Qr: *
Showing 76 - 100 of 1884 results
76.
Optogenetic-Controlled iPSC-Based Vaccines for Prophylactic and Therapeutic Tumor Suppression in Mice.
-
Qiao, L
-
Niu, L
-
Wang, Z
-
Dai, D
-
Tang, S
-
Ma, X
-
Deng, Z
-
Yu, G
-
Zhou, Y
-
Yan, T
-
Liu, X
-
Kong, D
-
Hu, L
-
Li, X
-
Zhao, J
-
Cai, F
-
Wang, M
-
Ye, H
Abstract:
Induced pluripotent stem cells (iPSCs) share similar cellular features and various antigens profiles with cancer cells. Leveraging these characteristics, iPSCs hold great promise for developing wide-spectrum vaccines against cancers. In practice, iPSCs are typically combined with immune adjuvants to enhance antitumor immune responses; however, traditional adjuvants lack controllability and can induce systemic toxicity, which has limited their broad application. Here, a red/far-red light-controlled iPSC-based vaccine (RIVA) based on the chimeric photosensory protein FnBphP and its interaction partner LDB3 is developed; RIVA preserves the intrinsic tumor antigens of iPSCs and enables optogenetic control of an immune adjuvant's (IFN-β) expression under red light illumination. Experiments in multiple mouse tumor models demonstrate that RIVA inhibits tumor growth and improves animal survival in prophylactic and therapeutic settings, including against pulmonary metastatic 4T1 breast cancer. RIVA efficiently stimulates dendritic cell maturation, eliciting innate immune activation effects through NK cells and elicit adaptive immune anti-tumor responses through CD4+ and CD8+ T cells. Moreover, RIVA protects animals against tumor re-challenge by inducing strong immunological memory, with minimal systemic toxicity. This study demonstrates RIVA as an effective optogenetic approach for developing safe multi-antigen vaccines for the prevention and treatment of cancer.
77.
Engineered bacteriophytochrome heterodimers for research and applications.
Abstract:
Many proteins are dimeric, functioning as complexes of two identical or different subunits. Bacteriophytochromes are homodimeric photoreceptor proteins that sense red/far-red light with a photosensory module (PSM) and convert it to a biological response via an output module, usually a histidine kinase (HK). Here, we generate monomeric bacteriophytochrome PSMs that form stable heterodimers once mixed by modifying two salt bridges at the dimerization interface of the Deinococcus radiodurans phytochrome (DrBphP). We confirm that these heterodimeric PSMs can control output HK module activity in response to red light and reveal that dimerization is required for kinase activity of the model HK FixL, but not necessarily for phosphatase activity of DrBphP. By applying the heterodimeric variants to a red light-regulated gene expression tool, we exemplify the combined control of cellular events using both heterodimerization and light. These results pave the way for new heterodimeric systems, for example, in receptor protein research and optogenetics.
78.
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.
79.
A simplified two-plasmid system for orthogonal control of mammalian gene expression using light-activated CRISPR effector.
Abstract:
Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.
80.
Optogenetic engineering of lipid droplet spatial organization for tumor suppression.
-
Bai, Q
-
Shao, X
-
Xia, Q
-
Yang, S
-
Gao, Y
-
Sun, K
-
Li, J
-
Wang, X
-
Tian, Z
-
Chen, X
-
Zhao, J
-
Diao, J
-
Chen, Q
Abstract:
In cancer cells, lipid droplets (LDs) establish extensive membrane contact sites (MCSs) with mitochondria to facilitate fatty acid transfer and sustain energy production, thus enabling cancer cell survival, in nutrient-deprived tumor microenvironments. However, effective strategies to disrupt these LD-mitochondria interactions remain unavailable. We engineered an optogenetic system to control LD intracellular organization through clustering. Upon blue light stimulation, the system induces LDs to undergo spatial reorganization and form clusters, thereby restricting LD accessibility by reducing the available surface area for mitochondrial interaction. Consequently, this clustering significantly diminishes the number of LD-mitochondria MCSs, suppresses fatty acid transport from LDs to mitochondria during starvation, and ultimately leads to cancer cell death in vitro and tumor growth inhibition in vivo. Collectively, our results demonstrate that optogenetically controlled LD clustering offers a novel approach to impede tumor progression by blocking nutrient flow from LDs to mitochondria.
81.
Stimulation of corticospinal neurons by optogenetic cAMP inductions promotes motor recovery after spinal cord injury in female rats via raphespinal tract modulation.
-
Martínez-Rojas, B
-
Martín-Pérez, S
-
Giraldo, E
-
Lopez-Mocholi, E
-
Alastrue, A
-
Andrade-Talavera, Y
-
Prius-Mengual, J
-
Paniagua, G
-
Pedraza, M
-
Hingorani, S
-
Rost, BR
-
Schmitz, D
-
Llansola, M
-
Felipo, V
-
Rodríguez-Moreno, A
-
Moreno-Manzano, V
Abstract:
After spinal cord injury (SCI), cyclic adenosine monophosphate (cAMP) levels drop in the spinal cord, cortex and brainstem, unlike in regenerating peripheral neurons. To address SCI recovery, we expressed photoactivatable adenylate cyclase (bPAC) in corticospinal neurons of female rats with dorsal hemisection for on-demand cAMP inductions. bPAC stimulation restored passive and firing properties of corticospinal neurons, promoted early and sustained locomotor recovery and increased corticospinal tract plasticity. Additionally, bPAC enhanced sparing of lumbar-projecting brainstem neurons after SCI, accompanied by activation of cAMP signaling in the raphe-reticular formation and increased excitatory/inhibitory neurotransmitter balance. Accordingly, augmented density of serotonergic tracts was found caudal to the injury in bPAC rats, correlating with enhanced functional performance. Serotonergic implication in motor recovery was further evidenced by selective depletion, resulting in the abrogation of bPAC-mediated recovery. Overall, our findings underscore that cAMP induction in corticospinal neurons enhances locomotion after SCI, through a cortical rerouting pathway via the serotonergic descending tract.
82.
Pharmacological interventions on GSK3β phosphorylation-mediated tau aggregation by modulating phase separation of tau proline-rich domain.
Abstract:
Tau pathological aggregation in neurofibrillary tangles is a hallmark of several neurodegenerative diseases, including Alzheimer's disease. Phase separation is a thermodynamic process that plays an important role in biomolecular membrane-less condensate formation, while abnormal phase separation of tau leads to pathological aggregate formation. However, the detailed molecular mechanism underlying tau condensation remains not fully understood. Moreover, whether condensation-based pharmacological intervention will be helpful for the treatment of tau-associated neurodegenerative diseases remains elusive. Here, we used an optogenetic tool (optoDroplets) in combination with cell biology and pharmacology to explore the contribution of different domains for tau condensation in cells, and we found that proline-rich domain (PRD) phosphorylation, which is mainly regulated by glycogen synthase kinase 3 β (GSK3β), plays important roles for tau condensation. Moreover, phosphorylation of tau PRD regulates its mis-localization on nuclear speckle. Interestingly and importantly, we found that pharmacological inhibition of GSK3β can impede abnormal tau condensation to slow down the tau-associated pathological process.
83.
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.
84.
Programmable genome engineering and gene modifications for plant biodesign.
-
Liu, J
-
Zhang, R
-
Chai, N
-
Su, L
-
Zheng, Z
-
Liu, T
-
Guo, Z
-
Ma, Y
-
Xie, Y
-
Xie, X
-
Lin, Q
-
Chen, L
-
Liu, YG
-
Zhu, Q
Abstract:
Plant science has entered a transformative era as genome editing enables precise DNA modifications to address global challenges such as climate adaptation and food security. These modifications are primarily driven by the integration of three modular components-DNA-targeting modules, effector modules, and control modules-that can be selectively activated or suppressed. The field has evolved from protein-based systems (e.g., zinc finger nucleases and transcription activator-like effector nucleases) to RNA-guided systems (e.g., CRISPR-Cas) that can control both genetic and epigenetic states. Modular pairing of DNA-targeting and effector domains, with or without inducible control, enables precise transcriptional regulation and chromatin remodeling. The present review examines these three modules and highlights strategies for their optimization. It also outlines innovative tools, such as optogenetic and receptor-integrated systems, that enable spatiotemporal control over genome editor expression. These modular approaches bypass traditional limitations and allow scientists to create plants with desirable traits, decipher complex gene networks, and promote sustainable agriculture.
85.
Optogenetics to biomolecular phase separation in neurodegenerative diseases.
Abstract:
Neurodegenerative diseases involve toxic protein aggregation. Recent evidence suggests that biomolecular phase separation, a process in which proteins and nucleic acids form dynamic, liquid-like condensates, plays a key role in this aggregation. Optogenetics, originally developed to control neuronal activity with light, has emerged as a powerful tool to investigate phase separation in living systems. This is achieved by fusing disease-associated proteins to light-sensitive oligomerization domains, enabling researchers to induce or reverse condensate formation with precise spatial and temporal control. This review highlights how optogenetic systems such as OptoDroplet are being used to dissect the mechanisms of neurodegenerative disease. We examine how these tools have been applied in models of neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's disease. These studies implicate small oligomeric aggregates as key drivers of toxicity and highlight new opportunities for therapeutic screening. Finally, we discuss advances in light-controlled dissolution of condensates and future directions for applying optogenetics to combat neurodegeneration. By enabling precise, dynamic control of protein phase behavior in living systems, optogenetic approaches provide a powerful framework for elucidating disease mechanisms and informing the development of targeted therapies.
86.
Optogenetic perturbation of lipid droplet localization affects lipid metabolism and development in Drosophila.
Abstract:
Lipid droplets (LDs) are dynamic organelles crucial for lipid storage and homeostasis. Despite extensive documentation of their importance, the causal relationship between LD localization and function in health and disease remains inadequately understood. Here, we developed optogenetics-based tools, termed "Opto-LDs," which facilitate the interaction between LDs and motor proteins in a light-dependent manner, enabling precise control of LD localization within cells. Utilizing these optogenetic modules, we demonstrated that light-induced relocation of LDs to the periphery of hepatocytes results in elevated very-low-density lipoprotein (VLDL) secretion, recapturing the beneficial effect of insulin in vitro. Furthermore, our studies in transgenic Drosophila revealed that proper LD localization is critical for embryonic development, with mistargeting of LDs significantly affecting egg hatching success. In summary, our work underscores the great importance of LD localization in lipid metabolism and development, and our developed tools offer valuable insights into the functions of LDs in health and disease.
87.
Potent optogenetic regulation of gene expression in mammalian cells for bioproduction and basic research.
Abstract:
Precise temporal and spatial control of gene expression greatly benefits the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. Beyond basic research, this could also prove decisive for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells, as expression levels and the degree of induction upon light stimulation are insufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter litoralis to the three transcriptional activator domains VP64, p65, and Rta in tandem. The resultant photoswitch, dubbed DEL-VPR, allows up to a 570-fold induction of target gene expression by blue light, thereby achieving expression levels of strong constitutive promoters. Here, we used DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression and increased yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to academic and industrial settings.
88.
RhoA activation promotes ordered membrane domain coalescence and suppresses neuronal excitability.
Abstract:
This study explores how the small GTPase RhoA modulates plasma membrane lipid nanodomains, particularly cholesterol-rich ordered membrane domains (OMDs). These nanodomains play a critical role in regulating ion channel activity and neuronal excitability. However, due to their nanoscale dimensions, OMDs remain challenging to visualize using conventional light microscopy. Here, we used fluorescently labeled cholera toxin B (CTxB) and the palmitoylated peptide Lck-10 (L10) as probes to visualize OMDs and quantified their size via confocal fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET). Pharmacological inhibition of RhoA significantly reduced OMD sizes in both human cell lines and dorsal root ganglion (DRG) neurons. To achieve better spatiotemporal control of specific RhoA activation, we employed an improved light-inducible dimerization (iLID) system. Optogenetic activation of RhoA rapidly increased FRET efficiency between CTxB probes, indicating OMD coalescence. Functionally, RhoA inhibition potentiated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity in nociceptive DRG neurons, increasing spontaneous action potential firing. Conversely, in a spared nerve injury rat model, RhoA activation expanded OMDs in nociceptive DRG neurons. Constitutive RhoA activation suppressed HCN channel activity and decreased membrane excitability. These findings support a neuroprotective role for RhoA activation, where it restores OMD size and suppresses pathological hyperexcitability in neuropathic pain.
89.
An Optical Approach to Modulating Membrane Protein Endocytosis Using a Light-Responsive Tag for Recruiting β-Arrestin.
Abstract:
Membrane receptors, particularly G protein-coupled receptors (GPCRs), are integral to numerous physiological processes. Precise control of the receptor endocytosis is essential for understanding cellular signaling pathways. In this study, we present the development of a broadly applicable optogenetic tool for light-inducible receptor internalization. This system, named E-fragment, leverages the CRY2-CIB photodimerization pair to enable blue-light-dependent recruitment of β-arrestin and subsequent receptor internalization. We showed that the E-fragment system is applicable across diverse membrane proteins, including multiple GPCRs. Furthermore, we investigated its impact on intracellular cAMP signaling in cells expressing dopamine receptor D1 and α2-adrenergic receptor. Quantitative analyses revealed that light-induced internalization led to reduced surface receptor expression and attenuated ligand-evoked cAMP responses. These findings demonstrate the versatility of the E-fragment system as a platform for studying membrane receptor function and suggest potential applications in therapeutic strategies targeting receptor trafficking and signaling modulation.
90.
Improving the Response of Microbial Fuel Cell-Based Biosensing through Optogenetic Enhancement of Electroactive Biofilms.
Abstract:
Early detection of pollutants in water discharge is an integral part of environmental monitoring. Electroactive biofilm (EAB)-enabled, microbial fuel cell (MFC)-based biosensors facilitate self-powered online pollutant detection. However, as EABs are highly dynamic, naturally formed EABs as sensing and transducing elements limit the performance of MFC-based biosensors. Here, we report a fast-response and sensitive MFC-based biosensor enabled by enhancing Shewanella oneidensis biofilms on the electrode using an optogenetic approach. We incorporated a near-infrared (NIR) light-responsive synthetic bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) module into S. oneidensis to promote biofilm formation on the anode under NIR light. The biosensors with enhanced EABs exhibited a rapid and sensitive response to Cr(VI), reducing the sensing time from approximately 30 min to just 3 min. This improved sensing performance was maintained over three sensing cycles, even with fluctuating Cr(VI) concentrations. Based on the analyses of the electrode biofilms and extracellular polymeric substance matrices, different Cr(VI) response mechanisms for the normal and enhanced EABs were proposed; enhanced EAB's massive dispersal by Cr(VI) was the cause of the improved response of the biosensors. Such improved response still held in the natural water matrix. This proof-of-concept study provides valuable insights into controlling electrode biofilm dynamics for the rapid and robust early detection of pollutants using MFC-based biosensors.
91.
Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter.
Abstract:
The in vivo continuous evolution system OrthoRep (orthogonal replication) is a powerful strategy for rapid enzyme evolution in Saccharomyces cerevisiae that diversifies genes at a rate exceeding the endogenous genome mutagenesis rate by several orders of magnitude. However, it is difficult to neofunctionalize genes using OrthoRep partly because of the way selection pressures are applied. Here we combine OrthoRep with optogenetics in a selection strategy we call OptoRep, which allows fine-tuning of selection pressure with light. With this capability, we evolved a truncated form of the endogenous monocarboxylate transporter JEN1 (JEN1t) into a de novo mevalonate importer. We demonstrate the functionality of the evolved JEN1t (JEN1tY180C/G) in the production of farnesene, a renewable aviation biofuel, from mevalonate fed to fermentation media or produced by microbial consortia. This study shows that the light-induced complementation of OptoRep may improve the ability to evolve functions not currently accessible for selection, while its fine tunability of selection pressure may allow the continuous evolution of genes whose desired function has a restrictive range between providing effective selection and cellular viability.
92.
Nanobody-Based Light-Controllable Systems for Investigating Biology.
Abstract:
Nanobodies, the camelid-derived single-chain variable domain of heavy-chain-only antibodies, are compact in size and exhibit high binding affinity and specificity to their binding partners. As innovative antibody modalities, nanobodies have garnered significant attention in medicine and biological research. To achieve higher spatiotemporal precision, nanobody-based light-controlled systems-such as photobody, optobody, photoactivatable nanobody conjugate inducers of dimerization, and others-have been developed. These systems enable optical control of biological processes while leveraging the advantages of nanobodies as a binding moiety. This concept, summarizes nanobody-based photoregulated systems for investigating biology through light, highlights their advantages and potential limitations, and discusses future directions in this emerging research area.
93.
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.
94.
Membranes arrest the coarsening of mitochondrial condensates.
Abstract:
Mitochondria contain double membranes that enclose their contents. Within their interior, the mitochondrial genome and its RNA products are condensed into ∼100 nm sized (ribo)nucleoprotein complexes. How these endogenous condensates maintain their roughly uniform size and spatial distributions within membranous mitochondria remains unclear. Here, we engineered an optogenetic tool (mt-optoIDR) that allowed for controlled formation of synthetic condensates upon light activation in live mitochondria. Using live cell super-resolution microscopy, we visualized the nucleation of small, yet elongated condensates (mt-opto-condensates), which recapitulated the morphologies of endogenous mitochondrial condensates. We decoupled the contribution of the double membranes from the environment within the matrix by overexpressing the dominant negative mutant of a membrane fusion protein (Drp1K38A). The resulting bulbous mitochondria had significantly more dynamic condensates that coarsened into a single, prominent droplet. These observations inform how mitochondrial membranes can limit the growth and dynamics of the condensates they enclose, without the need of additional regulatory mechanisms.
95.
Single-cell characterization of bacterial optogenetic Cre recombinases.
Abstract:
Microbial optogenetic tools can regulate gene expression with high spatial and temporal precision, offering excellent potential for single-cell resolution studies. However, bacterial optogenetic systems have primarily been deployed for population-level experiments. It is not always clear how these tools perform in single cells, where stochastic effects can be substantial. In this study, we focus on optogenetic Cre recombinase and systematically compare the performance of three variants (OptoCre-REDMAP, OptoCre-Vvd, and PA-Cre) for their population-level and single-cell activity. We quantify recombination efficiency, expression variability, and activation dynamics using reporters which produce changes in fluorescence or antibiotic resistance following light-induced Cre activity. Our results indicate that optogenetic recombinase performance can be reporter-dependent, suggesting that this is an important consideration in system design. Further, our single-cell analysis reveals highly heterogeneous activity across cells. Although general trends match expectations for mean levels of light-dependent recombination, we found substantial variation in this behavior across individual cells. In addition, our results show that the timing of recombinase activity is highly variable from cell to cell. These findings suggest critical criteria for selecting appropriate optogenetic recombinase systems and indicate areas for optimization to improve the single-cell capabilities of bacterial optogenetic tools.
96.
The pioneer transcription factor Zelda controls the exit from regeneration and restoration of patterning in Drosophila.
Abstract:
Many animals can regenerate tissues after injury. While the initiation of regeneration has been studied extensively, how the damage response ends and normal gene expression returns is unclear. We found that in Drosophila wing imaginal discs, the pioneer transcription factor Zelda controls the exit from regeneration and return to normal gene expression. Optogenetic inactivation of Zelda during regeneration disrupted patterning, induced cell fate errors, and caused morphological defects yet had no effect on normal wing development. Using Cleavage Under Targets & Release Using Nuclease, we identified targets of Zelda important for the end of regeneration, including genes that control wing margin and vein specification, compartment identity, and cell adhesion. We also found that GAGA factor and Fork head similarly coordinate patterning after regeneration and that chromatin regions bound by Zelda increase in accessibility during regeneration. Thus, Zelda orchestrates the transition from regeneration to normal gene expression, highlighting a fundamental difference between developmental and regeneration patterning in the wing disc.
97.
KIF2C condensation concentrates PLK1 and phosphorylated BRCA2 on kinetochore microtubules in mitosis.
-
Skobelkina , A
-
Julien, M
-
Jeannin, S
-
Miron, S
-
Egger, T
-
Chaaban, R
-
Bouvignies, G
-
Alghoul, E
-
Ghouil, R
-
Friel, C
-
Busso, D
-
Cañas, JC
-
Theillet, FX
-
Le Bars, R
-
Carreira, A
-
Constantinou, A
-
Basbous, J
-
Zinn-Justin, S
Abstract:
During mitosis, the microtubule depolymerase KIF2C, the tumor suppressor BRCA2, and the kinase PLK1 contribute to the control of kinetochore-microtubule attachments. Both KIF2C and BRCA2 are phosphorylated by PLK1, and BRCA2 phosphorylated at T207 (BRCA2-pT207) serves as a docking site for PLK1. Reducing this interaction results in unstable microtubule-kinetochore attachments. Here we identified that KIF2C also directly interacts with BRCA2-pT207. Indeed, the N-terminal domain of KIF2C adopts a Tudor/PWWP/MBT fold that unexpectedly binds to phosphorylated motifs. Using an optogenetic platform, we found that KIF2C forms membrane-less organelles that assemble through interactions mediated by this phospho-binding domain. KIF2C condensation does not depend on BRCA2-pT207 but requires active Aurora B and PLK1 kinases. Moreover, it concentrates PLK1 and BRCA2-pT207 in an Aurora B-dependent manner. Finally, KIF2C depolymerase activity promotes the formation of KIF2C condensates, but strikingly, KIF2C condensates exclude tubulin: they are located on microtubules, especially at their extremities. Altogether, our results suggest that, during the attachment of kinetochores to microtubules, the assembly of KIF2C condensates amplifies PLK1 and KIF2C catalytic activities and spatially concentrates BRCA2-pT207 at the extremities of microtubules. We propose that this novel and highly regulated mechanism contributes to the control of microtubule-kinetochore attachments, chromosome alignment, and stability.
98.
Combining light-induced aggregation and biotin proximity labeling implicates endolysosomal proteins in early α-synuclein oligomerization.
Abstract:
Alpha-synuclein (α-syn) aggregation is a defining feature of Parkinson's disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding α-syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the light-inducible protein aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify α-syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 α-syn-interacting proteins, including both established and previously unreported candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of α-syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.
99.
Light-Driven Enzyme Catalysis: Ultrafast Mechanisms and Biochemical Implications.
Abstract:
Light-activated enzymes are an important class of biocatalysts in which light energy is directly converted into biochemical activity. In most cases the light absorbing group is the isoalloxazine ring of an embedded flavin cofactor and in general two types of mechanism are in operation depending on whether the excited chromophore directly participates in catalysis or where photoexcitation triggers conformational changes that modulate the activity of a downstream output partner. This review will summarize studies on DNA photolyase, fatty acid photodecarboxylase (FAP), the monooxygenase PqsL, and flavin-dependent ene-reductases, where flavin radicals generated by excitation are directly used in the reactions catalyzed by these enzymes, and the blue light using FAD (BLUF) and light oxygen voltage (LOV) domain photoreceptors where flavin excitation drives ultrafast structural changes that ultimately result in enzyme activation. Recent advances in methods such as time-resolved spectroscopy and structural imaging have enabled unprecedented insight into the ultrafast dynamics that underly the mechanism of light-activated enzymes, and here we highlight how understanding ultrafast protein dynamics not only provides valuable insights into natural phototransduction processes but also opens new avenues for enzyme engineering and consequent applications in fields such as optogenetics.
100.
Balancing Doses of EL222 and Light Improves Optogenetic Induction of Protein Production in Komagataella phaffii.
Abstract:
Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab-scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol-inducible bioprocesses using K. phaffii.
