical properties of the chromatin and its compartmentalization into condensates contribute to the nuclear adaptation to external forces, while preserving its integrity. By interrogating the effects of MLL4 loss-of-function in Kabuki Syndrome, we found that the balancing of transcriptional and Polycomb condensates tunes the nuclear responsiveness to external mechanical forces. We showed that MLL4 acts as a chromatin mechano-sensor by clustering into condensates through its Prion-like domain, and its response was regulated by the chromatin context. Furthermore, the mechano-sensing activity of MLL4 condensates is instrumental to withstand the physical challenges that nuclei experience during cell confinement and migration by preserving their integrity. In Kabuki Syndrome persistent rupture of nuclear envelope triggers cGAS-STING activation, which leads to programmed cell death. Ultimately, these results demonstrate the critical role chromatin compartments play in mechano-responses and how they impact pathological conditions by stimulating cGAS-STING signaling.

Abstract: Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer’s disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. This intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

Abstract: Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Abstract: The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of β-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.

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 split 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: Protein engineering is important for creating novel variants from natural proteins, enabling a wide range of applications. Approaches such as rational design and directed evolution are routinely used to make new protein variants. Computational tools like de novo design can introduce new protein folds. Expanding the amino acid repertoire to include unnatural amino acids with non-canonical side chains in vitro by native chemical ligation and in vivo via codon expansion methods broadens sequence and structural possibilities. Circular permutation (CP) is an invaluable approach to redesigning a protein by rearranging the amino acid sequence, where the connectivity of the secondary structural elements is altered without changing the overall structure of the protein. Artificial CP proteins (CPs) are employed in various applications such as biocatalysis, sensing of small molecules by fluorescence, genome editing, ligand-binding protein switches, and optogenetic engineering. Many studies have shown that CP can lead to either reduced or enhanced stability or catalytic efficiency. The effects of CP on a protein's energy landscape cannot be predicted a priori. Thus, it is important to understand how CP can affect the thermodynamic and kinetic stability of a protein. In this review, we discuss the discovery and advancement of techniques to create protein CP, and existing reviews on CP. We delve into the plethora of biological applications for designed CP proteins. We subsequently discuss the experimental and computational reports on the effects of CP on the thermodynamic and kinetic stabilities of proteins of various topologies. An understanding of the various aspects of CP will allow the reader to design robust CP proteins for their specific purposes.

Abstract: OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side_x0002_chain of the criticalQ48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates.

Abstract: Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1β and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.

Abstract: Auditory receptors can be motile to actively amplify their mechanical input. Here we describe a novel and different type of motility that, residing in supporting cells, shapes physiological responses of mechanoreceptor cells. In Drosophila larvae, supporting cap cells transmit mechanical stimuli to proprioceptive chordotonal neurons. We found that the cap cells are strongly pre-stretched at rest to twice their relaxed length. The tension in these cells is modulated by non-muscle myosin-II motors. Activating the motors optogenetically causes contractions of the cap cells. Cap-cell-specific knockdown of the regulatory light chain of myosin-II alters mechanically evoked receptor neuron responses, converting them from phasic to more tonic, impairing sensory adaptation. Hence, two motile mechanisms seem to operate in concert in insect chordotonal organs, one in the sensory receptor neurons, based on dynein, and the other in supporting cells, based on myosin.

Abstract: Liquid-liquid phase separation (LLPS) has emerged as a major organizing principle in cells. Recent work showed that multiple components of integrin-mediated focal adhesions including p130Cas can form LLPS, which govern adhesion dynamics and related cell behaviors. In this study, we found that the focal adhesion protein p130Cas drives formation of structures with the characteristics of LLPS that bud from focal adhesions into the cytoplasm. Condensing concentrated cytoplasm around p130Cas-coated beads allowed their isolation, which were enriched in a subset of focal adhesion proteins, mRNAs and RNA binding proteins, including those implicated in inhibiting mRNA translation. Plating cells on very high concentrations of fibronectin to induce large focal adhesions inhibited message translation which required p130Cas and correlated with droplet formation. Photo-induction of p130Cas condensates using the Cry2 system also reduced translation. These results identify a novel regulatory mechanism in which high adhesion limits message translation via induction of p130Cas-dependent cytoplasmic LLPS. This mechanism may contribute to the quiescent state of very strongly adhesive myofibroblasts and senescent cells.

Abstract: Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.

Abstract: For cells to thrive, they must make appropriate fate decisions based on a myriad of internal and external stimuli. But how do they integrate these different forms of information to contextualise their decisions? Old yeast cells showed an ability to dampen their proliferation as they entered senescence. Conversely, they had an enhanced ability to promote proliferation during escape from pheromone stimulation. A network of nucleoprotein condensation states involving processing bodies (P-bodies) and the prion-like RNA-binding protein, Whi3, controlled these opposing fate decisions. In old but not in young cells, condensation of Whi3 was both necessary and sufficient for senescence entry. In old cells, Whi3 localised to age-dependent P-bodies. Preventing their formation stopped Whi3 condensation from driving senescence entry. Challenging old cells with an external stimulus, pheromone, revealed that the condensates had a second function: potentiating the cell's ability to trigger escape from the mating pheromone response. These findings identify biomolecular condensation as an integrator of contextual information as cells make decisions, enabling them to navigate overlapping life events.

Abstract: Biomolecular condensates, often assembled through phase transition mechanisms, play key roles in organizing diverse cellular activities. The material properties of condensates, ranging from liquid droplets to solid-like glasses or gels, are key features impacting the way resident components associate with one another. However, it remains unclear whether and how different material properties would influence specific cellular functions of condensates. Here, we combine optogenetic control of phase separation with single-molecule mRNA imaging to study relations between phase behaviors and functional performance of condensates. Using light-activated condensation, we show that sequestering target mRNAs into condensates causes translation inhibition. Orthogonal mRNA imaging reveals highly transient nature of interactions between individual mRNAs and condensates. Tuning condensate composition and material property towards more solid-like states leads to stronger translational repression, concomitant with a decrease in molecular mobility. We further demonstrate that β-actin mRNA sequestration in neurons suppresses spine enlargement during chemically induced long-term potentiation. Our work highlights how the material properties of condensates can modulate functions, a mechanism that may play a role in fine-tuning the output of condensate-driven cellular activities.

Abstract: During mitosis, the human microtubule depolymerase KIF2C increases the turnover of kinetochore-microtubule attachments. This facilitates the correction of attachment errors. Moreover, BRCA2 phosphorylated at Thr207 by PLK1 (BRCA2-pT207) assembles a complex including PLK1, PP2A and BUBR1 that contributes to the stability of the kinetochore-microtubule attachments. PLK1, together with Aurora B, critically regulate the accurate segregation of chromosomes. Here we demonstrate that KIF2C contains an N-terminal domain that binds directly to several phosphorylated peptides, including BRCA2-pT207. Using an optogenetic platform, we reveal that KIF2C assembles into membrane-less compartments or biomolecular condensates that are located next to microtubules. We provide evidence that condensate assembly depends on the presence of the newly defined N-terminal phospho-binding domain of KIF2C and on the kinase activities of Aurora B and PLK1. Moreover, KIF2C condensates concentrate active PLK1 and colocalize with BRCA2-pT207. We propose that, because of its phospho-dependent binding and oligomerization capacities, KIF2C forms biomolecular condensates that partition PLK1 and locally amplify its kinase activity during mitosis.

Abstract: The transition of bacteria from an individualistic to a biofilm lifestyle profoundly alters their biology. During biofilm development, the bacterial cell-cell adhesions are a major determinant of initial microcolonies, which serve as kernels for the subsequent microscopic and mesoscopic structure of the biofilm, and determine the resulting functionality. In this study, the significance of bacterial cell-cell adhesion dynamics on bacterial aggregation and biofilm maturation is elucidated. Using photoswitchable adhesins between bacteria, modifying the dynamics of bacterial cell-cell adhesions with periodic dark-light cycles is systematic. Dynamic cell-cell adhesions with liquid-like behavior improve bacterial aggregation and produce more compact microcolonies than static adhesions with solid-like behavior in both experiments and individual-based simulations. Consequently, dynamic cell-cell adhesions give rise to earlier quorum sensing activation, better intermixing of different bacterial populations, improved biofilm maturation, changes in the growth of cocultures, and higher yields in fermentation. The here presented approach of tuning bacterial cell-cell adhesion dynamics opens the door for regulating the structure and function of biofilms and cocultures with potential biotechnological applications.

Abstract: A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creating designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.

Abstract: Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Abstract: Synthetic Sc2.0 yeast strains contain hundreds to thousands of loxPsym recombination sites that allow restructuring of the Saccharomyces cerevisiae genome by SCRaMbLE. Thus, a highly diverse yeast population can arise from a single genotype. The selection of genetically diverse candidates with rearranged synthetic chromosomes for downstream analysis requires an efficient and straightforward workflow. Here we present loxTags, a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations after SCRaMbLE. To cope with the large number of amplicons, we generated qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based genotyping and long-read sequencing, we show that light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or a circular version of synthetic chromosome III.

Abstract: Light-dependent adaptations of organismal physiology, development, and behavior abound in nature and depend on sensory photoreceptors. As one class, light–oxygen–voltage (LOV) photoreceptors harness flavin-nucleotide chromophores to sense blue light. Photon absorption drives the LOV receptor to its signaling state, characterized by a metastable thioadduct between the flavin and a conserved cysteine residue. With this cysteine absent, LOV receptors instead undergo photoreduction to the flavin semiquinone which however can still elicit downstream physiological responses. Irrespective of the cysteine presence, the LOV photochemical response thus entails a formal reduction of the flavin. Against this backdrop, we here investigate the reduction midpoint potential E0 in the paradigmatic LOV2 domain from Avena sativa phototropin 1 (AsLOV2), and how it can be deliberately varied. Replacements of residues at different sites near the flavin by methionine consistently increase E0 from its value of around −280 mV by up to 40 mV. Moreover, methionine introduction invariably impairs photoactivation efficiency and thus renders the resultant AsLOV2 variants less light-sensitive. Although individual methionine substitutions also affect the stability of the signaling state and downstream allosteric responses, no clear-cut correlation with the redox properties emerges. With a reduction midpoint potential near −280 mV, AsLOV2 and, by inference, other LOV receptors may be partially reduced inside cells which directly affects their light responsiveness. The targeted modification of the chromophore environment, as presently demonstrated, may mitigate this effect and enables the design of LOV receptors with stratified redox sensitivities.

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: Diabetes mellitus and its associated secondary complications have become a pressing global healthcare issue. The current integrated theranostic plan involves a glucometer-tandem pump. However, external condition-responsive insulin delivery systems utilizing rigid glucose sensors pose challenges in on-demand, long-term insulin administration. To overcome these challenges, we present a novel model of antidiabetic management based on printable metallo-nucleotide hydrogels and optogenetic engineering. The conductive hydrogels were self-assembled by bioorthogonal chemistry using oligonucleotides, carbon nanotubes, and glucose oxidase, enabling continuous glucose monitoring in a broad range (0.5-40 mM). The optogenetically engineered cells were enabled glucose regulation in type I diabetic mice via a far-red light-induced transgenic expression of insulin with a month-long avidity. Combining with a microchip-integrated microneedle patch, a prototyped close-loop system was constructed. The glucose levels detected by the sensor were received and converted by a wireless controller to modulate far-infrared light, thereby achieving on-demand insulin expression for several weeks. This study sheds new light on developing next-generation diagnostic and therapy systems for personalized and digitalized precision medicine.

Abstract: Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

Abstract: Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

Abstract: Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

Abstract: The ability of cells to perceive and respond to mechanical cues is essential for numerous biological activities. Emerging evidence indicates important contributions of organelles to cellular mechanosensitivity and mechanotransduction. However, whether and how the endoplasmic reticulum (ER) senses and reacts to mechanical forces remains elusive. To fill the knowledge gap, after developing a light-inducible ER-specific mechanostimulator (LIMER), we identify that mechanostimulation of ER elicits a transient, rapid efflux of Ca2+ from ER in monkey kidney COS-7 cells, which is dependent on the cation channels transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and polycystin-2 (PKD2) in an additive manner. This ER Ca2+ release can be repeatedly stimulated and tuned by varying the intensity and duration of force application. Moreover, ER-specific mechanostimulation inhibits ER-to-Golgi trafficking. Sustained mechanostimuli increase the levels of binding-immunoglobulin protein (BiP) expression and phosphorylated eIF2α, two markers for ER stress. Our results provide direct evidence for ER mechanosensitivity and tight mechanoregulation of ER functions, placing ER as an important player on the intricate map of cellular mechanotransduction.