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 553 results
1.

Optogenetic dissection of mitotic spindle positioning in vivo.

blue TULIP C. elegans in vivo Control of cytoskeleton / cell motility / cell shape Cell cycle control
Elife, 15 Aug 2018 DOI: 10.7554/elife.38198 Link to full text
Abstract: The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα-GDP, Gα-GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.
2.

Spatiotemporal control of zebrafish (Danio rerio) gene expression using a light-activated CRISPR activation system.

blue CRY2/CIB1 HEK293T ZF4 Transgene expression
Gene, 1 Aug 2018 DOI: 10.1016/j.gene.2018.07.077 Link to full text
Abstract: CRISPR activation (CRISPRa) system is the convenient tool for targeted-gene activation, it has been developed and combined with a lighting-based system that can control transcription initiation spatially and temporally by utilizing photoreceptor derived from plant Arabidopsis thaliana. A blue light photoreceptor the Cryptochrome 2 (CRY2), and its binding partner CIB1 will dimerize by exposure to the blue light and it has been applied to human cells. However, the application of a combination of these two systems to zebrafish cell is still not explored. We performed zebrafish gene activation using p65 and VP64 activators in the zebrafish cells (ZF4). Our study demonstrated that we have successfully controlled the transcription level of ASCL1a, BCL6a, and HSP70 genes using blue light-activated CRISPR activation system. The result showed that using this system, mRNA level expression of ASCL1a, BCL6a, and HSP70 genes increased after irradiated under blue light for several hours and significantly different to those which treated in the dark.
3.

Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast.

blue NcWC1-LOV VVD S. cerevisiae Transgene expression Control of cell-cell / cell-material interactions
MBio, 31 Jul 2018 DOI: 10.1128/mbio.00626-18 Link to full text
Abstract: Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.
4.

A compendium of chemical and genetic approaches to light-regulated gene transcription.

blue cyan green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Crit Rev Biochem Mol Biol, 24 Jul 2018 DOI: 10.1080/10409238.2018.1487382 Link to full text
Abstract: On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.
5.

Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics across Scales.

near-infrared Phytochromes Review
Trends Biotechnol, 21 Jul 2018 DOI: 10.1016/j.tibtech.2018.06.011 Link to full text
Abstract: Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescent proteins (FPs) engineered from bacterial phytochromes have become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.
6.

Oscillatory Control of Notch Signaling in Development.

blue LOV domains Review
Adv Exp Med Biol, 19 Jul 2018 DOI: 10.1007/978-3-319-89512-3_13 Link to full text
Abstract: The Notch effectors Hes1 and Hes7 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner during neurogenesis and somitogenesis. These two biological events exhibit different types of oscillations: anti-/out-of-phase oscillation in neural stem cells during neurogenesis and in-phase oscillation in presomitic mesoderm (PSM) cells during somitogenesis. Accelerated or delayed Dll1 expression by shortening or elongating the size of the Dll1 gene, respectively, dampens or quenches Dll1 oscillation at intermediate levels, a phenomenon known as "amplitude/oscillation death" of coupled oscillators. Under this condition, both Hes1 oscillation in neural stem cells and Hes7 oscillation in PSM cells are also dampened. As a result, maintenance of neural stem cells is impaired, leading to microcephaly, while somite segmentation is impaired, leading to severe fusion of somites and their derivatives, such as vertebrae and ribs. Thus, the appropriate timing of Dll1 expression is critical for the oscillatory expression in Notch signaling and normal processes of neurogenesis and somitogenesis. Optogenetic analysis indicated that Dll1 oscillations transfer the oscillatory information between neighboring cells, which may induce anti-/out-of-phase and in-phase oscillations depending on the delay in signaling transmission. These oscillatory dynamics can be described in a unified manner by mathematical modeling.
7.

Illuminating pathogen-host intimacy through optogenetics.

blue green red BLUF domains Cryptochromes LOV domains Opsins Phytochromes Review
PLoS Pathog, 12 Jul 2018 DOI: 10.1371/journal.ppat.1007046 Link to full text
Abstract: The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host-pathogen interactions in a way that has not been feasible otherwise.
8.

Shining light on spindle positioning.

blue LOV domains Review
Elife, 9 Jul 2018 DOI: 10.7554/elife.38748 Link to full text
Abstract: Optogenetic approaches are leading to a better understanding of the forces that determine the plane of cell division.
9.

Blue-Light Receptors for Optogenetics.

blue green red UV BLUF domains Cryptochromes Fluorescent proteins LOV domains Opsins Phytochromes UV receptors Review
Chem Rev, 9 Jul 2018 DOI: 10.1021/acs.chemrev.8b00163 Link to full text
Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.
10.

Optical activation of TrkA signaling.

blue CRY2/CIB1 CRY2/CRY2 NIH/3T3 PC-12 Signaling cascade control Cell differentiation
ACS Synth Biol, 5 Jul 2018 DOI: 10.1021/acssynbio.8b00126 Link to full text
Abstract: Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.
11.

Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.

blue red BphS CRY2/CIB1 HEK293 mouse in vivo Cell differentiation Endogenous gene expression Immediate control of second messengers
Proc Natl Acad Sci USA, 2 Jul 2018 DOI: 10.1073/pnas.1802448115 Link to full text
Abstract: The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.
12.

"Rho"ing a Cellular Boat with Rearward Membrane Flow.

blue LOV domains Review
Dev Cell, 2 Jul 2018 DOI: 10.1016/j.devcel.2018.06.008 Link to full text
Abstract: The physicist Edward Purcell wrote in 1977 about mechanisms that cells could use to propel themselves in a low Reynolds number environment. Reporting in Developmental Cell, O'Neill et al. (2018) provide direct evidence for one of these mechanisms by optogenetically driving the migration of cells suspended in liquid through RhoA activation.
13.

Four Key Steps Control Glycolytic Flux in Mammalian Cells.

red PhyB/PIF6 NIH/3T3 Signaling cascade control
Cell Syst, 26 Jun 2018 DOI: 10.1016/j.cels.2018.06.003 Link to full text
Abstract: Altered glycolysis is a hallmark of diseases including diabetes and cancer. Despite intensive study of the contributions of individual glycolytic enzymes, systems-level analyses of flux control through glycolysis remain limited. Here, we overexpress in two mammalian cell lines the individual enzymes catalyzing each of the 12 steps linking extracellular glucose to excreted lactate, and find substantial flux control at four steps: glucose import, hexokinase, phosphofructokinase, and lactate export (and not at any steps of lower glycolysis). The four flux-controlling steps are specifically upregulated by the Ras oncogene: optogenetic Ras activation rapidly induces the transcription of isozymes catalyzing these four steps and enhances glycolysis. At least one isozyme catalyzing each of these four steps is consistently elevated in human tumors. Thus, in the studied contexts, flux control in glycolysis is concentrated in four key enzymatic steps. Upregulation of these steps in tumors likely underlies the Warburg effect.
14.

An Optogenetic approach to control protein localization during embryogenesis of the sea urchin.

blue TULIP sea urchin in vivo Developmental processes
Dev Biol, 26 Jun 2018 DOI: 10.1016/j.ydbio.2018.06.015 Link to full text
Abstract: Light inducible protein-protein interactions have been used to manipulate protein localization and function in the cell with utmost spatial and temporal precision. In this technical report, we use a recently developed optogenetic approach to manipulate protein localization in the developing sea urchin embryo. A photosensitive LOV domain from Avena sativa phototropin1 cages a small peptide that binds the engineered PDZ domain (ePDZ) upon blue light irradiation. Using this system, mCherry tagged proteins fused with the LOV domain were recruited to ectopic sub-cellular regions such as the membrane, microtubules, or actin by GFP tagged proteins fused with the ePDZ domain upon blue light irradiation within 1~3 minutes in the sea urchin embryo. The efficiency and speed of recruitment of each protein to its respective subcellular region appeared to be dependent on the power and duration of laser irradiation, as well as the respective level of affinity to the tagged location. Controlled laser irradiation allowed partial recruitment of the spindle to the membrane, and resulted in cell blebbing. Vasa, a cell cycle and germline factor that localizes on the spindle and enriches in the micromeres at 8-16 cell stage was recruited to ectopic sites, preventing normal enrichment. Continuous blue light activation with a regular blue aquarium light over two days of culture successfully induced LOV-ePDZ binding in the developing embryos, resulting in continued ectopic recruitment of Vasa and failure in gastrulation at Day 2. Although some cytotoxicity was observed with prolonged blue light irradiation, this optogenetic system provides a promising approach to test the sub-cellular activities of developmental factors, as well as to alter protein localization and development during embryogenesis.
15.

Membrane Flow Drives an Adhesion-Independent Amoeboid Cell Migration Mode.

blue iLID RAW264.7 Control of cytoskeleton / cell motility / cell shape
Dev Cell, 21 Jun 2018 DOI: 10.1016/j.devcel.2018.05.029 Link to full text
Abstract: Cells migrate by applying rearward forces against extracellular media. It is unclear how this is achieved in amoeboid migration, which lacks adhesions typical of lamellipodia-driven mesenchymal migration. To address this question, we developed optogenetically controlled models of lamellipodia-driven and amoeboid migration. On a two-dimensional surface, migration speeds in both modes were similar. However, when suspended in liquid, only amoeboid cells exhibited rapid migration accompanied by rearward membrane flow. These cells exhibited increased endocytosis at the back and membrane trafficking from back to front. Genetic or pharmacological perturbation of this polarized trafficking inhibited migration. The ratio of cell migration and membrane flow speeds matched the predicted value from a model where viscous forces tangential to the cell-liquid interface propel the cell forward. Since this mechanism does not require specific molecular interactions with the surrounding medium, it can facilitate amoeboid migration observed in diverse microenvironments during immune function and cancer metastasis.
16.

Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins.

blue iLID Magnets in vitro
ACS Synth Biol, 21 Jun 2018 DOI: 10.1021/acssynbio.8b00250 Link to full text
Abstract: Towards the bottom-up assembly of synthetic cells from molecular building blocks it is an ongoing challenge to assemble micrometer sized compartments that host different processes into precise multicompartmental assemblies, also called prototissues. The difficulty lies in controlling interactions between different compartments dynamically both in space and time, as these interactions determine how they organize with respect to each other and how they work together. In this study, we have been able to control the self-assembly and social self-sorting of four different types of colloids, which we use as a model for synthetic cells, into two separate families with visible light. For this purpose we used two photoswitchable protein pairs (iLID/Nano and nHagHigh/pMagHigh) that both reversibly heterodimerize upon blue light exposure and dissociate from each other in the dark. These photoswitchable proteins provide non-invasive, dynamic and reversible remote control under biocompatible conditions over the self-assembly process with unprecedented spatial and temporal precision. In addition, each protein pair brings together specifically two different types of colloids. The orthogonality of the two protein pairs enables social self-sorting of a four component mixture into two distinct families of colloidal aggregates with controlled arrangements. These results will ultimately pave the way for the bottom-up assembly of multicompartment synthetic prototissues of a higher complexity, enabling us to control precisely and dynamically the organization of different compartments in space and time.
17.

OptoBase: A web platform for molecular optogenetics.

ACS Synth Biol, 18 Jun 2018 DOI: 10.1021/acssynbio.8b00120 Link to full text
Abstract: OptoBase is an online platform for molecular optogenetics. At its core is a hand-annotated and ontology-supported database that aims to cover all existing optogenetic switches and publications, which is further complemented with a collection of convenient optogenetics-related web tools. OptoBase is meant for both expert optogeneticists, to easily keep track of the field, as well as for all researchers who find optogenetics inviting as a powerful tool to address their biological questions of interest. It is available at https://www.optobase.org. This work also presents OptoBase-based analysis of the trends in molecular optogenetics.
18.

Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis.

blue CRY2/CIB1 D. melanogaster in vivo Developmental processes
Nat Commun, 18 Jun 2018 DOI: 10.1038/s41467-018-04754-z Link to full text
Abstract: During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. Here we use optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. We show that precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes.
19.

Independent Control over Multiple Cell Types in Space and Time Using Orthogonal Blue and Red Light Switchable Cell Interactions.

blue red CRY2/CIB1 PhyB/PIF6 MDA-MB-231 Control of cell-cell / cell-material interactions
Adv Sci, 17 Jun 2018 DOI: 10.1002/advs.201800446 Link to full text
Abstract: Independent control over multiple cell–material interactions with high spatiotemporal resolution is a key for many biomedical applications and understanding cell biology, as different cell types can perform different tasks in a multicellular context. In this study, the binding of two different cell types to materials is orthogonally controlled with blue and red light providing independent regulation in space and time. Cells expressing the photoswitchable protein cryptochrome 2 (CRY2) on cell surface bind to N‐truncated CRY‐interacting basic helix–loop–helix protein 1 (CIBN)‐immobilized substrates under blue light and cells expressing the photoswitchable protein phytochrome B (PhyB ) on cell surface bind to phytochrome interaction factor 6 (PIF6)‐immobilized substrates under red light, respectively. These light‐switchable cell interactions provide orthogonal and noninvasive control using two wavelengths of visible light. Moreover, both cell–material interactions are dynamically switched on under light and reversible in the dark. The specificity of the CRY2/CIBN and PhyB/PIF6 interactions and their response to different wavelengths of light allow selectively activating the binding of one cell type with blue and the other cell type with red light in the presence of the other cell type.
20.

LOV Domains in the Design of Photoresponsive Enzymes.

blue LOV domains Review
ACS Chem Biol, 15 Jun 2018 DOI: 10.1021/acschembio.8b00159 Link to full text
Abstract: In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occuring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.
21.

A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging.

blue CRY2/CIB1 HeLa Signaling cascade control
Sci Rep, 12 Jun 2018 DOI: 10.1038/s41598-018-27174-x Link to full text
Abstract: Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.
22.

Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B.

red PhyB/PIF6 HEK293
Photochem Photobiol, 12 Jun 2018 DOI: 10.1111/php.12955 Link to full text
Abstract: Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor need to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments. This article is protected by copyright. All rights reserved.
23.

Engaging myosin VI tunes motility, morphology, and identity in endocytosis.

blue TULIP HeLa Organelle manipulation
Traffic, 4 Jun 2018 DOI: 10.1111/tra.12583 Link to full text
Abstract: While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself. In this study, we use a combination of optogenetic and chemical-dimerization strategies to target exogenous myosin VI to early endosomes, and probe its influence on organelle motility, morphology, and identity. Our analysis across time scales suggests a model wherein the artificial engagement of myosin VI motility on early endosomes restricts microtubule-based motion, followed by morphological changes characterized by the rapid condensation and disintegration of organelles, ultimately leading to the enhanced overlap of markers that demarcate endosomal compartments. Together, our findings show that synthetic engagement of myosin VI motility is sufficient to alter organelle homeostasis in the endocytic pathway. This article is protected by copyright. All rights reserved.
24.

Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble.

blue iLID HCT116 Control of cytoskeleton / cell motility / cell shape
Elife, 31 May 2018 DOI: 10.7554/elife.36559 Link to full text
Abstract: To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.
25.

Regulation of cell cycle progression by cell-cell and cell-matrix forces.

blue CRY2/CIB1 MDCK Control of cytoskeleton / cell motility / cell shape Cell cycle control
Nat Cell Biol, 25 May 2018 DOI: 10.1038/s41556-018-0107-2 Link to full text
Abstract: It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces1-12. However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression is unknown. Here, we quantified cell-cell tension and cell-ECM traction throughout the complete cycle of a large cell population in a growing epithelium. These measurements unveil temporal mechanical patterns that span the entire cell cycle and regulate its duration, the G1-S transition and mitotic rounding. Cells subjected to higher intercellular tension exhibit a higher probability to transition from G1 to S, as well as shorter G1 and S-G2-M phases. Moreover, we show that tension and mechanical energy are better predictors of the duration of G1 than measured geometric properties. Tension increases during the cell cycle but decreases 3 hours before mitosis. Using optogenetic control of contractility, we show that this tension drop favours mitotic rounding. Our results establish that cell cycle progression is regulated cooperatively by forces between the dividing cell and its neighbours.
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