Qr: application:"Signaling cascade control"
Showing 51 - 75 of 250 results
51.
Engineering of an Optogenetic T Cell Receptor Compatible with Fluorescence-Based Readouts.
Abstract:
Optogenetics offers a set of tools for the precise manipulation of signaling pathways. Here we exploit optogenetics to experimentally change the kinetics of protein-protein interactions on demand. We had developed a system in which the interaction of a modified T cell receptor (TCR) with an engineered ligand can be controlled by light. The ligand was the plant photoreceptor phytochrome B (PhyB) and the TCR included a TCRβ chain fused to GFP and a mutated PhyB-interacting factor (PIFS), resulting in the GFP-PIFS-TCR. We failed to engineer a nonfluorescent PIFS-fused TCR, since PIFS did not bind to PhyB when omitting GFP. Here we tested nine different versions of PIFS-fused TCRs. We found that the SNAP-PIFS-TCR was expressed well on the surface, bound to PhyB, and subsequently elicited activation signals. This receptor could be combined with a GFP reporter system in which the expression of GFP is driven by the transcription factor NF-AT.
52.
Local negative feedback of Rac activity at the leading edge underlies a pilot pseudopod-like program for amoeboid cell guidance.
Abstract:
To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to "lock" onto a particular direction, limiting the ability of cells to reorient. We use spatially defined optogenetic control of a leading edge organizer (PI3K) to probe how neutrophil-like HL-60 cells balance "decisiveness" needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibition process that destabilizes the leading edge to promote exploration. We show that this local inhibition enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.
53.
Light-inducible T cell engagers trigger, tune, and shape the activation of primary T cells.
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Jaeger, M
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Anastasio, A
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Chamy, L
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Brustlein, S
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Vincentelli, R
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Durbesson, F
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Gigan, J
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Thépaut, M
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Char, R
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Boussand, M
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Lechelon, M
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Argüello, RJ
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Marguet, D
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He, HT
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Lasserre, R
Abstract:
To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.
54.
Photoswitchable binders enable temporal dissection of endogenous protein function.
Abstract:
General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Here, we report robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. We created topological variants of the DARPin scaffold by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following EGF-activation of EGFR, Ras is required for sustained EGFR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function.
55.
Optogenetic engineering of STING signaling allows remote immunomodulation to enhance cancer immunotherapy.
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Dou, Y
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Chen, R
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Liu, S
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Lee, YT
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Jing, J
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Liu, X
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Ke, Y
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Wang, R
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Zhou, Y
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Huang, Y
Abstract:
The cGAS-STING signaling pathway has emerged as a promising target for immunotherapy development. Here, we introduce a light-sensitive optogenetic device for control of the cGAS/STING signaling to conditionally modulate innate immunity, called 'light-inducible SMOC-like repeats' (LiSmore). We demonstrate that photo-activated LiSmore boosts dendritic cell (DC) maturation and antigen presentation with high spatiotemporal precision. This non-invasive approach photo-sensitizes cytotoxic T lymphocytes to engage tumor antigens, leading to a sustained antitumor immune response. When combined with an immune checkpoint blocker (ICB), LiSmore improves antitumor efficacy in an immunosuppressive lung cancer model that is otherwise unresponsive to conventional ICB treatment. Additionally, LiSmore exhibits an abscopal effect by effectively suppressing tumor growth in a distal site in a bilateral mouse model of melanoma. Collectively, our findings establish the potential of targeted optogenetic activation of the STING signaling pathway for remote immunomodulation in mice.
56.
Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.
Abstract:
Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.
57.
Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor.
Abstract:
Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.
58.
Automatic detection of spatio-temporal signalling patterns in cell collectives.
Abstract:
An increasing experimental evidence points to physiological importance of space-time correlations in signalling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of bio-molecules between cells allows the collectives to perform more complex tasks and better tackle environmental challenges. To understand this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signalling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We make a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells induce ERK activity waves with different size, duration, and frequency. The open-source implementations of ARCOS are available as R and Python packages, and as a plugin for napari image viewer to interactively quantify collective phenomena without prior programming experience.
59.
Senescence-induced cellular reprogramming drives cnidarian whole-body regeneration.
Abstract:
Cell fate stability is essential to maintaining “law and order” in complex animals. However, high stability comes at the cost of reduced plasticity and, by extension, poor regenerative ability. This evolutionary trade-off has resulted in most modern animals being rather simple and regenerative or complex and non-regenerative. The mechanisms mediating cellular plasticity and allowing for regeneration remain unknown. We show that signals emitted by senescent cells can destabilize the differentiated state of neighboring somatic cells, reprogramming them into stem cells that are capable of driving whole-body regeneration in the cnidarian Hydractinia symbiolongicarpus. Pharmacological or genetic inhibition of senescence prevents reprogramming and regeneration. Conversely, induction of transient ectopic senescence in a regenerative context results in supernumerary stem cells and faster regeneration. We propose that senescence signaling is an ancient mechanism mediating cellular plasticity. Understanding the senescence environment that promotes cellular reprogramming could provide an avenue to enhance regeneration.
60.
Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.
Abstract:
The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.
61.
Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength.
Abstract:
The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.
62.
Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons.
Abstract:
RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.
63.
Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture.
Abstract:
In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging, and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition, and TGF-β signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish an hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.
64.
An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells.
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Zhou, W
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Li, W
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Wang, S
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Salovska, B
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Hu, Z
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Tao, B
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Di, Y
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Punyamurtula, U
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Turk, BE
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Sessa, WC
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Liu, Y
Abstract:
The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.
65.
Synthetic Frizzled agonist and LRP antagonist for high-efficiency Wnt/β-catenin signaling manipulation in organoid cultures and in vivo.
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Dai, D
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Wang, W
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Lin, L
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Yu, Y
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Yang, Y
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Zhang, Z
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Li, L
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Zeng, Z
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Hu, H
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Ni, N
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Zhao, Z
Abstract:
Wnt/β-catenin signaling and its dysregulation play critical roles in the fate determination of stem cells and the pathology of various diseases. However, the application of translated Wnt ligand in regenerative medicine is hampered by its hydrophobicity and cross-reactivity with Frizzled (FZD) receptors. Here, we generate an engineered water-soluble, FZD subtype-specific agonist, RRP-pbFn, for high-efficiency Wnt/β-catenin signaling activation. In the absence of direct binding to LRP5/6, RRP-pbFn stimulates Wnt/β-catenin signaling more potently than surrogate Wnt. RRP-pbFn supports the growth of a variety of mouse and human organoids, and induces the expansion of liver and intestine progenitors in vivo. Meanwhile, we develop a synthetic LRP antagonist, RRP-Dkk1c, which exhibits heightened effectiveness in attenuating Wnt/β-catenin signaling activity compared to Dkk1, thereby abolishing the formation of CT26-derived colon cancer xenograft in vivo. Together, these two paired Wnt/β-catenin signaling manipulators hold great promise for biomedical research and potential therapeutics.
66.
The MAPK/ERK channel capacity exceeds 6 bit/hour.
Abstract:
Living cells utilize signaling pathways to sense, transduce, and process information. As the extracellular stimulation often has rich temporal characteristics which may govern dynamic cellular responses, it is important to quantify the rate of information flow through the signaling pathways. In this study, we used an epithelial cell line expressing a light-activatable FGF receptor and an ERK activity reporter to assess the ability of the MAPK/ERK pathway to transduce signal encoded in a sequence of pulses. By stimulating the cells with random light pulse trains, we demonstrated that the MAPK/ERK channel capacity is at least 6 bits per hour. The input reconstruction algorithm detects the light pulses with 1-min accuracy 5 min after their occurrence. The high information transmission rate may enable the pathway to coordinate multiple processes including cell movement and respond to rapidly varying stimuli such as chemoattracting gradients created by other cells.
67.
Actuation of single downstream nodes in growth factor network steers immune cell migration.
Abstract:
Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.
68.
Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases.
Abstract:
During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.
69.
Optogenetic manipulation identifies the roles of ERK and AKT dynamics in controlling mouse embryonic stem cell exit from pluripotency.
Abstract:
ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues.
70.
OptIC Notch reveals mechanism that regulates receptor interactions with CSL.
Abstract:
Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.
71.
Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-β signaling.
Abstract:
In tissue development and homeostasis, transforming growth factor (TGF)-β signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-β signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-β signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-β gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-β signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.
72.
Optogenetic control of YAP can enhance the rate of wound healing.
Abstract:
Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.
73.
Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation.
Abstract:
Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of Gα protein – Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαi signaling.
74.
Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.
Abstract:
By acting both upstream of and downstream from biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTOR complex 2 (mTORC2) programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading-edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input phosphatidylinositol (3,4,5)-trisphosphate to robustly amplify mTORC2 activation. Our results suggest that different signaling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.
75.
Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis.
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Kawamura, G
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Kokaji, T
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Kawata, K
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Sekine, Y
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Suzuki, Y
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Soga, T
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Ueda, Y
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Endo, M
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Kuroda, S
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Ozawa, T
Abstract:
Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.
