Showing 1 - 25 of 202 results
Optogenetic control of spine-head JNK reveals a role in dendritic spine regression.
In this study, we use an optogenetic inhibitor of JNK in dendritic spine sub-compartments of rat hippocampal neurons. JNK inhibition exerts rapid (within seconds) reorganisation of actin in the spine-head. Using real-time FRET to measure JNK activity, we find that either excitotoxic insult (NMDA) or endocrine stress (corticosterone), activate spine-head JNK causing internalization of AMPARs and spine retraction. Both events are prevented upon optogenetic inhibition of JNK, and rescued by JNK inhibition even 2 h after insult. Moreover, we identify that the fast-acting anti-depressant ketamine reduces JNK activity in hippocampal neurons suggesting that JNK inhibition may be a downstream mediator of its anti-depressant effect. In conclusion, we show that JNK activation plays a role in triggering spine elimination by NMDA or corticosterone stress, whereas inhibition of JNK facilitates regrowth of spines even in the continued presence of glucocorticoid. This identifies that JNK acts locally in the spine-head to promote AMPAR internalization and spine shrinkage following stress, and reveals a protective function for JNK inhibition in preventing spine regression.SIGNIFICANCE STATEMENT Identifying mechanisms that underlie dendritic spine elimination is important if we are to understand maladaptive changes that contribute to psychiatric disease. Compartment-specific, fast-acting tools can expedite this endeavor. Here we use a light-activated inhibitor of JNK to control kinase activity specifically in dendritic spines. Light-activation of the JNK inhibitor reduces AMPA receptor removal and spine regression in response to corticosterone and NMDA stress. Furthermore, we find that the anti-depressant drug ketamine lowers JNK activity in hippocampal neurons and prevents spine regression, though direct JNK inhibition is more effective. This study identifies a role for JNK in spine regression and may be relevant for endocrine control of synaptic strength and for conditions where chronic glucocorticoid stress leads to spine elimination.
Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.
Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.
Strategies for Engineering and Rewiring Kinase Regulation.
Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.
Visualization of a blue light transmission area in living animals using light-induced nuclear translocation of fluorescent proteins.
Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.
Designing protein structures and complexes with the molecular modeling program Rosetta.
Proteins perform an amazingly diverse set of functions in all aspects of life. Critical to the function of many proteins are the highly specific three-dimensional structures they adopt. For this reason, there is strong interest in learning how to rationally design proteins that adopt user-defined structures. Over the last 25-years there has been significant progress in the field of computational protein design as rotamer-based sequence optimization protocols have enabled accurate design of protein tertiary and quaternary structure. In this award article I will summarize how the molecular modeling program Rosetta is used to design new protein structures and describe how we have taken advantage of this capability to create proteins that have important applications in research and medicine. I will highlight three protein design stories: the use of protein interface design to create therapeutic bispecific antibodies, the engineering of light-inducible proteins that can be used to recruit proteins to specific locations in the cell, and the de novo design of new protein structures from pieces of naturally occurring proteins.
Structural Basis of Design and Engineering for Advanced Plant Optogenetics.
In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.
Single-Molecule Analysis and Engineering of DNA Motors.
Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.
Locally Activating TrkB Receptor Generates Actin Waves and Specifies Axonal Fate.
Actin waves are filamentous actin (F-actin)-rich structures that initiate in the somato-neuritic area and move toward neurite ends. The upstream cues that initiate actin waves are poorly understood. Here, using an optogenetic approach (Opto-cytTrkB), we found that local activation of the TrkB receptor around the neurite end initiates actin waves and triggers neurite elongation. During actin wave generation, locally activated TrkB signaling in the distal neurite was functionally connected with preferentially localized Rac1 and its signaling pathways in the proximal region. Moreover, TrkB activity changed the location of ankyrinG--the master organizer of the axonal initial segment-and initiated the stimulated neurite to acquire axonal characteristics. Taken together, these findings suggest that local Opto-cytTrkB activation switches the fate from minor to major axonal neurite during neuronal polarization by generating actin waves.
Chemokine Receptor CCR7 Triggers an Endomembrane Signaling Complex for Spatial Rac Activation.
Chemokine-guided cell migration is pivotal for many immunological and developmental processes. How chemokine receptor signaling persists to guarantee sustained directional migration despite receptor desensitization and internalization remains poorly understood. Here, we uncover a function for an intracellular pool of the chemokine receptor CCR7 present in human dendritic cells and cellular model systems. We find that CCR7 signaling, initiated at the plasma membrane, is translocated by joint trafficking of β-arrestin and Src kinase to endomembrane-residing CCR7. There, Src tyrosine phosphorylates CCR7, required for the recruitment of Vav1 to form an endomembrane-residing multi-protein signaling complex comprising CCR7, the RhoGEF Vav1, and its effector, Rac1. Interfering with vesicular trafficking affects CCR7-driven cell migration, whereas CCR7:Vav1 interaction at endomembranes is essential for local Rac1 recruitment to CCR7. Photoactivation of Rac1 at endomembranes leads to lamellipodia formation at the cell's leading edge, supporting the role of sustained endomembrane signaling in guiding cell migration.
Principles and applications of optogenetics in developmental biology.
The development of multicellular organisms is controlled by highly dynamic molecular and cellular processes organized in spatially restricted patterns. Recent advances in optogenetics are allowing protein function to be controlled with the precision of a pulse of laser light in vivo, providing a powerful new tool to perturb developmental processes at a wide range of spatiotemporal scales. In this Primer, we describe the most commonly used optogenetic tools, their application in developmental biology and in the nascent field of synthetic morphogenesis.
Optogenetics sheds new light on tissue engineering and regenerative medicine.
Optogenetics has demonstrated great potential in the fields of tissue engineering and regenerative medicine, from basic research to clinical applications. Spatiotemporal encoding during individual development has been widely identified and is considered a novel strategy for regeneration. A as a noninvasive method with high spatiotemporal resolution, optogenetics are suitable for this strategy. In this review, we discuss roles of dynamic signal coding in cell physiology and embryonic development. Several optogenetic systems are introduced as ideal optogenetic tools, and their features are compared. In addition, potential applications of optogenetics for tissue engineering are discussed, including light-controlled genetic engineering and regulation of signaling pathways. Furthermore, we present how emerging biomaterials and photoelectric technologies have greatly promoted the clinical application of optogenetics and inspired new concepts for optically controlled therapies. Our summation of currently available data conclusively demonstrates that optogenetic tools are a promising method for elucidating and simulating developmental processes, thus providing vast prospects for tissue engineering and regenerative medicine applications.
Repurposing protein degradation for optogenetic modulation of protein activities.
Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellu-lar signal-regulated kinase (ERK) pathway, as well as a constitutively active form of MEK (CA MEK), a positive regulator of the same pathway. Kinetics study showed that light-induced protein stabilization could be achieved within 30 minutes of blue light stimulation. GLIMPSe enables target-independent optogenetic control of protein activities and therefore minimizes the systematic variation embedded within different photoactivatable proteins. Overall, GLIMPSe promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.
Optogenetic Repressors of Gene Expression in Yeasts Using Light-Controlled Nuclear Localization.
Introduction: Controlling gene expression is a fundamental goal of basic and synthetic biology because it allows insight into cellular function and control of cellular activity. We explored the possibility of generating an optogenetic repressor of gene expression in the model organism Saccharomyces cerevisiae by using light to control the nuclear localization of nuclease-dead Cas9, dCas9. Methods: The dCas9 protein acts as a repressor for a gene of interest when localized to the nucleus in the presence of an appropriate guide RNA (sgRNA). We engineered dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool to control nuclear localization (LINuS) as parts in an existing yeast optogenetic toolkit. This allowed expression cassettes containing novel dCas9 repressor configurations and guide RNAs to be rapidly constructed and integrated into yeast. Results: Our library of repressors displays a range of basal repression without the need for inducers or promoter modification. Populations of cells containing these repressors can be combined to generate a heterogeneous population of yeast with a 100-fold expression range. We find that repression can be dialed modestly in a light dose- and intensity-dependent manner. We used this library to repress expression of the lanosterol 14-alpha-demethylase Erg11, generating yeast with a range of sensitivity to the important antifungal drug fluconazole. Conclusions: This toolkit will be useful for spatiotemporal perturbation of gene expression in Saccharomyces cerevisiae. Additionally, we believe that the simplicity of our scheme will allow these repressors to be easily modified to control gene expression in medically relevant fungi, such as pathogenic yeasts.
FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics.
Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.
Synthetic biology approaches for targeted protein degradation.
Protein degradation is an effective native mechanism used in modulating intracellular information, and thus it plays an essential role in maintaining cellular homeostasis. Repurposing native protein degradation in a synthetic context is gaining attention as a new strategy to manipulate cellular behavior rapidly for a wide range of applications including disease detection and therapy. This review examines the native mechanisms and machineries by which mammalian cells degrade their own proteins including the sequence of events from identifying a candidate for degradation to the protein's destruction. Next, it explores engineering efforts to degrade both exogenous and native proteins with high specificity and control by targeting proteins into the degradation cascade. A complete understanding of design rules with an ability to use cellular information as signals will allow control over the cellular behavior in a well-defined manner.
Optogenetic control of protein binding using light-switchable nanobodies.
A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins.
Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II.
Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While non-muscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin co-localize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo. Movie S1 Movie S1 Live imaging of border cell specification and delamination from anterior epithelium From Figure 1D-I. Slbo promoter driving Lifeact-GFP (green) marks border cells, Upd-Gal4, UAS-DsRed.nls (red) mark polar cell nuclei. Hoechst 33342 (blue) marks DNA. Time resolution is 4 min. Movie S2 Movie S2 Representative Z-projected and registered live imaging of Sqh-mCherry accumulating in cortical junctions (flashing arrows) during border cell migration. From Figure 3J-K. Time resolution is 25 sec. Movie S3 Movie S3 Representative Z-projected and registered live imaging of E-cad-GFP during border cell migration. From Figure 3M-N. Time resolution is 60 sec. Movie S4 Movie S4 Representative Z-projection of control flpout cells from hs-Flp;, Slbo>Lifeact-GFP; AyGal4, UAS-RFP. Clonal cells are marked by magenta nuclei (nls-RFP). Time resolution is 2.5 min. From Supp. Figure 3 A-D. Movie S5 Movie S5 Representative Z-projection of Sqh-RNAi flpout cells from hs-Flp;, Slbo>Lifeact-GFP; AyGal4, UAS-RFP, UAS-sqh-RNAi. Clonal cells are marked by magenta nuclei (nls-RFP). Time resolution is 2.5 min. From Supp. Figure 3 E-H. Movie S6 Movie S6 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-white RNAi. Time resolution is 2 min. From Supp. Figure 4 A-D. Movie S7 Movie S7 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-sqh-RNAi showing frequent side protrusions. Time resolution is 2 min. From Supp. Figure 4 E-H. White arrows indicate ectopic side and rear protrusions. Movie S8 Movie S8 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-sqh-RNAi showing long lived side protrusions. Time resolution is 2 min. From Supp. Figure 4 I-L. Movie S9 Movie S9 Representative Z-projected live imaging of c306-Gal4 driving UAS-white-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during periods of protrusive and round migration phases. From Figure 6A-D. 25 min corresponds to 6A and B and 1hr:25 min corresponds to 6C and D. Time resolution is 2.5 min. Movie S10 Movie S10 Sqh-mCherry (magenta) channel from Supplementary Movie 9. From Figure 6A-D. 25 min corresponds to 6A and B and 1hr:25 min corresponds to 6C and D. Time resolution is 2.5 min. Movie S11 Movie S11 Representative Z-projected live imaging of c306-Gal4 driving UAS-Ecad-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during a protrusive phase of migration. From Figure 6E-F. Time resolution is 2.5 min. Movie S12 Movie S12 Sqh-mCherry (magenta) channel from Supplementary Movie 11. From Figure 6E-F. Time resolution is 2.5 min. Movie S13 Movie S13 Representative Z-projected live imaging of c306-Gal4 driving UAS-Ecad-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during a rounded phase of migration. From Figure 6G-H. Time resolution is 2.5 min. Movie S14 Movie S14 Sqh-mCherry (magenta) channel from Supplementary Movie 13. From Figure 6G-H. Time resolution is 2.5 min. Movie S15 Movie S15 Example segmentation analysis from a representative Z-projected time lapse of a cluster expressing c306-Gal4 driving UAS-white-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during migration. Time lapse analyzed in Imaris by 1. segmentation of the cluster using Lifeact-GFP, 2. Rendering of Sqh-mCherry by masking the inside of the Life-act surface, 3. performing a distance transformation using the masked Sqh-mCherry that is color coded for distance from membrane (dark colors are short distances and bright/white colors are more distant), 4. combining the distance transformation with the Sqh-mCherry mask to only include the cortical 2 μm of the original Sqh-mCherry signal for quantification in Figure 6I. Movie S16 Movie S16 Representative Z-projected time lapse of Lifeact-GFP and Sqh-mCherry expressing clusters used for quantification of Figure 7B-C during protrusion/retractions cycles. Time resolution is 2 min. Movie S17 Movie S17 Sqh-mCherry channel from Supplementary movie 16. Time resolution is 2 min. Movie S18 Movie S18 Representative Z-projections of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-Sqh-E20E21 migrating border cells clusters that split. Time resolution is 2 min. Movie S19 Movie S19 Representative Z-projections of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-LifeactGFP and UAS-Sqh-E20E21 migrating border cells clusters during protrusive phase. Time resolution is 2 min. Movie S20 Movie S20 Representative Z-projection of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-Sqh-E20E21 border cells cluster at the oocyte border during a blebbing phase. Time resolution is 2 min. Movie S21 Movie S21 Representative Z-projection of control cluster expressing slbo-Gal4; UAS-PLCδ1-PH-GFP. Time resolution is 2 min. Movie S22 Movie S22 Representative Z-projection of cluster expressing slbo-Gal4; UAS-PLCδ1-PH-GFP, UAS-Rho1V14. Blebs are marked by white arrows. Time resolution is 2 min.
Reversible photocontrol of oxidase activity by inserting a photosensitive domain into the oxidase.
Photocontrol of protein activity has become a helpful strategy for regulating biological pathways. Herein, a method for the precise and reversible photocontrol of oxidase activity was developed by using the conformational change of the AsLOV2 domain.
The AsLOV2 domain was inserted into the nonconserved sites exposed on the surface of the AdhP protein, and the alov9 fusion was successfully screened for subsequent optical experiments under the assumption that neither of these actions affected the original activity of AdhP protein. The activity of alov9 was noticeably inhibited when the fusion was exposed to 470 nm blue light and recovered within 30 min. As a result, we could precisely and reversibly photocontrol alov9 activity through the optimization of several parameters, including cofactor concentration, light intensity, and illumination time.
An efficient method was developed for the photoinhibition of enzymatic activity based on the insertion of the light-sensitive AsLOV2 domain, providing new ideas for photocontrolling metabolic pathways without carriers in the future.
Versatile cell ablation tools and their applications to study loss of cell functions.
Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.
Light-induced dimerization approaches to control cellular processes.
Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.
VIEW-MOD: a versatile illumination engine with a modular optical design for fluorescence microscopy.
We developed VIEW-MOD (Versatile Illumination Engine with a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.
Optical control of transcription - genetically encoded photoswitchable variants of T7 RNA polymerase.
Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much notice for engineering new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions as well as reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For successful application of these tools it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase since both of its termini are involved in catalysis. We show here that it is possible to covalently insert LOV domains into the polymerase protein while preserving its activity and generating new light-responsive allosteric coupling.
Regulation of signaling proteins in the brain by light.
In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.
A molecular toolbox for interrogation of membrane contact sites.
Membrane contact sites (MCSs) are specialized subcellular compartments formed by closely apposed membranes from two organelles. The intermembrane gap is separated by a distance ranging from 10 to 35 nm. MCSs are typically maintained through dynamic protein-protein and protein-lipid interactions. These intermembrane contact sites constitute important intracellular signalling hotspots to mediate a plethora of cellular processes, including calcium homeostasis, lipid metabolism, membrane biogenesis and organelle remodelling. In recent years, a series of genetically encoded probes and chemogenetic or optogenetic actuators have been invented to aid the visualization and interrogation of MCSs in both fixed and living cells. These molecular tools have greatly accelerated the pace of mechanistic dissection of membrane contact sites at the molecular level. In this review, we present an overview on the latest progress in this endeavour, and provide a general guide to the selection of methods and molecular tools for probing interorganellar membrane contact sites.
Engineering Optogenetic Control of Endogenous p53 Protein Levels.
The transcription factor p53 is a stress sensor that turns specific sets of genes on to allow the cell to respond to the stress depending on its severity and type. p53 is classified as tumor suppressor because its function is to maintain genome integrity promoting cell cycle arrest, apoptosis, or senescence to avoid proliferation of cells with damaged DNA. While in many human cancers the p53 gene is itself mutated, there are some in which the dysfunction of the p53 pathway is caused by the overexpression of negative regulators of p53, such as Mdm2, that keep it at low levels at all times. Here we develop an optogenetic approach to control endogenous p53 levels with blue light. Specifically, we control the nuclear localization of the Mmd2-binding PMI peptide using the light-inducible export system LEXY. In the dark, the PMI-LEXY fusion is nuclear and binds to Mdm2, consenting to p53 to accumulate and transcribe the target gene p21. Blue light exposure leads to the export of the PMI-LEXY fusion into the cytosol, thereby Mdm2 is able to degrade p53 as in the absence of the peptide. This approach may be useful to study the effect of localized p53 activation within a tissue or organ.