Qr: journal:"Cell"
Showing 1 - 25 of 229 results
1.
Optogenetic engineered macrophages for light-induced M1 polarization and enhanced chemo-immunotherapy in melanoma models.
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He, K
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Jiang, H
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Zhang, W
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Yang, N
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Li, S
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Wang, Y
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Zhang, J
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Li, X
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Tan, L
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Yang, G
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Li, H
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Lu, Y
Abstract:
Macrophage-based adoptive cell therapies hold promise for solid tumors, but spatiotemporally controlling macrophage polarization within the immunosuppressive tumor microenvironment remains challenging. Here, we aimed to validate an optogenetic strategy using the LOV2-STIM1 system to achieve light-induced, sustained M1 polarization of macrophages. Upon blue light stimulation, engineered macrophages robustly exhibited M1 phenotypes, suppressed melanoma cell proliferation, migration, and invasion in vitro, and recapitulated the antitumor functions of M1 macrophages. Notably, combining light-activated engineered macrophages with temozolomide in melanoma models resulted in synergistic inhibition of tumor growth. This synergy is accompanied by a profound remodeling of the tumor immune microenvironment, characterized by M1-driven reversal of chemoresistance and enhanced infiltration of cytotoxic CD8+ T cells. Our findings establish a proof-of-concept for optogenetic regulation of macrophage polarization and demonstrate its feasibility for enhancing antitumor effects and chemosensitivity in melanoma models, providing a promising and controllable platform for macrophage-based immunotherapy.
2.
Optogenetic stimulation of Lbc GEF-mediated Rho activity dynamics promotes cell invasion.
Abstract:
Cancer cell invasion relies on dynamic cell shape changes, which originate from protrusive and contractile intracellular forces. Previous studies revealed that contractile forces are controlled by positive-feedback amplification of the contraction regulator Rho by Lbc GEFs. These GEFs were previously linked to tumor progression; however, the underlying mechanisms are poorly understood. Here, we generated a mouse melanoma model in which cytosolic levels of the Lbc GEF GEF-H1 are controlled by light. Using this model, we found that increased GEF-H1 levels strongly stimulate cell contraction dynamics. Interestingly, increased contraction dynamics rapidly induced expansion of tumor spheroids via a focal adhesion kinase-dependent mechanism. Furthermore, long-term stimulation led to the escape of individual cells from spheroids. These findings reveal new insights into the oncogenic roles of Lbc GEFs and how they might promote tumor cell invasion. We propose a mechanism in which increased cell contraction dynamics result in asymmetric pulling forces at the tumor border, promoting the detachment and escape of individual cells.
3.
Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation.
Abstract:
Localized translation broadly enables spatiotemporal control of gene expression. Here, we present LOV-domain-controlled ligase for translation localization (LOCL-TL), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially localized translation revealed that ∼20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic-origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-kinase anchoring protein 1 (AKAP1) in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM.
4.
Investigating morphogen and patterning dynamics with optogenetic control of morphogen production.
Abstract:
Morphogen gradients provide the patterning cues that instruct cell fate decisions during development. Here, we establish an optogenetic system for the precise spatiotemporal control in vitro of Sonic hedgehog (Shh) morphogen production. Using a tunable light-inducible gene expression system, we generate long-range Shh gradients that pattern mouse neural progenitors into spatially distinct domains, mimicking neural tube development. We investigate how biochemical features of Shh and Shh-interacting proteins affect patterning length scales. By measuring clearance rates, we determine that Shh has an extracellular half-life below 1.5 h, substantially shorter than downstream gene expression dynamics, indicating gradients are continually renewed during patterning. We provide evidence that progenitor identity acquisition and maintenance depend on both Shh concentration and exposure duration. Together, this approach provides a quantitative framework for investigating morphogen patterning, enabling reproducible control of morphogen dynamics to dissect the interplay between biochemical cues, gradient formation biophysics, and transcriptional programs underlying developmental patterning.
5.
Optogenetics-enabled discovery of integrated stress response modulators.
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Wong, F
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Li, A
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Omori, S
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Lach, RS
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Nunez, J
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Ren, Y
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Brown, SP
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Singhal, V
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Lyda, BR
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Batjargal, T
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Dickson, E
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Rodrigues Reyes, JR
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Uruena Vargas, JM
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Wahane, S
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Kim, H
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Collins, JJ
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Wilson, MZ
Abstract:
The integrated stress response (ISR) is a conserved stress response that maintains homeostasis in eukaryotic cells. Modulating the ISR holds therapeutic potential for diseases including viral infection, cancer, and neurodegeneration, but few known compounds can do so without toxicity. Here, we present an optogenetic platform for the discovery of compounds that selectively modulate the ISR. Optogenetic clustering of PKR induces ISR-mediated cell death, enabling the high-throughput screening of 370,830 compounds. We identify compounds that potentiate cell death without cytotoxicity across diverse cell types and stressors. Mechanistic studies reveal that these compounds upregulate activating transcription factor 4 (ATF4), sensitizing cells to stress and apoptosis, and identify GCN2 as a molecular target. Additionally, these compounds exhibit antiviral activity, and one compound reduced viral titers in a mouse model of herpesvirus infection. Structure-activity and toxicology studies highlight opportunities to optimize therapeutic efficacy. This work demonstrates an optogenetic approach to drug discovery and introduces ISR potentiators with therapeutic potential.
6.
Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments.
Abstract:
Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but it reduced cell stiffness. Mitochondria and endoplasmic reticulum (ER) sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.
7.
Opto-p53: A light-controllable activation of p53 signaling pathway.
Abstract:
p53 protein, a crucial transcription factor in cellular responses to a wide variety of stress, regulates multiple target genes involved in tumor suppression, senescence induction, and metabolic functions. To characterize the context-dependent roles of p53, it is still needed to develop an experimental system that enables selective activation of p53 in cells and tissues. In this study, we developed an optogenetic tool, Opto-p53, to control p53 signaling by light. Opto-p53 was designed to trigger p53 signaling by reconstituting p53 N-terminal and C-terminal fragments with a light-inducible dimerization (LID) system. Upon light exposure, cells expressing Opto-p53 demonstrated p53 transcriptional activation, resulting in cell death and cell cycle arrest. We further enhanced the efficacy of light-induced p53 activation by introducing specific mutations into Opto-p53 fragments. Our findings unveil the capability of Opto-p53 to serve as a powerful tool for dissecting the complex roles of p53 in cellular processes, thereby contributing to the field of synthetic biology and providing general design principles for optogenetic tools using endogenous transcription factors.Key words: synthetic biology, transcriptional factor, p53, optogenetics.
8.
zHORSE as an optogenetic zebrafish strain for precise spatiotemporal control over gene expression during development.
Abstract:
Proper vertebrate development is dependent on tightly regulated expression of genes at the correct time and place. To identify normal but also dysregulated development leading to disease, in vivo interrogation methods with high spatiotemporal resolution are required. Recently, optogenetic tools to manipulate gene expression with spatiotemporal control have emerged, but their in vivo applications remain challenging. Here, we present a transgenic zebrafish strain termed zebrafish for heat-shock-inducible optogenetic recombinase expression (zHORSE) with inducible expression of a light-activatable Cre recombinase. We demonstrate that zHORSE endows robust spatiotemporal control over gene expression down to single-cell level at different developmental stages. We apply zHORSE for lineage tracing to identify caudal fin progenitors and for targeted expression of oncogenes. Surprisingly, one oncogene, EWS::FLI1, can cause ectopic fin formation when induced in permissive environments. zHORSE is compatible with existing loxP zebrafish effector strains and will enable many applications ranging from dissecting and precisely manipulating development to clonal cancer modeling.
9.
Chip (Ldb1) is a putative cofactor of Zelda forming a functional bridge to CBP during zygotic genome activation.
Abstract:
The cofactor LIM-domain-binding protein 1 (Ldb1) is linked to many processes in gene regulation, including enhancer-promoter communication, interchromosomal interactions, and enhanceosome-cofactor-like activity. However, its functional requirement and molecular role during embryogenesis remain unclear. Here, we used optogenetics (iLEXY) to rapidly deplete Drosophila Ldb1 (Chip) from the nucleus at precise time windows. Remarkably, this pinpointed the essential window of Chip's function to just 1 h of embryogenesis, overlapping zygotic genome activation (ZGA). We show that Zelda, a pioneer factor essential for ZGA, recruits Chip to chromatin, and both factors regulate concordant changes in gene expression, suggesting that Chip is a cofactor of Zelda. Chip does not significantly impact chromatin architecture at these stages, but instead recruits CBP, and is essential for H3K27ac deposition at enhancers and promoters, and for the proper expression of co-regulated genes. These data identify Chip as a functional bridge between Zelda and the coactivator CBP to regulate gene expression in early embryogenesis.
10.
Neighbor cells restrain furrowing during Xenopus epithelial cytokinesis.
Abstract:
Cytokinesis challenges epithelial tissue homeostasis by generating forces that pull on neighboring cells. Junction reinforcement at the furrow in Xenopus epithelia regulates the speed of furrowing, suggesting that cytokinesis is subject to resistive forces from epithelial neighbors. We show that contractility factors accumulate near the furrow in neighboring cells, and increasing neighbor cell stiffness slows furrowing. Optogenetically increasing contractility in one or both neighbor cells slows furrowing or induces cytokinetic failure. Uncoupling mechanotransduction between dividing cells and their neighbors increases the furrow ingression rate, alters topological cell packing following cytokinesis, and impairs barrier function at the furrow. Computational modeling validates our findings and provides additional insights about epithelial mechanics during cytokinesis. We conclude that forces from the cytokinetic array must be carefully balanced with restraining forces generated by neighbor cells to regulate the speed and success of cytokinesis and maintain epithelial homeostasis.
11.
Inward transport of organelles drives outward migration of the spindle during C. elegans meiosis.
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Aquino, AP
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Li, W
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Lele, A
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Lazureanu, D
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Hampton, MF
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Do, RM
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Lafrades, MC
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Barajas, MG
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Batres, AA
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McNally, FJ
Abstract:
Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex, while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesize that the inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generate a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packs mitochondria into a tight ball and efficiently moves the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.
12.
An improved FLARE system for recording and manipulating neuronal activity.
Abstract:
To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.
13.
Tissue sculpting with light.
Abstract:
While optogenetic tools have recently opened new avenues for controlling and understanding cellular behavior, Suh et al.1 present an effective strategy to regulate tissue densification and outgrowth through optogenetic control of EGFR. Their work ultimately uncovers fundamental principles that pave the way for improved tissue engineering approaches.
14.
Large-scale control over collective cell migration using light-activated epidermal growth factor receptors.
Abstract:
Receptor tyrosine kinases (RTKs) play key roles in coordinating cell movement at both single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggests that these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled epidermal growth factor (EGF) receptor (OptoEGFR) can be deployed in epithelial cells for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by phosphoinositide 3-kinase (PI3K) signaling, rather than diffusible ligands, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications, including wound healing and tissue morphogenesis.
15.
Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3.
Abstract:
Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase Rap1 bound to GTP (Rap1-GTP) and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate the interaction of talin with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin-1 (TLN1; herein referred to as talin) to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first, by releasing Rap1-GTP from its sequestration by SHANK3, and second, by serving as the relevant Rap1 effector for integrin activation.
16.
Live imaging of paracrine signaling: Advances in visualization and tracking techniques.
Abstract:
Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.
17.
Spatiotemporal dissection of collective cell migration and tissue morphogenesis during development by optogenetics.
Abstract:
Collective cell migration and tissue morphogenesis play a variety of important roles in the development of many species. Tissue morphogenesis often generates mechanical forces that alter cell shapes and arrangements, resembling collective cell migration-like behaviors. Genetic methods have been widely used to study collective cell migration and its like behavior, advancing our understanding of these processes during development. However, a growing body of research shows that collective cell migration during development is not a simple behavior but is often combined with other cellular and tissue processes. In addition, different surrounding environments can also influence migrating cells, further complicating collective cell migration during development. Due to the complexity of developmental processes and tissues, traditional genetic approaches often encounter challenges and limitations. Thus, some methods with spatiotemporal control become urgent in dissecting collective cell migration and tissue morphogenesis during development. Optogenetics is a method that combines optics and genetics, providing a perfect strategy for spatiotemporally controlling corresponding protein activity in subcellular, cellular or tissue levels. In this review, we introduce the basic mechanisms underlying different optogenetic tools. Then, we demonstrate how optogenetic methods have been applied in vivo to dissect collective cell migration and tissue morphogenesis during development. Additionally, we describe some promising optogenetic approaches for advancing this field. Together, this review will guide and facilitate future studies of collective cell migration in vivo and tissue morphogenesis by optogenetics.
18.
Cell-cell junctions in focus - imaging junctional architectures and dynamics at high resolution.
Abstract:
Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.
19.
Photo-tunable hydrogels reveal cellular sensing of rapid rigidity changes through the accumulation of mechanical signaling molecules.
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Yang, J
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Wang, P
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Zhang, Y
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Zhang, M
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Sun, Q
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Chen, H
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Dong, L
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Chu, Z
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Xue, B
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Hoff, WD
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Zhao, C
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Wang, W
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Wei, Q
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Cao, Y
Abstract:
Cells use traction forces to sense mechanical cues in their environment. While the molecular clutch model effectively explains how cells exert more forces on stiffer substrates, it falls short in addressing their adaptation to dynamic mechanical fluctuations prevalent in tissues and organs. Here, using hydrogel with photo-responsive rigidity, we show that cells' response to rigidity changes is frequency dependent. Strikingly, at certain frequencies, cellular traction forces exceed those on static substrates 4-fold stiffer, challenging the established molecular clutch model. We discover that the discrepancy between the rapid adaptation of traction forces and the slower deactivation of mechanotransduction signaling proteins results in their accumulation, thereby enhancing long-term cellular traction in dynamic settings. Consequently, we propose a new model that melds immediate mechanosensing with extended mechanical signaling. Our study underscores the significance of dynamic rigidity in the development of synthetic biomaterials, emphasizing the importance of considering both immediate and prolonged cellular responses.
20.
Light-induced targeting enables proteomics on endogenous condensates.
Abstract:
Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass spectrometry and other proteomics profiling. Here, we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light-dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.
21.
RNA G-quadruplexes form scaffolds that promote neuropathological α-synuclein aggregation.
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Matsuo, K
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Asamitsu, S
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Maeda, K
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Suzuki, H
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Kawakubo, K
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Komiya, G
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Kudo, K
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Sakai, Y
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Hori, K
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Ikenoshita, S
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Usuki, S
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Funahashi, S
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Oizumi, H
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Takeda, A
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Kawata, Y
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Mizobata, T
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Shioda, N
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Yabuki, Y
Abstract:
Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are triggered by α-synuclein aggregation, triggering progressive neurodegeneration. However, the intracellular α-synuclein aggregation mechanism remains unclear. Herein, we demonstrate that RNA G-quadruplex assembly forms scaffolds for α-synuclein aggregation, contributing to neurodegeneration. Purified α-synuclein binds RNA G-quadruplexes directly through the N terminus. RNA G-quadruplexes undergo Ca2+-induced phase separation and assembly, accelerating α-synuclein sol-gel phase transition. In α-synuclein preformed fibril-treated neurons, RNA G-quadruplex assembly comprising synaptic mRNAs co-aggregates with α-synuclein upon excess cytoplasmic Ca2+ influx, eliciting synaptic dysfunction. Forced RNA G-quadruplex assembly using an optogenetic approach evokes α-synuclein aggregation, causing neuronal dysfunction and neurodegeneration. The administration of 5-aminolevulinic acid, a protoporphyrin IX prodrug, prevents RNA G-quadruplex phase separation, thereby attenuating α-synuclein aggregation, neurodegeneration, and progressive motor deficits in α-synuclein preformed fibril-injected synucleinopathic mice. Therefore, Ca2+ influx-induced RNA G-quadruplex assembly accelerates α-synuclein phase transition and aggregation, potentially contributing to synucleinopathies.
22.
Precision in situ cryo-correlative light and electron microscopy of optogenetically-positioned organelles.
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Tillu, VA
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Redpath, GMI
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Rae, J
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Ruan, J
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Yao, Y
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Cagigas, ML
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Whan, R
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Hardeman, EC
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Gunning, PW
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Ananthanarayanan, V
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Parton, RG
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Ariotti, NA
Abstract:
Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles otherwise positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.
23.
Plant Phytochrome Interactions Decode Light and Temperature Signals.
Abstract:
Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications.
24.
ERK synchronizes embryonic cleavages in Drosophila.
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Yang, L
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Zhu, A
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Aman, JM
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Denberg, D
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Kilwein, MD
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Marmion, RA
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Johnson, ANT
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Veraksa, A
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Singh, M
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Wühr, M
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Shvartsman, SY
Abstract:
Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.
25.
Optogenetic control of kinesin-1, -2, -3 and dynein reveals their specific roles in vesicular transport.
Abstract:
Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.
