Showing 1 - 25 of 658 results
Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos.
The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.
Optogenetic downregulation of protein levels with an ultrasensitive switch.
Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, e.g. for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycycline-dependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low light-fluxes as well as fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.
Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.
The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.
Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling.
T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.
Reversible induction of mitophagy by an optogenetic bimodular system.
Autophagy-mediated degradation of mitochondria (mitophagy) is a key process in cellular quality control. Although mitophagy impairment is involved in several patho-physiological conditions, valuable methods to induce mitophagy with low toxicity in vivo are still lacking. Herein, we describe a new optogenetic tool to stimulate mitophagy, based on light-dependent recruitment of pro-autophagy protein AMBRA1 to mitochondrial surface. Upon illumination, AMBRA1-RFP-sspB is efficiently relocated from the cytosol to mitochondria, where it reversibly mediates mito-aggresome formation and reduction of mitochondrial mass. Finally, as a proof of concept of the biomedical relevance of this method, we induced mitophagy in an in vitro model of neurotoxicity, fully preventing cell death, as well as in human T lymphocytes and in zebrafish in vivo. Given the unique features of this tool, we think it may turn out to be very useful for a wide range of both therapeutic and research applications.
Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth.
Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation.
Cortical mitochondria regulate insulin secretion by local Ca2+ buffering.
Mitochondria play an essential role in regulating insulin secretion from beta cells by providing ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine if the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria 2-fold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely important for the generation of Ca2+ microdomains required for efficient insulin granule release.
Dronpa: a light-switchable fluorescent protein for opto-biomechanics.
Since the development of GFP, fluorescent proteins (FP) are indispensable tools in molecular biology. Some FPs change their structure under illumination, which affects their interaction with other biomolecules or proteins. Especially, FPs that are able to form switchable dimers became an important tool in the field of optogenetics. They are widely used for the investigation of signaling pathways, the control of surface recruitment as well as enzyme and gene regulation. However, optogenetics did not yet develop tools for the investigation of biomechanical processes. This could be leveraged if one could find a light-switchable FP dimer, that is able to withstand sufficiently high forces. In this work we measure the rupture force of the switchable interface in pdDronpa1.2 dimers using atomic force microscopy based single molecule force spectroscopy. The most probable dimer rupture force amounts to around 80 pN at a pulling speed of 1600 nm/s. After switching of the dimer using illumination at 488 nm there are hardly any measurable interface interactions, which indicates the successful dissociation of the dimers. Hence this Dronpa dimer could expand the current toolbox in optogenetics with new opto-biomechanical applications like the control of tension in adhesion processes.
Direct observation and analysis of the dynamics of the photoresponsive transcription factor GAL4.
We report direct visualization of the dynamic behavior of transcription factor GAL4 with photo-switching function (GAL4-VVD) in the DNA origami structure. Using high-speed atomic force microscopy (HS-AFM), we observed photo-induced complex formation of GAL4-VVD and substrate DNAs. Dynamic behaviors of GAL4-VVD such as binding, sliding, stalling, and dissociation with two substrate DNA strands, containing specific GAL4 binding sites, were observed. We also observed inter-strand hopping on two double-stranded (ds) DNAs. On a long substrate DNA strand that contained five binding sites, a series of GAL4-VVD/DNA interactions including binding, sliding, stalling, and dissociation could be identified while interacting with the surface. We also found the clear difference in the movement of GAL4-VVD between sliding and stalling in the AFM images. Detailed analysis revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping for rapidly searching specific binding sites, and then stalled to the specific sites for the stable complex formation. The results suggest the existence of the different conformational mode of the protein for sliding and stalling. This single-molecule imaging system at the nanoscale resolution provides the insight of the searching mechanism of the DNA binding proteins.
A yeast system for discovering optogenetic inhibitors of eukaryotic translation initiation.
The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, LOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photo-activated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate of human eIF4E-depednednt translation initiation in a mechanistically defined manner.
Advances in optogenetic regulation of gene expression in mammalian cells using cryptochrome 2 (CRY2).
Synthetic regulation of gene expression provides a powerful approach to reprogram molecular and cellular processes and test the function of specific genes and gene products. In the last decade, optogenetic systems that allow light-dependent gene regulation have become valuable tools, providing tight spatiotemporal control of protein levels. Here we discuss and build on recent optogenetic approaches for regulating gene expression in mammalian cells using cryptochrome 2 (CRY2), a photoreceptor protein from Arabidopsis. We provide detailed protocols for using light to manipulate activity of a CRY2-based engineered photoactivatable Cre DNA recombinase, and to induce or disrupt transcription factor function. In addition, we provide instructions and software for building an inexpensive Rasberry-Pi-based programable LED device for optogenetic experiments, delivering pulsed light with customized control of illumination duration, frequency, and intensity.
Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology.
Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.
Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions.
Optogenetics enables the spatio-temporally precise control of cell and animal behaviour. Many optogenetic tools are driven by light-controlled protein-protein-interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPI to new target proteins is challenging because it is difficult to predict whether one the many available LSDs will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Optically inducible membrane recruitment and signaling systems.
Optical induction of intracellular signaling by membrane-associated and integral membrane proteins allows spatiotemporally precise control over second messenger signaling and cytoskeletal rearrangements that are important to cell migration, development, and proliferation. Optogenetic membrane recruitment of a protein-of-interest to control its signaling by altering subcellular localization is a versatile means to these ends. Here, we summarize the signaling characteristics and underlying structure-function of RGS-LOV photoreceptors as single-component membrane recruitment tools that rapidly, reversibly, and efficiently carry protein cargo from the cytoplasm to the plasma membrane by a light-regulated electrostatic interaction with the membrane itself. We place the technology-relevant features of these recently described natural photosensory proteins in context of summarized protein engineering and design strategies for optically controlling membrane protein signaling.
Membrane-Associated, Not Cytoplasmic or Nuclear, FGFR1 Induces Neuronal Differentiation.
The intracellular transport of receptor tyrosine kinases results in the differential activation of various signaling pathways. In this study, optogenetic stimulation of fibroblast growth factor receptor type 1 (FGFR1) was performed to study the effects of subcellular targeting of receptor kinases on signaling and neurite outgrowth. The catalytic domain of FGFR1 fused to the algal light-oxygen-voltage-sensing (LOV) domain was directed to different cellular compartments (plasma membrane, cytoplasm and nucleus) in human embryonic kidney (HEK293) and pheochromocytoma (PC12) cells. Blue light stimulation elevated the pERK and pPLCγ1 levels in membrane-opto-FGFR1-transfected cells similarly to ligand-induced receptor activation; however, no changes in pAKT levels were observed. PC12 cells transfected with membrane-opto-FGFR1 exhibited significantly longer neurites after light stimulation than after growth factor treatment, and significantly more neurites extended from their cell bodies. The activation of cytoplasmic FGFR1 kinase enhanced ERK signaling in HEK293 cells but not in PC12 cells and did not induce neuronal differentiation. The stimulation of FGFR1 kinase in the nucleus also did not result in signaling changes or neurite outgrowth. We conclude that FGFR1 kinase needs to be associated with membranes to induce the differentiation of PC12 cells mainly via ERK activation.
Bacteriophytochromes - from informative model systems of phytochrome function to powerful tools in cell biology.
Bacteriophytochromes are a subfamily of the diverse light responsive phytochrome photoreceptors. Considering their preferential interaction with biliverdin IXα as endogenous cofactor, they have recently been used for creating optogenetic tools and engineering fluorescent probes. Ideal absorption characteristics for the activation of bacteriophytochrome-based systems in the therapeutic near-infrared window as well the availability of biliverdin in mammalian tissues have resulted in tremendous progress in re-engineering bacteriophytochromes for diverse applications. At the same time, both the structural analysis and the functional characterization of diverse naturally occurring bacteriophytochrome systems have unraveled remarkable differences in signaling mechanisms and have so far only touched the surface of the evolutionary diversity within the family of bacteriophytochromes. This review highlights recent findings and future challenges.
Design, construction, and validation of optogenetic proteins.
Cellular optogenetics employs light-regulated, genetically encoded protein actuators to perturb cellular signaling with unprecedented spatial and temporal control. Here, we present a potentially generalized approach for transforming a given protein of interest (POI) into an optogenetic species. We describe the rational and methods by which we developed three different optogenetic POIs utilizing the Cry2-Cib photodimerizing pair. The process pipeline is highlighted by (1) developing a low level, constitutively active POI that is independent of endogenous regulation, (2) fusion of the mutant protein of interest to an optogenetic photodimerizing system, and (3) light-mediated recruitment of the light-responsive POI to specific subcellular regions.
Neurotrophin receptor tyrosine kinases regulated with near-infrared light.
Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.
B12-based photoreceptors: from structure and function to applications in optogenetics and synthetic biology.
Vitamin B12-based photoreceptor proteins sense ultraviolet (UV), blue or green light using 5'-deoxyadenosylcobalamin (AdoCbl). The prototype of this widespread bacterial photoreceptor family, CarH, controls light-dependent gene expression in photoprotective cellular responses. It represses transcription in the dark by binding to operator DNA as an AdoCbl-bound tetramer, whose disruption by light relieves operator binding to allow transcription. Structures of the 'dark' (free and DNA-bound) and 'light' CarH states and studies on the unusual AdoCbl photochemistry have provided fundamental insights into these photoreceptors. We highlight these, the plasticity within a conserved mode of action among CarH homologs, their distribution, and their promising applications in optogenetics and synthetic biology.
Controlling protein conformation with light.
Optogenetics, genetically encoded engineering of proteins to respond to light, has enabled precise control of the timing and localization of protein activity in live cells and for specific cell types in animals. Light-sensitive ion channels have become well established tools in neurobiology, and a host of new methods have recently enabled the control of other diverse protein structures as well. This review focuses on approaches to switch proteins between physiologically relevant, naturally occurring conformations using light, accomplished by incorporating light-responsive engineered domains that sterically and allosterically control the active site.
RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43.
TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.
Light-Controlled Afﬁnity Puriﬁcation of Protein Complexes Exempliﬁed by the Resting ZAP70 Interactome.
Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the know interaction partners, and could filter out all other proteins.
Photodimerization systems for regulating protein-protein interactions with light.
Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.
Physical Plasma Membrane Perturbation Using Subcellular Optogenetics Drives Integrin-Activated Cell Migration.
Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.
Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells.
Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells.