Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 143 results

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation.

blue cpLOV2 HEK293 mouse in vivo primary mouse cortical neurons Transgene expression
bioRxiv, 31 Aug 2023 DOI: 10.1101/2023.08.30.554643 Link to full text
Abstract: The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is one of the primary triggers for initiating cap-dependent translation. Amongst its functions, mTORC1 phosphorylates eIF4E-binding proteins (4E-BPs), which prevents them from binding to eIF4E and thereby enables translation initiation. mTORC1 signaling is required for multiple forms of protein synthesis- dependent synaptic plasticity and various forms of long-term memory (LTM), including associative threat memory. However, the approaches used thus far to target mTORC1 and its effectors, such as pharmacological inhibitors or genetic knockouts, lack fine spatial and temporal control. The development of a conditional and inducible eIF4E knockdown mouse line partially solved the issue of spatial control, but still lacked optimal temporal control to study memory consolidation. Here, we have designed a novel optogenetic tool (Opto4E-BP) for cell type-specific, light-dependent regulation of eIF4E in the brain. We show that light-activation of Opto4E-BP decreases protein synthesis in HEK cells and primary mouse neurons. In situ, light-activation of Opto4E-BP in excitatory neurons decreased protein synthesis in acute amygdala slices. Finally, light activation of Opto4E-BP in principal excitatory neurons in the lateral amygdala (LA) of mice after training blocked the consolidation of LTM. The development of this novel optogenetic tool to modulate eIF4E-dependent translation with spatiotemporal precision will permit future studies to unravel the complex relationship between protein synthesis and the consolidation of LTM.

An optogenetic approach to control and monitor inflammasome activation.

blue CRY2/CRY2 iBMDM MEF-1 Cell death
bioRxiv, 28 Aug 2023 DOI: 10.1101/2023.07.25.550490 Link to full text
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.

Spatiotemporal optical control of Gαq-PLCβ interactions.

blue CRY2/CIB1 iLID HeLa RAW264.7 Signaling cascade control
bioRxiv, 12 Aug 2023 DOI: 10.1101/2023.08.10.552801 Link to full text
Abstract: Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington’s Disease, and Alzheimer’s Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Optogenetic strategies for optimizing the performance of biosensors of membrane phospholipids in live cells.

blue cpLOV2 CRY2/CIB1 CRY2/CRY2 LOVTRAP HEK293T HeLa Organelle manipulation
bioRxiv, 4 Aug 2023 DOI: 10.1101/2023.08.03.551799 Link to full text
Abstract: High-performance biosensors are crucial for elucidating the spatiotemporal regulatory roles and dynamics of membrane lipids, but there is a lack of improvement strategies for biosensors with low sensitivity and low-content substrates detection. Here we developed universal optogenetic strategies to improve a set of membrane biosensors by trapping them into specific region and further reducing the background signal, or by optically-controlled phase separation for membrane lipids detection and tracking. These improved biosensors were superior to typical tools and light simulation would enhance their detection performance and resolution, which might contribute to the design and optimization of other biosensors.

RNA G-quadruplexes forming scaffolds for alpha-synuclein aggregation lead to progressive neurodegeneration.

blue CRY2olig mouse in vivo Neuro-2a primary mouse cortical neurons Cell death Organelle manipulation
bioRxiv, 11 Jul 2023 DOI: 10.1101/2023.07.10.548322 Link to full text
Abstract: Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are primarily neurodegenerative diseases with progressive decline in motor function. Aggregates composed of alpha-synuclein, which are known as Lewy bodies, are a neuropathological hallmark of synucleinopathies; their pathogenesis has been attributed to neuronal loss owing to intracellular alpha-synuclein accumulation. However, the mechanism of alpha-synuclein aggregation remains unclear. Here we show that the RNA G-quadruplexes assembly forms scaffolds for alpha-synuclein aggregation, thereby contributing to neurodegeneration. RNA G-quadruplexes undergo phase separation and form scaffolds for co-aggregation with & alpha-synuclein. Upon pathogenic alpha-synuclein seeds-induced cellular stress and an optogenetic assembly of RNA G-quadruplexes, phase-separated RNA G-quadruplexes served as scaffolds for & alpha-synuclein phase transition, and the co-aggregates initiated synaptic dysfunction and Parkinsonism in mice. Treatment with 5-aminolevulinic acid and protoporphyrin IX, which prevents RNA G-quadruplexes phase separation, attenuates alpha-synuclein phase transition, neurodegeneration, and motor deficits in synucleinopathy model mice. Together, the RNA G-quadruplexes assembly accelerates alpha-synuclein phase transition and aggregation owing to intracellular Ca2+ homeostasis, thereby contributing to the pathogenesis of synucleinopathies.

Mechanosensitive dynamics of lysosomes along microtubules regulate leader cell emergence in collective cell migration.

blue CRY2/CIB1 MDCK Control of cytoskeleton / cell motility / cell shape
bioRxiv, 4 Jul 2023 DOI: 10.1101/2022.08.03.502740 Link to full text
Abstract: Collective cell migration during embryonic development, wound healing, and cancer metastasis entails the emergence of leader cells at the migration front. These cells with conspicuous lamellipodial structures provide directional guidance to the collective. Despite their physiological relevance, the mechanisms underlying the emergence of leader cells remain elusive. Here we report that in diverse model systems for wound healing, including cultured epithelial monolayer, Drosophila embryo, and mouse embryonic skin, leader cells display a peripheral accumulation of lysosomes. This accumulation appears essential for leader cell emergence, involves lysosomal movement along microtubules, and depends on the actomyosin contractility-generated cellular forces. Peripheral lysosomes associate with inactive Rac1 molecules to remove them from the leading periphery, which increases local Rac1-activity, triggering actin polymerization and promoting lamellipodium formation. Taken together, we demonstrate that beyond their catabolic role, lysosomes act as the intracellular platform that links mechanical and biochemical signals to control the emergence of leader cells.

All-optical mapping of cAMP transport reveals rules of sub-cellular localization.

blue bPAC (BlaC) HEK293T MDCK rat hippocampal neurons Immediate control of second messengers
bioRxiv, 29 Jun 2023 DOI: 10.1101/2023.06.27.546633 Link to full text
Abstract: Cyclic adenosine monophosphate (cAMP) is a second messenger that mediates diverse intracellular signals. Studies of cAMP transport in cells have produced wildly different results, from reports of nearly free diffusion to reports that cAMP remains localized in nanometer-scale domains. We developed an all-optical toolkit, termed cAMP-SITES, to locally perturb and map cAMP transport. In MDCK cells and in cultured neurons, cAMP had a diffusion coefficient of ~120 μm2/s, similar to the diffusion coefficients of other small molecules in cytoplasm. In neuronal dendrites, a balance between diffusion and degradation led to cAMP domains with a length scale of ~30 μm. Geometrical confinement by membranes led to subcellular variations in cAMP concentration, but we found no evidence of nanoscale domains or of distinct membrane-local and cytoplasmic pools. We introduce theoretical relations between cell geometry and small-molecule reaction-diffusion dynamics and transport to explain our observations.

A cytokinetic ring-driven cell rotation achieves Hertwig’s rule in early development.

blue TULIP C. elegans in vivo Control of cytoskeleton / cell motility / cell shape Cell cycle control
bioRxiv, 27 Jun 2023 DOI: 10.1101/2023.06.23.546115 Link to full text
Abstract: Cells tend to divide along the direction in which they are longest, as famously stated by Oscar Hertwig in 1884 in his long axis rule. The orientation of the mitotic spindle determines the cell division axis, and the long axis rule is usually ensured by forces stemming from microtubules. Pulling on the spindle from the cell cortex can give rise to unstable behaviors, and we here set out to understand how the long axis rule is realized in early embryonic divisions where cortical pulling forces are prevalent. We focus on early C. elegans development, where we compressed embryos to reveal that cortical pulling forces favor an alignment of the spindle with the short axis of the cell. Strikingly, we find that this misalignment is corrected by an actomyosin-based mechanism that rotates the entire cell, including the mitotic spindle. We uncover that myosin-driven contractility in the cytokinetic ring generates inward forces that align it with the short axis, and thereby the spindle with the long axis. A theoretical model together with experiments using slightly compressed mouse zygotes suggest that a constricting cytokinetic ring can ensure the long axis rule in cells that are free to rotate inside a confining structure, thereby generalizing the underlying principle.

A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation.

blue EL222 HEK293 HeLa mouse in vivo Transgene expression
bioRxiv, 27 Jun 2023 DOI: 10.1101/2023.06.25.546469 Link to full text
Abstract: We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.

A non-invasive photoactivatable split-Cre recombinase system for genome engineering in zebrafish.

blue Magnets zebrafish in vivo Nucleic acid editing
bioRxiv, 25 Jun 2023 DOI: 10.1101/2023.06.23.546268 Link to full text
Abstract: The cyclic recombinase (Cre)/loxP recombination system is a powerful technique for in vivo cell labeling and tracking. However, achieving high spatiotemporal precision in cell tracking using this system is challenging due to the requirement for reliable tissue-specific promoters. In contrast, light-inducible systems offer superior regional confinement, tunability and non-invasiveness compared to conventional lineage tracing methods. Here, we took advantage of the unique strengths of the zebrafish to develop an easy-to-use highly efficient, genetically encoded, Magnets-based, light-inducible transgenic Cre/loxP system. Our system relies on the reassembly of split Cre fragments driven by the affinity of the Magnets and is controlled by the zebrafish ubiquitin promoter. We demonstrate that our system does not exhibit phototoxicity or leakiness in the dark, and it enables efficient and robust Cre/loxP recombination in various tissues and cell types at different developmental stages through noninvasive illumination with blue light. Our newly developed tool is expected to open novel opportunities for light-controlled tracking of cell fate and migration in vivo.

Fluorogenesis: Inducing Fluorescence in a Non-Fluorescent Protein Through Photoinduced Chromophore Transfer of a Genetically Encoded Chromophore.

violet PhoCl in vitro
bioRxiv, 25 Jun 2023 DOI: 10.1101/2023.06.24.546416 Link to full text
Abstract: Fluorescent proteins, while essential for bioimaging, are limited to visualizing cellular localization without offering additional functionality. We report for the first time a strategy to expand the chemical, structural, and functional diversity of fluorescent proteins by harnessing light to induce red fluorescence in a previously non-fluorescent protein. We accomplish this by inducing the transfer of the genetically encoded chromophore from a photocleavable protein (PhoCl1) to a non-fluorescent kinase (MjRibK) inducing red fluorescence in the latter. We have employed analytical and spectroscopic techniques to validate the presence of red fluorescence in MjRibK. Furthermore, molecular dynamics simulations were carried out to investigate the amino acid residues of MjRibK involved in the generation of red fluorescence. Finally, we demonstrate the ability of the red fluorescent MjRibK to operate as a cyclable high-temperature sensor. We anticipate that this light-induced chromophore transfer strategy will open new possibilities for developing multifunctional genetically encoded fluorescent sensors.

Light-induced condensates show accumulation-prone and less dynamic properties in the nucleus compared to the cytoplasm.

blue CRY2olig Neuro-2a Organelle manipulation
bioRxiv, 23 Jun 2023 DOI: 10.1101/2023.06.07.544154 Link to full text
Abstract: Biomolecular condensates, including membraneless organelles, are ubiquitously observed in subcellular compartments. However, the accumulation and dynamic properties of arbitrarily in-duced condensates remain elusive. Here, we show the size, amount, and dynamic properties of subcellular condensates using various fluorescence spectroscopic imaging analyses. Spatial image correlation spectroscopy showed that the size of blue-light-induced condensates of cryptochrome 2-derived oligomerization tag (CRY2olig) tagged with a red fluorescent protein in the nucleus was not different from that in the cytoplasm. Fluorescence intensity measurements showed that the condensates in the nucleus were more prone to accumulation than those in the cytoplasm. Sin-gle-particle tracking analysis showed that the condensates in the nucleus are predisposed to be stationary dynamics compared to those in the cytoplasm. Therefore, the subcellular compartment may, in part, affect the characteristics of self-recruitment of biomolecules in the condensates and their movement property.

Synthetic Frizzled agonist and LRP antagonist for high-efficiency Wnt/β-catenin signaling manipulation in organoid cultures and in vivo.

blue Magnets HEK293T Signaling cascade control
bioRxiv, 22 Jun 2023 DOI: 10.1101/2023.06.21.545860 Link to full text
Abstract: Wnt/β-catenin signaling and its dysregulation play critical roles in the fate determination of stem cells and the pathology of various diseases. However, the application of translated Wnt ligand in regenerative medicine is hampered by its hydrophobicity and cross-reactivity with Frizzled (FZD) receptors. Here, we generate an engineered water-soluble, FZD subtype-specific agonist, RRP-pbFn, for high-efficiency Wnt/β-catenin signaling activation. In the absence of direct binding to LRP5/6, RRP-pbFn stimulates Wnt/β-catenin signaling more potently than surrogate Wnt. RRP-pbFn supports the growth of a variety of mouse and human organoids, and induces the expansion of liver and intestine progenitors in vivo. Meanwhile, we develop a synthetic LRP antagonist, RRP-Dkk1c, which exhibits heightened effectiveness in attenuating Wnt/β-catenin signaling activity compared to Dkk1, thereby abolishing the formation of CT26-derived colon cancer xenograft in vivo. Together, these two paired Wnt/β-catenin signaling manipulators hold great promise for biomedical research and potential therapeutics.

mRNA condensation fluidizes the cytoplasm.

blue CRY2/CRY2 U-2 OS Organelle manipulation
bioRxiv, 31 May 2023 DOI: 10.1101/2023.05.30.542963 Link to full text
Abstract: The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.

A photoreceptor-based hydrogel with red light-responsive reversible sol-gel transition as transient cellular matrix.

red PhyB/PIF6 in vitro Extracellular optogenetics
bioRxiv, 23 May 2023 DOI: 10.1101/2023.04.04.535523 Link to full text
Abstract: Hydrogels with adjustable mechanical properties have been engineered as matrices for mammalian cells and allow the dynamic, mechano-responsive manipulation of cell fate and function. Recent research yielded hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provided important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Here, we overcome this limitation by developing a photoreceptor-based hydrogel with reversibly adjustable stiffness from 800 Pa to the sol state. The hydrogel is based on star-shaped polyethylene glycol, functionalized with the red/far-red light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor 6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6, thus crosslinking the polymers and resulting in gelation. However, upon illumination with far-red light, the proteins dissociate and trigger a complete gel-to-sol transition. We comprehensively characterize the hydrogel’s light-responsive mechanical properties and apply it as reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. We anticipate that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.

Optogenetic spatial patterning of cooperation in yeast populations.

blue EL222 S. cerevisiae Transgene expression
bioRxiv, 15 May 2023 DOI: 10.1101/2023.05.15.540783 Link to full text
Abstract: Microbial communities are a siege of complex metabolic interactions such as cooperation and competition for resources. Methods to control such interactions could lead to major advances in our ability to engineer microbial consortia for bioproduction and synthetic biology applications. Here, we used optogenetics to control invertase production in yeast, thereby creating landscapes of cooperator and cheater cells. Yeast cells behave as cooperators (i.e., transform sucrose into glucose, a public “good”) upon blue light illumination or cheaters (i.e., consume glucose produced by cooperators to grow) in the dark. We show that cooperators benefit best from the hexoses they produce when their domain size is constrained between two cut-off length-scales. From an engineering point of view, the system behaves as a band pass filter. The lower limit is the trace of cheaters’ competition for hexoses, while the upper limit is defined by cooperators’ competition for sucrose. Hence, cooperation mostly occurs at the frontiers with cheater cells, which not only compete for hexoses but also cooperate passively by letting sucrose reach cooperators. We anticipate that this optogenetic method could be applied to shape metabolic interactions in a variety of microbial ecosystems.

Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation.

blue CRY2/CIB1 C. elegans in vivo D. melanogaster in vivo HEK293A rat dorsal root ganglion NSCs zebrafish in vivo Signaling cascade control Developmental processes
bioRxiv, 8 May 2023 DOI: 10.1101/2023.05.06.539674 Link to full text
Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of Gα protein – Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαi signaling.

Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions.

bioRxiv, 6 May 2023 DOI: 10.1101/2022.10.22.513249 Link to full text
Abstract: Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the resolution and accuracy of proximity labeling methods are limited by a lack of control over the enzymatic labeling process. Here, we present a high spatial and temporal resolution technology that can be activated on demand using light, for high accuracy proximity labeling. Our system, called Light Activated BioID (LAB), is generated by fusing the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. Using live cell imaging, immunofluorescence, western blotting, and mass spectrometry, we show that upon exposure to blue light, CRY2 and CIB1 dimerize, reconstitute the split-TurboID enzyme, and biotinylate proximate proteins. Turning off the light halts the biotinylation reaction. We validate LAB in different cell types and demonstrate that it can identify known binding partners of proteins while reducing background labeling and false positives.

Optogenetic control of kinesins -1, -2, -3 and dynein reveals their specific roles in vesicular transport.

blue LOVTRAP Cos-7 U-2 OS Control of vesicular transport
bioRxiv, 29 Apr 2023 DOI: 10.1101/2023.04.18.537380 Link to full text
Abstract: Each cargo in a cell employs a unique set of motor proteins for its transport. Often multiple types of kinesins are bound to the same cargo. It is puzzling why several types of motors are required for robust transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of kinesin-1, -2, -3 and dynein. This system allows us to control the activity of the endogenous set of motor proteins that are bound to intracellular cargoes. We examined the effect of optogenetic inhibition of kinesins-1, -2, and -3 and dynein on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, kinesin-3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. In agreement with previous studies, sustained inhibition of either kinesins or dynein results in reduced motility in both directions. However, transient, optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. In contrast, optogenetic inhibition of kinesin-2 reduces the motility of late endosomes and lysosomes, and inhibition of kinesin-3 reduces the motility of endosomes and lysosomes. These results suggest that the directionality of transport is likely controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, motility can be directed by modulating the activity of a single type of motor on the cargo.

Focal adhesions are controlled by microtubules through local contractility regulation.

blue iLID FAK-/- HT-1080 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 17 Apr 2023 DOI: 10.1101/2023.04.17.535593 Link to full text
Abstract: Microtubules regulate cell polarity and migration by local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with the major component of focal adhesions, talin. Local optogenetic activation of KANK1-mediated links which promoted microtubule targeting to individual focal adhesion resulting in its centripetal sliding and rapid disassembly. The sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesion upon KANK activation. Other players participating in microtubule-driven KANK-dependent focal adhesion disassembly include kinases ROCK and PAK, as well as microtubules/focal adhesions associated proteins Kinesin-1, APC and αTAT. Finally, we propose a physical model of a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK dependent activation of contractility which is consistent with experimental data.

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.

blue iLID D. melanogaster in vivo Signaling cascade control Developmental processes
bioRxiv, 10 Mar 2023 DOI: 10.1101/2023.03.09.531972 Link to full text
Abstract: Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal – a gradient of ERK kinase activity – and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be ‘blurred’ through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.

Light inducible protein degradation in E. coli with LOVtag.

blue AsLOV2 EL222 E. coli
bioRxiv, 26 Feb 2023 DOI: 10.1101/2023.02.25.530042 Link to full text
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 LOVtag, a protein tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVtag 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 LOVtag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVtag system. Finally, we use the LOVtag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVtag system, and introduce a powerful new tool for bacterial optogenetics.

Calcium transients trigger switch-like discharge of prostaglandin E2 (PGE2) in an ERK-dependent manner.

blue CRY2clust MDCK Immediate control of second messengers
bioRxiv, 23 Feb 2023 DOI: 10.1101/2023.02.01.526734 Link to full text
Abstract: Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The ERK MAP kinase activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.

Crosstalk between Rac and Rho GTPase activity mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

blue AsLOV2 A-431 HeLa Neuro-2a NIH/3T3 U-2 OS Control of cytoskeleton / cell motility / cell shape
bioRxiv, 20 Feb 2023 DOI: 10.1101/2023.02.20.529203 Link to full text
Abstract: Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by an increase in migration directionality and reduced migration velocity. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.

Rac negative feedback links local PIP3 rate-of-change to dynamic control of neutrophil guidance.

blue iLID HL-60 Control of cytoskeleton / cell motility / cell shape Transgene expression
bioRxiv, 5 Jan 2023 DOI: 10.1101/2022.12.30.521706 Link to full text
Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to “lock” onto a particular direction, limiting the ability of cells to reorient. We use spatially-defined optogenetic control of a leading edge organizer (PI3K) to probe how cells balance “decisiveness” needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibitor that destabilizes the leading edge to promote exploration. We show that this local inhibitor enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.
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