Showing 1 - 25 of 1687 results
1.
Light-Guided Rabies Virus Tracing for Neural Circuit Analysis.
Abstract:
Neuronal tracing methods are essential tools to understand the fundamental architecture of neural circuits and their connection to the overall functional behavior of the brain. Viral vectors used to map these transsynaptic connections are capable of cell-type-specific and directional-specific labeling of the neuronal connections. Herein, we describe a novel approach to guide the transsynaptic spreading of the Rabies Virus (RV) retrograde tracer using light. We built a Baculovirus (BV) as a helper virus to deliver all the functional components necessary and sufficient for a nontoxic RV to spread from neuron to neuron, with a light-actuated gene switch to control the RV polymerase, the L gene. This design should allow for precisely controlled polysynaptic viral tracing with minimal viral toxicity. To use this system in a highly scalable and automated manner, we built optoelectronics for controlling this system in vitro with a large field of view using an off-the-shelf CMOS sensor, OLED display panel, and microcontrollers. We describe the assembly of these genetic circuits using the uLoop DNA assembly method and a library of genetic parts designed for the uLoop system. Combining these tools provides a framework for increasing the capabilities of nontoxic tracing through multiple synapses and increasing the throughput of neural tracing using viruses.
2.
Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility.
Abstract:
Endogenous biomolecular condensates, composed of a multitude of proteins and RNAs, can organize into multiphasic structures with compositionally distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here we use in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein results in enhanced immiscibility and multiphase formation. Interestingly, we find that oligomerization tunes the miscibility of intrinsically disordered proteins in an asymmetric manner, with the effect being more pronounced when the intrinsically disordered protein, exhibiting stronger homotypic interactions, is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism that cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.
3.
Optogenetic Regulation of EphA1 RTK Activation and Signaling.
Abstract:
Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic strategies could be applied to circumvent this requirement for ligand activation and enable selective activation of the EphA1 subtype. In this work, we designed and tested several iterations of an optogenetic EphA1 - Cryptochrome 2 (Cry2) fusion, investigating their capacity to mimic EphA1-dependent signaling in response to light activation. We then characterized the key cell signaling target of MAPK phosphorylation activated in response to light stimulation. The optogenetic regulation of Eph receptor RTK signaling without the need for external stimulus promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions.
4.
Light-Mediated Enhancement of Glucose-Stimulated Insulin Release of Optogenetically Engineered Human Pancreatic Beta-Cells.
Abstract:
Enhancement of glucose-stimulated insulin secretion (GSIS) in exogenously delivered pancreatic β-cells is desirable, for example, to overcome the insulin resistance manifested in type 2 diabetes or to reduce the number of β-cells for supporting homeostasis of blood sugar in type 1 diabetes. Optogenetically engineered cells can potentiate their function with exposure to light. Given that cyclic adenosine monophosphate (cAMP) mediates GSIS, we surmised that optoamplification of GSIS is feasible in human β-cells carrying a photoactivatable adenylyl cyclase (PAC). To this end, human EndoC-βH3 cells were engineered to express a blue-light-activated PAC, and a workflow was established combining the scalable manufacturing of pseudoislets (PIs) with efficient adenoviral transduction, resulting in over 80% of cells carrying PAC. Changes in intracellular cAMP and GSIS were determined with the photoactivation of PAC in vitro as well as after encapsulation and implantation in mice with streptozotocin-induced diabetes. cAMP rapidly rose in β-cells expressing PAC with illumination and quickly declined upon its termination. Light-induced amplification in cAMP was concomitant with a greater than 2-fold GSIS vs β-cells without PAC in elevated glucose. The enhanced GSIS retained its biphasic pattern, and the rate of oxygen consumption remained unchanged. Diabetic mice receiving the engineered β-cell PIs exhibited improved glucose tolerance upon illumination compared to those kept in the dark or not receiving cells. The findings support the use of optogenetics for molecular customization of the β-cells toward better treatments for diabetes without the adverse effects of pharmacological approaches.
5.
A temperature-inducible protein module for control of mammalian cell fate.
Abstract:
Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. Using custom devices for rapid and high-throughput temperature control during live-cell microscopy, we find that the original Melt variant fully switches states between 28-32°C, and state changes can be observed within minutes of temperature changes. Melt was highly modular, permitting thermal control over diverse intracellular processes including signaling, proteolysis, and nuclear shuttling through straightforward end-to-end fusions with no further engineering. Melt was also highly tunable, giving rise to a library of Melt variants with switch point temperatures ranging from 30-40°C. The variants with higher switch points allowed control of molecular circuits between 37°C-41°C, a well-tolerated range for mammalian cells. Finally, Melt could thermally regulate important cell decisions over this range, including cytoskeletal rearrangement and apoptosis. Thus Melt represents a versatile thermogenetic module that provides straightforward, temperature-based, real-time control of mammalian cells with broad potential for biotechnology and biomedicine.
6.
OptoREACT: Optogenetic Receptor Activation on Nonengineered Human T Cells.
-
Armbruster, A
-
Ehret, AK
-
Russ, M
-
Idstein, V
-
Klenzendorf, M
-
Gaspar, D
-
Juraske, C
-
Yousefi, OS
-
Schamel, WW
-
Weber, W
-
Hörner, M
Abstract:
Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.
7.
Epithelial folding through local degradation of an elastic basement membrane plate.
Abstract:
Epithelia are polarised layers of cells that line the outer and inner surfaces of organs. At the basal side, the epithelial cell layer is supported by a basement membrane, which is a thin polymeric layer of self-assembled extracellular matrix (ECM) that tightly adheres to the basal cell surface. Proper shaping of epithelial layers is an important prerequisite for the development of healthy organs during the morphogenesis of an organism. Experimental evidence indicates that local degradation of the basement membrane drives epithelial folding. Here, we present a coarse-grained plate theory model of the basement membrane that assumes force balance between i) cell-transduced active forces and ii) deformation-induced elastic forces. We verify key assumptions of this model through experiments in the Drosophila wing disc epithelium and demonstrate that the model can explain the emergence of outward epithelial folds upon local plate degradation. Our model accounts for local degradation of the basement membrane as a mechanism for the generation of epithelial folds in the absence of epithelial growth.
8.
Optogenetic control of pheromone gradients reveals functional limits of mating behavior in budding yeast.
Abstract:
Cell-cell communication through diffusible signals allows distant cells to coordinate biological functions. Such coordination depends on the signal landscapes generated by emitter cells and the sensory capacities of receiver cells. In contrast to morphogen gradients in embryonic development, microbial signal landscapes occur in open space with variable cell densities, spatial distributions, and physical environments. How do microbes shape signal landscapes to communicate robustly under such circumstances remains an unanswered question. Here we combined quantitative spatial optogenetics with biophysical theory to show that in the mating system of budding yeast— where two mates communicate to fuse—signal landscapes convey demographic or positional information depending on the spatial organization of mating populations. This happens because α-factor pheromone and its mate-produced protease Bar1 have characteristic wide and narrow diffusion profiles, respectively. Functionally, MATα populations signal their presence as collectives, but not their position as individuals, and Bar1 is a sink of alpha-factor, capable of both density-dependent global attenuation and local gradient amplification. We anticipate that optogenetic control of signal landscapes will be instrumental to quantitatively understand the spatial behavior of natural and engineered cell-cell communication systems.
9.
Interplay of condensation and chromatin binding underlies BRD4 targeting.
-
Strom, AR
-
Eeftens, JM
-
Polyachenko, Y
-
Weaver, CJ
-
Watanabe, HF
-
Bracha, D
-
Orlovsky, ND
-
Jumper, CC
-
Jacobs, WM
-
Brangwynne, CP
Abstract:
Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is under-explored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcription factor BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.
10.
Enhancing high-throughput optogenetics: Integration of LITOS with Lustro enables simultaneous light stimulation and shaking.
Abstract:
Optogenetics is a powerful tool that uses light to control cellular behavior. Here we enhance high-throughput characterization of optogenetic experiments through the integration of the LED Illumination Tool for Optogenetic Stimulation (LITOS) with the previously published automated platform Lustro. Lustro enables efficient high-throughput screening and characterization of optogenetic systems. The initial iteration of Lustro used the optoPlate illumination device for light induction, with the robot periodically moving the plate over to a shaking device to resuspend cell cultures. Here, we designed a 3D-printed adaptor, rendering LITOS compatible with the BioShake 3000-T ELM used in Lustro. This novel setup allows for concurrent light stimulation and culture agitation, streamlining experiments. Our study demonstrates comparable growth rates between constant and intermittent shaking of Saccharomyces cerevisiae liquid cultures. While the light intensity of the LITOS is not as bright as the optoPlate used in the previous iteration of Lustro, the constant shaking increased the maturation rate of the mScarlet-I fluorescent reporter used. Only a marginal increase in temperature was observed when using the modified LITOS equipped with the 3D-printed adaptor. Our findings show that the integration of LITOS onto a plate shaker allows for constant culture shaking and illumination compatible with laboratory automation platforms, such as Lustro.
11.
Epstein-Barr Virus Promotes Tumorigenicity and Worsens Hodgkin Lymphoma Prognosis by Activating JAK/STAT and NF-κB Signaling Pathways.
-
Alabiad, MA
-
Said, WMM
-
Saad, RHF
-
Balata, R
-
Mahmoud, AA
-
Metwally, EA
-
Shalaby, AM
-
Samy, W
-
Yehia, AM
-
Yahia, AIO
-
Alorini, M
-
Abdelrahman, DI
Abstract:
Epstein-Barr virus (EBV) is detected in 40% of patients with Hodgkin lymphoma (HL). During latency, EBV induces epigenetic alterations to the host genome and decreases the expression of pro-apoptotic proteins. The present study aimed to evaluate the expression levels of mRNA molecules and the end product of proteins for the JAK/STAT and NF-κB pathways, and their association with clinicopathological and prognostic parameters in patients with EBV-positive and -negative classical Hodgkin lymphoma (CHL).
12.
Using split protein reassembly strategy to optically control PLD enzymatic activity.
-
Yao, Y
-
Lou, X
-
Jianxu, L
-
Zhu, M
-
Qian, X
-
Liu, J
-
Zhang, L
-
Zhang, P
-
He, L
-
Li, H
-
Xu, Y
Abstract:
Phospholipase D (PLD) and phosphatidic acid (PA) play a spatio-temporal role in regulating diverse cellular activities. Although current methodologies enable optical control of the subcellular localization of PLD and by which influence local PLD enzyme activity, the overexpression of PLD elevates the basal PLD enzyme activity and further leads to increased PA levels in cells. In this study, we employed a split protein reassembly strategy and optogenetic techniques to modify superPLD (a PLDPMF variant with a high basal activity). We splited this variants into two HKD domains and fused these domains with optogenetic elements and by which we achieved light-mediated dimerization of the two HKD proteins and then restored the PLD enzymatic activity.
13.
Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling
Abstract:
Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light–oxygen–voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.
14.
Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid.
Abstract:
Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3',5'-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 µm2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.
15.
Optogenetic generation of leader cells reveals a force-velocity relation for collective cell migration.
Abstract:
The front of migratory cellular clusters during development, wound healing and cancer invasion is typically populated with highly protrusive cells that are called leader cells. Leader cells are thought to physically pull and direct their cohort of followers, but how leaders and followers are mechanically organized to migrate collectively remains controversial. One possibility is that the autonomous local action of a leader cell is sufficient to drive migration of the group. Yet another possibility is that a global mechanical organization is required for the group to move cohesively. Here we show that the effectiveness of leader-follower organization is proportional to the asymmetry of traction and tension within the cellular cluster. By combining hydrogel micropatterning and optogenetic activation of Rac1, we locally generate highly protrusive leaders at the edge of minimal cell groups. We find that the induced leader can robustly drag one follower but is generally unable to direct larger groups. By measuring traction forces and tension propagation in groups of increasing size, we establish a quantitative relationship between group velocity and the asymmetry of the traction and tension profiles. We propose a model of the motile cluster as an active polar fluid that explains this force-velocity relationship in terms of asymmetries in the distribution of active tractions. Our results challenge the notion of autonomous leader cells by showing that collective cell migration requires a global mechanical organization within the cluster.
16.
Programmable RNA base editing with photoactivatable CRISPR-Cas13.
Abstract:
CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.
17.
Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.
Abstract:
Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature: light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below ∼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.
18.
Crucial Residue for Tuning Thermal Relaxation Kinetics in the Biliverdin-binding Cyanobacteriochrome Photoreceptor Revealed by Site-saturation Mutagenesis.
Abstract:
Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.
19.
Development of an optogenetics tool, Opto-RANK, for control of osteoclast differentiation using blue light.
Abstract:
Optogenetics enables precise regulation of intracellular signaling in target cells. However, the application of optogenetics to induce the differentiation of precursor cells and generate mature cells with specific functions has not yet been fully explored. Here, we focused on osteoclasts, which play an important role in bone remodeling, to develop a novel optogenetics tool, Opto-RANK, which can manipulate intracellular signals involved in osteoclast differentiation and maturation using blue light. We engineered Opto-RANK variants, Opto-RANKc and Opto-RANKm, and generated stable cell lines through retroviral transduction. Differentiation was induced by blue light, and various assays were conducted for functional analysis. Osteoclast precursor cells expressing Opto-RANK differentiated into multinucleated giant cells on light exposure and displayed upregulation of genes normally induced in differentiated osteoclasts. Furthermore, the differentiated cells exhibited bone-resorbing activities, with the possibility of spatial control of the resorption by targeted light illumination. These results suggested that Opto-RANK cells differentiated by light possess the features of osteoclasts, both morphological and functional. Thus, Opto-RANK should be useful for detailed spatiotemporal analysis of intracellular signaling during osteoclast differentiation and the development of new therapies for various bone diseases.
20.
Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.
Abstract:
Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344-28354 (2020); and M. Frieman et al., J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.
21.
Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag.
Abstract:
Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function—dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.
22.
An RNA Motif That Enables Optozyme Control and Light-Dependent Gene Expression in Bacteria and Mammalian Cells.
-
Pietruschka, G
-
Ranzani, AT
-
Weber, A
-
Patwari, T
-
Pilsl, S
-
Renzl, C
-
Otte, DM
-
Pyka, D
-
Möglich, A
-
Mayer, G
Abstract:
The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.
23.
Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe.
Abstract:
The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.
24.
A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow during Drosophila gastrulation.
Abstract:
Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.
25.
Neuropeptidergic regulation of neuromuscular signaling in larval zebrafish alters swimming behavior and synaptic transmission.
Abstract:
The regulation of synaptic transmission is crucial for plasticity, homeostasis and learning. Chemical synaptic transmission is thus modulated to accommodate different activity levels, which also enables homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cAMP signaling enhances cholinergic neuron output, and these neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish. Optogenetic stimulation by photoactivated adenylyl cyclase (bPAC) resulted in elevated locomotion as measured in behavioural assays. Furthermore, post-synaptic patch-clamp recordings revealed that in bPAC transgenics, the frequency of miniature excitatory postsynaptic currents (mEPSCs) was increased after photostimulation. These results suggested that cAMP-mediated activation of ZF motor neurons leads to increased fusion of SVs, consequently resulting in enhanced neuromuscular activity. We generated mutants lacking the neuropeptide processing enzyme carboxypeptidase E (cpe), and the most abundant neuropeptide precursor in motor neurons, tachykinin (tac1). Both mutants showed exaggerated locomotion after photostimulation. cpe mutants exhibit lower mEPSC frequency during photostimulation and less large-amplitude mEPSCs. In tac1 mutants mEPSC frequency was not affected but amplitudes were significantly smaller. Exaggerated locomotion in the mutants thus reflected upscaling of postsynaptic excitability. cpe and tac1 mutant muscles expressed more nicotinic acetylcholine receptors (nAChR) on their surface. Thus, neuropeptide signaling regulates synaptic transmitter output in zebrafish motor neurons, and muscle cells homeostatically regulate nAChR surface expression, compensating reduced presynaptic input. This mechanism may be widely conserved in the animal kingdom.