Qr: switch:"CRY2/CRY2"
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1.
Astrocytic ankyrin-2 enables memory persistence in the mouse hippocampus.
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Kim, H
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Lim, J
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Kim, J
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Ozkan, E
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Kim, GH
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Park, H
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Park, MG
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Joo, B
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Lee, S
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Lee, KJ
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Kaang, BK
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Lee, CJ
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Koh, W
Abstract:
Memory persistence, the ability to retain information over time, is a fundamental feature of long-term memory. Although astrocytes contribute to synaptic plasticity, the molecular mechanisms by which they support memory persistence remain unclear. Here we show that astrocytic ankyrin-2 (Ank2) is required for memory persistence in adult mice. Astrocyte-specific deletion of Ank2 impaired remote memory without affecting recent memory and disrupted the maintenance of long-term potentiation. Loss of Ank2 reduced astrocyte contacts with engram neurons and impaired astrocyte morphogenesis driven by brain-derived neurotrophic factor (BDNF) signaling through the truncated tropomyosin receptor kinase B receptor (TrkB.T1) and inositol 1,4,5-trisphosphate receptor type 2 (IP3R2). Consistent with this mechanism, astrocytic Ank2 was required for the enhancement of memory persistence by hippocampal BDNF infusion. Furthermore, selective optogenetic activation of astrocytic TrkB.T1 signaling enhanced remote memory, demonstrating that astrocytic BDNF signaling is sufficient to promote memory persistence. These findings identify astrocytic Ank2 as a key regulator of long-term memory persistence.
2.
Linear ubiquitin chain assembly complex contributes to NLRP3-mediated pyroptotic cell death.
Abstract:
Activation of the NLRP3 inflammasome by infectious or sterile insults culminates in pyroptosis, a lytic and highly inflammatory form of programmed cell death. A safeguarded two-step process tightly regulates pyroptosis: priming, which drives NF-κB signaling, followed by execution, ultimately leading to plasma membrane rupture. Linear (Met1-linked) ubiquitination, catalyzed by the E3 ligase complex LUBAC, was previously shown to participate in pyroptosis, but the underlying mechanisms are not fully understood. In this study, we show that Met1-linked ubiquitin chains can assemble during both priming and execution phases, independently of the inflammasome sensor NLRP3. Genetic deletion of the LUBAC enzymes or pharmacological inhibition impairs pyroptosis. Conversely, cell death is enhanced without the deubiquitinase OTULIN, which selectively removes linear ubiquitination. Finally, using an optogenetic model to bypass priming, we demonstrate that Met-1-linked ubiquitination is required for the execution phase of pyroptosis. These findings offer insights into the regulation of pyroptotic cell death by linear ubiquitination.
3.
Engineering of genetically encoded programmable calcium channel inhibitory binders.
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Liu, X
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Ali, S
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Lan, TH
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Wang, D
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McKee, B
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Nonomura, T
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Liu, S
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Zhao, F
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Zhu, MX
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Huang, Y
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Deng, Q
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Ma, G
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Zhou, Y
Abstract:
Store-operated Ca2+ release-activated Ca2+ (CRAC) channels, composed of STIM and ORAI, are essential for immune and developmental processes, and their dysregulation underlies channelopathies such as Stormorken syndrome. Here, we report the engineering of genetically encoded CRAC channel inhibitory binders (CRABs) derived from the ORAI C-terminal tail. Guided by deep mutational scanning, we optimize a membrane-anchored CRAB variant that potently inhibits Ca2+ influx and NFAT signaling, and rescues thrombocytopenia-like phenotypes in a zebrafish model of Stormorken syndrome. To enable tunable inhibition, we further design oligomeric, optogenetic (Opto-CRAB), and chemogenetic (Chemo-CRAB) variants, providing graded and real-time control of CRAC activity. Chemo-CRAB further suppresses Ca2+ signaling downstream of RTKs, GPCRs, and CAR-T cell activation, establishing broad applicability across physiological and synthetic contexts. Together, these programmable peptide-based inhibitors provide a versatile platform to dissect SOCE dynamics and hold promise as a therapeutic strategy against autoimmune, inflammatory, and neoplastic disorders driven by CRAC channel hyperactivity.
4.
Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling.
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Prahl, LS
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Canlla, R
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Huang, AZ
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Alber, DS
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Shefter, SL
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Davis, SN
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Grindel, SH
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Huang, ZD
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Mumford, TR
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Benman, W
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Bugaj, LJ
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McCracken, KW
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Hughes, AJ
Abstract:
The mammalian kidney relies on a branched network of collecting ducts for fluid transport and homeostasis. Replicating this network in vitro would parallelize function in synthetic replacement kidneys, yet current organoids have limited branching capacity. Here, we establish a developmentally-informed strategy to control organoid budding through optogenetic control of a receptor tyrosine kinase, RET. We first show pharmacological manipulation of RET signaling controls the extent of branching in mouse embryonic kidneys and human stem cell-derived kidney organoids. Next, we develop an optogenetic RET receptor (optoRET) that signals in a ligand-independent manner via blue light-mediated clustering. Epithelial cells expressing optoRET reproduce stereotyped RET signaling, scattering, and symmetry breaking in response to blue light. Human kidney organoids undergo budding with controllable orientation in response to spatially patterned optoRET stimulation. Our results establish ligand-free optogenetic control of branching and inspire new synthetic biology strategies for epithelial organoid design.
5.
Optimizing information transmission in optogenetic Wnt signaling.
Abstract:
Populations of cells regulate gene expression in response to external signals, but their ability to make reliable collective decisions is limited by both intrinsic noise in molecular signaling and variability between individual cells. In this work, we use optogenetic control of the canonical Wnt pathway as an example to study how reliably information about an external signal is transmitted to a population of cells, and determine an optimal encoding strategy to maximize information transmission from Wnt signals to gene expression. We find that it is possible to reach an information capacity beyond 1 bit only through an appropriate, discrete encoding of signals: using no Wnt, a short Wnt pulse, or a sustained Wnt signal. By averaging over an increasing number of outputs, we systematically vary the effective noise in the pathway. As the effective noise decreases, the optimal encoding comprises more discrete input signals. These signals do not need to be fine-tuned to achieve near-optimal information transmission. The optimal code transitions into a continuous code in the small-noise limit, which can be shown to be consistent with the Jeffreys prior. We visualize the performance of different signal encodings using decoding maps. Our results suggest that optogenetic Wnt signaling allows for regulatory control beyond a simple binary switch and provide a framework to apply ideas from information processing to single-cell in vitro experiments.
6.
Optogenetic WNT signaling drives germ layer self-organization in a human gastruloid model.
Abstract:
In vitro stem cell models of human gastrulation have been an advance for developmental biology, though elucidating mechanisms of germ layer formation remains challenging. While investigating whether spatially-patterned signaling is required for germ layer formation, we tested a "salt-and-pepper" signaling strategy in which WNT was optogenetically activated in a subset of human pluripotent stem cells (hPSC) uniformly mixed into an aggregate. Following mesendodermal specification, WNT-activated cells spatially segregated into a hemisphere, then underwent further differentiation and organization into mesoderm and endoderm. RNAseq-based lineage analysis revealed that WNT activation non-autonomously induced TGFβ/BMP signaling, leading to robust emergence of an anterior visceral endoderm-like population that patterned adjacent neural and mesendodermal fates. Transcriptional profiles and trajectories closely mirrored those observed during human gastrulation. Moreover, TGFβ or cadherin perturbation disrupted germ layer formation or spatial organization, respectively. This simple model thus enables mechanistic dissection of complex human lineage specifications and organization during gastrulation.
7.
p62/SQSTM1 Condensation Modulates Mitochondrial Clustering to Participate in Mitochondrial Quality Control.
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Sun, S
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Xin, J
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Zhang, Y
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Yang, B
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Su, D
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Ni, R
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Ma, Q
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Li, N
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Ma, G
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Peng, Q
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Chen, S
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Prehn, JHM
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Tam, KY
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Wang, H
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Ying, Z
Abstract:
Mitochondrial quality control is tightly associated with aging-related neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Previous studies reported that ALS/FTD-associated protein p62 drives "mitochondrial clustering" (perinuclear clustering of fragmented and swollen mitochondria) during PINK1/Parkin-mediated mitophagy, but the underlying molecular mechanism, especially the precise role of p62 in mitochondrial clustering during mitophagy and the potential relationship between the mitochondrial quality control mediated by p62 and disease pathogenesis of ALS/FTD, remains unclear. Here, using cell biology in combination with an optogenetic tool, we show that the phase separation (condensation) of p62 mediates the clustering of damaged mitochondria to form "grape-like" clusters during PINK1/Parkin-mediated mitophagy, which is tightly associated with aging-related neurodegenerative diseases. In addition, our data suggest this mitochondrial clustering process is an arrest mechanism driven by p62 condensation (beyond the function of other autophagy receptors in mitophagy), which acts as a "brake" to reduce the surface area of dysfunctional mitochondria within cytoplasm for minimizing mitochondrial turnover in cells. Moreover, ALS/FTD-related pathological mutations perturb p62 condensation, thereby inhibiting mitochondrial clustering and destroying the "brake" machinery of mitochondrial quality control. Together, our data highlight how p62 condensation modulates organelle quality control in cell biology, and the important role of p62 condensation in both physiology and pathology.
8.
Optogenetic Translocation to Subcellular Compartments through Regulation of Protein Avidity.
Abstract:
Inducible translocation to subcellular compartments is a common strategy for protein switches that control a variety of cell behaviors. However, existing switches achieve translocation through induced dimerization, requiring constitutive anchoring of one component into the target compartment and optimization of relative expression levels between the two components. We present a simpler, single-component strategy called Avidity-assisted targeting (Aviatar). Aviatar achieves translocation with only a single protein by converting low-affinity monomers into high-avidity assemblies through inducible clustering. We demonstrated the Aviatar concept and its generality using optogenetic clustering to drive translocation to the plasma membrane, endosomes, golgi, endoplasmic reticulum, and microtubules using binding domains for lipids or endogenous proteins that were specific to those compartments. Aviatar recruitment regulated actin polymerization at the cell periphery and revealed compartment-specific signaling of receptor tyrosine kinase fusions associated with cancer. Finally, GFP-targeting Aviatar probes allowed inducible localization to any GFP-tagged target, including endogenously tagged stress granule proteins. Aviatar is a straightforward platform that can be rapidly adapted to a broad array of targets without the need for their prior modification or disruption.
9.
Anti-resonance in developmental signaling regulates cell fate decisions.
Abstract:
Cells process dynamic signaling inputs to regulate fate decisions during development. While oscillations or waves in key developmental pathways, such as Wnt, have been widely observed, the principles governing how cells decode these signals remain unclear. By leveraging optogenetic control of the Wnt signaling pathway in both HEK293T cells and H9 human embryonic stem cells, we systematically map the relationship between signal frequency and downstream pathway activation. We find that cells exhibit a minimal response to Wnt at certain frequencies, a behavior we term anti-resonance. We developed both detailed biochemical and simplified hidden variable models that explain how anti-resonance emerges from the interplay between fast and slow pathway dynamics. Remarkably, we find that frequency directly influences cell fate decisions involved in human gastrulation; signals delivered at anti-resonant frequencies result in dramatically reduced mesoderm differentiation. Our work reveals a previously unknown mechanism of how cells decode dynamic signals and how anti-resonance may filter against spurious activation. These findings establish new insights into how cells decode dynamic signals with implications for tissue engineering, regenerative medicine, and cancer biology.
10.
The membrane transition strongly enhances biopolymer condensation through prewetting.
Abstract:
Biopolymers that separate into condensed and dilute phases in solution also prewet membranes when one or more components couple to membrane lipids. Here we demonstrate that this prewetting transition becomes exquisitely sensitive to lipid composition when membranes have compositions near the boundary of liquid-ordered/liquid-disordered phase coexistence in both simulation and in reconstitution when polyelectrolytes are coupled to model membranes. In cells, we use an optogenetic tool to characterize prewetting at both the plasma membrane (PM) and the endoplasmic reticulum (ER) and find that prewetting is potentiated or inhibited by perturbations of membrane composition. Prewetting can also mediate membrane adhesion, with avidity dependent on membrane composition, as demonstrated in cells through the potentiation or inhibition of ER-PM contact sites. The strong correspondence of results in simulation, reconstitution and cells reveals a new role for membrane lipids in regulating the recruitment and assembly of soluble proteins.
11.
The G3BP stress-granule proteins reinforce the integrated stress response translation programme.
Abstract:
When mammalian cells are exposed to stress, they co-ordinate the condensation of stress granules (SGs) through the action of proteins G3BP1 and G3BP2 (G3BPs) and, simultaneously, undergo a massive reduction in translation. Although SGs and G3BPs have been linked to this translation response, their overall impact has been unclear. Here we investigate the question of how, and indeed whether, G3BPs and SGs shape the stress translation response. We find that SGs are enriched for mRNAs that are resistant to the stress-induced translation shutdown. Although the accurate recruitment of these stress-resistant mRNAs does require the context of stress, a combination of optogenetic tools and spike-normalized ribosome profiling demonstrates that G3BPs and SGs are necessary and sufficient to both help prioritize the translation of their enriched mRNAs and help suppress cytosolic translation. Together, these results support a model in which G3BPs and SGs reinforce the stress translation programme by prioritizing the translation of their resident mRNAs.
12.
Optogenetic control of biomolecular organization reveals distinct roles of phase separation in RTK signaling.
Abstract:
Multimerization and phase separation represent two paradigms for organizing receptor tyrosine kinases (RTKs). However, their functional distinctions from the perspective of biomolecular organization remain unclear. Here, we present CORdensate, a light-controllable condensation system combining two synergistic photoactuators: oligomeric Cry2 and heterodimeric LOVpep/ePDZ. Engineering single-chain photoswitches, we achieve four biomolecular organization patterns ranging from monomerization to phase separation. CORdensate exhibits constant assembly and disassembly kinetics. Applying CORdensate to mimic pathogenic RTK granules establishes the role of phase separation in activating ALK and RET. Moreover, assembling ALK and RET through varying organization patterns, we highlight the superior organizational ability of phase separation over multimerization. Additionally, CORdensate-based RTK granules suggest that phase separation broadly and robustly activates RTKs. This study introduces a optogenetic tool for investigating biomolecular condensation.
13.
FLASH-AWAY: Intrabody-Directed Targeting of Optogenetic Tools for Protein Degradation.
Abstract:
Protein homeostasis, or proteostasis, is essential for cellular proteins to function properly. The buildup of abnormal proteins (such as damaged, misfolded, or aggregated proteins) is associated with many diseases, including cancer. Therefore, maintaining proteostasis is critical for cellular health. Currently, genetic methods for modulating proteostasis, such as RNA interference and CRISPR knockout, lack spatial and temporal precision. They are also not suitable for depleting already-synthesized proteins. Similarly, molecular tools like PROTACs and molecular glue face challenges in drug design and discovery. To directly control targeted protein degradation within cells, we introduce an intrabody-based optogenetic toolbox named Flash-Away. Flash-Away integrates the light-responsive ubiquitination activity of the RING domain of TRIM21 for protein degradation, coupled with specific intrabodies for precise targeting. Upon exposure to blue light, Flash-Away enables rapid and targeted degradation of selected proteins. This versatility is demonstrated through successful application to diverse protein targets, including actin, MLKL, and ALFA-tag fused proteins. This innovative light-inducible protein degradation system offers a powerful approach to investigate the functions of specific proteins within physiological contexts. Moreover, Flash-Away presents potential opportunities for clinical translational research and precise medical interventions, advancing the prospects of precision medicine.
14.
Quantifying cancer- and drug-induced changes in Shannon information capacity of RTK signaling.
Abstract:
Cancer can result from abnormal regulation of cells by their environment, potentially because cancer cells may misperceive environmental cues. However, the magnitude to which the oncogenic state alters cellular information processing has not been quantified. Here, we apply pseudorandom pulsatile optogenetic stimulation, live-cell imaging, and information theory to compare the information capacity of receptor tyrosine kinase (RTK) signaling pathways in EML4-ALK-driven lung cancer (STE-1) and in non-transformed (BEAS-2B) cells. The average information rate through RTK/ERK signaling in STE-1 cells was less than 0.5 bit/hour, compared to 7 bit/hour in BEAS-2B cells, but increased to 3 bit/hour after oncogene inhibition. Information was transmitted by 50-70% of cells, whose channel capacity (maximum information rate) was estimated through in silico protocol optimization. In BEAS-2B cells, channel capacity of the parallel RTK/calcineurin pathway surpassed that of the RTK/ERK pathway. This study highlights information capacity as a sensitive metric for identifying disease-associated dysfunction and evaluating the effects of targeted interventions.
15.
Shining light on drug discovery: optogenetic screening for TopBP1 biomolecular condensate inhibitors.
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Morano, L
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Vie, N
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Aissanou, A
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Hodroj, D
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Garambois, V
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Fauvre, A
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Promonet, A
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Egger, T
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Bordignon, B
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Hassen-Khodja, C
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Fiachetti, S
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Basbous, J
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Gongora, C
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Constantinou, A
Abstract:
Human topoisomerase IIβ binding protein 1 (TopBP1) is a scaffold protein involved in DNA replication initiation, DNA repair, transcription regulation, and checkpoint activation. TopBP1 forms nuclear condensates that act as a molecular switch to amplify ATR activity and promote the activation of the checkpoint effector kinase Chk1. In cancer cells, ATR activity is crucial to tolerate the intrinsically high level of DNA lesions and obstacles that block replication fork progression. Thus, ATR inhibitors are currently tested in clinical trials, often in combination with chemotherapy drugs. However, resistance and toxicity are still major issues. The weak interactions that hold TopBP1 condensates together are highly sensitive to changes in the cellular milieu, suggesting that small molecules may alter the formation of TopBP1 condensates. Here, we developed a high-throughput screening system to identify TopBP1 condensation modulators. This system allowed us to identify FDA-approved drugs, including thimerosal and quinacrine, that inhibit TopBP1 condensation and block the activation of ATR/Chk1 signaling. Mechanistically, quinacrine impaired TopBP1's ability to associate with chromatin, thereby interfering with its capacity to form condensates. Furthermore, quinacrine enhanced the therapeutic efficacy of 5-fluorouracil and irinotecan, components of the clinically used FOLFIRI regimen in a mouse model of peritoneal carcinomatosis from colorectal cancer.
16.
A single-component optogenetic toolkit for programmable control of microtubule.
Abstract:
Microtubules (MTs) form dynamic cytoskeletal scaffolds essential for intracellular transport, organelle positioning, and spatial organization of signaling. Their architecture and function are continuously remodeled through the concerted actions of microtubule-associated proteins (MAPs), post-translational modifications (PTMs), and molecular motors. To precisely interrogate these processes in living systems, we developed a genetically encoded optogenetic toolkit for spatiotemporal control of MT organization and dynamics. By replacing native multimerization motifs with a blue light-responsive oligoermization domain, we have engineered single-component probes, OptoMT and OptoTIP, that reversibly label MT polymers or track plus-ends with tunable kinetics from seconds to minutes. When coupled to enzymatic effectors, these modules enable localized tubulin acetylation or detyrosination, directly linking PTMs to MT stability. We further engineered OptoMotor, a light-activatable kinesin platform that reconstitutes tail-dependent cargo transport along MTs, and OptoSAW, a light-triggered severing actuator for controlled MT disassembly. Using these tools, we reveal how local MT integrity governs lysosomal trafficking and ER-associated signaling dynamics. Collectively, this versatile single-component toolkit bridges molecular design with cytoskeletal function, offering new avenues to illuminate how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.
17.
Modulating inter-mitochondrial contacts to increase membrane potential for mitigating blue light damage.
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Wang, Y
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Qui, K
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Zou, W
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Amom, P
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Ganjawala, TH
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Lee, E
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Tian, Z
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Xu, X
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Huang, T
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Tsai, NP
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Shi, D
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Kang, P
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Bai, H
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Zacharias, AL
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Zhang, K
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Diao, J
Abstract:
Mitochondrial membrane potential (MMP) is essential for mitochondrial functions, yet current methods for modulating MMP lack precise spatial and temporal control. Here, we present an optogenetic system that enables reversible formation of inter-mitochondrial contacts (mito-contacts) with high spatiotemporal precision. Blue light stimulation induces rapid formation of mito-contacts, which fully dissipate upon cessation of illumination. These light-induced mito-contacts can enhance MMP, leading to increased ATP production under stress conditions. Moreover, in human retinal cells and C. elegans, high MMP induced by mito-contacts alleviates the deleterious effects of prolonged blue light exposure, restoring energy metabolism and extending organismal lifespan. This optogenetic approach provides a powerful tool for modulating MMP and offers potential therapeutic applications for diseases linked to mitochondrial dysfunction.
18.
OptoBarrier: An Optogenetic Platform for Modulating Endothelial Barriers In Vitro.
Abstract:
Organ-on-a-chip platforms have emerged as promising human tissue models for drug screening and mechanistic studies, offering alternatives to traditional animal models. Integration of vascular structures into these platforms is pivotal for creating physiologically faithful models of individual organs and studying interorgan crosstalk. However, most vascular structures grown in vitro do not account for organ-specific endothelial permeability or its modulation in response to disease. Here, we present optoBarrier, an optogenetic organ-on-a-chip platform designed to modulate endothelial barrier permeability through light stimulation. By optically activating RhoA signaling in engineered optogenetic endothelial cells, we demonstrate the formation of stress fibers, disruption of vascular endothelial cadherin (VE-cadherin) and increased barrier permeability. We further show that permeability is tunable in a reversible and dose-dependent manner in response to light. We therefore propose that optoBarrier offers a user-defined, controlled and simple method to manipulate endothelial permeability for in vitro studies of human vasculature.
19.
Activation of NF-κB Signaling by Optogenetic Clustering of IKKα and β.
Abstract:
Molecular optogenetics allows the control of molecular signaling pathways in response to light. This enables the analysis of the kinetics of signal activation and propagation in a spatially and temporally resolved manner. A key strategy for such control is the light-inducible clustering of signaling molecules, which leads to their activation and subsequent downstream signaling. In this work, an optogenetic approach is developed for inducing graded clustering of different proteins that are fused to eGFP, a widely used protein tag. To this aim, an eGFP-specific nanobody is fused to Cryptochrome 2 variants engineered for different orders of cluster formation. This is exemplified by clustering eGFP-IKKα and eGFP-IKKβ, thereby achieving potent and reversible activation of NF-κB signaling. It is demonstrated that this approach can activate downstream signaling via the endogenous NF-κB pathway and is thereby capable of activating both an NF-κB-responsive reporter construct as well as endogenous NF-κB-responsive target genes as analyzed by RNA sequencing. The generic design of this system is likely transferable to other signaling pathways to analyze the kinetics of signal activation and propagation.
20.
Optogenetic engineering of lipid droplet spatial organization for tumor suppression.
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Bai, Q
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Shao, X
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Xia, Q
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Yang, S
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Gao, Y
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Sun, K
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Li, J
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Wang, X
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Tian, Z
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Chen, X
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Zhao, J
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Diao, J
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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.
21.
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.
22.
KIF2C condensation concentrates PLK1 and phosphorylated BRCA2 on kinetochore microtubules in mitosis.
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Skobelkina , A
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Julien, M
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Jeannin, S
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Miron, S
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Egger, T
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Chaaban, R
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Bouvignies, G
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Alghoul, E
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Ghouil, R
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Friel, C
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Busso, D
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Cañas, JC
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Theillet, FX
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Le Bars, R
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Carreira, A
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Constantinou, A
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Basbous, J
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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.
23.
β-Arrestin Condensates Regulate G Protein-Coupled Receptor Function.
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Anderson, PJ
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Xiao, P
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Zhong, Y
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Kaakati, A
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Alfonso-DeSouza, J
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Zhang, T
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Zhang, C
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Yu, K
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Qi, L
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Ding, W
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Liu, S
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Pani, B
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Krishnan, A
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Chen, O
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Jassal, C
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Strawn, J
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Sun, JP
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Rajagopal, S
Abstract:
G protein-coupled receptors (GPCRs) are the largest class of receptors in the genome and control many signaling cascades essential for survival. GPCR signaling is regulated by β-arrestins, multifunctional adapter proteins that direct receptor desensitization, internalization, and signaling. While at many GPCRs, β-arrestins interact with a wide array of signaling effectors, it is unclear how β-arrestins promote such varied functions. Here we show that β-arrestins undergo liquid-liquid phase separation (LLPS) to form condensates that regulate GPCR function. We demonstrate that β-arrestin oligomerization occurs in proximity to the GPCR and regulates GPCR functions such as internalization and signaling. This model is supported by a cryoEM structure of the adhesion receptor ADGRE1 in a 2:2 complex with β-arrestin 1, with a β-arrestin orientation that can promote oligomerization. Our work provides a paradigm for β-arrestin condensates as regulators of GPCR function, with LLPS serving as an important promoter of signaling compartmentalization at GPCRs.
24.
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.
25.
Optogenetically Activatable MLKL as a Standalone Functional Module for Necroptosis and Therapeutic Applications in Antitumoral Immunity.
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Jeong, DH
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Kim, S
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Park, HH
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Woo, KJ
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Choi, JI
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Choi, M
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Shin, J
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Park, SH
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Seon, MW
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Lee, D
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Cha, JH
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Kim, YS
Abstract:
Necroptosis plays a crucial role in the progression of various diseases and has gained substantial attention for its potential to activate antitumor immunity. However, the complex signaling networks that regulate necroptosis have made it challenging to fully understand its mechanisms and translate this knowledge into therapeutic applications. To address these challenges, an optogenetically activatable necroptosis system is developed that allows for precise spatiotemporal control of key necroptosis regulators, bypassing complex upstream signaling processes. The system, specifically featuring optoMLKL, demonstrates that it can rapidly assemble into functional higher-order "hotspots" within cellular membrane compartments, independent of RIPK3-mediated phosphorylation. Moreover, the functional module of optoMLKL significantly enhances innate immune responses by promoting the release of iDAMPs and cDAMPs, which are critical for initiating antitumor immunity. Furthermore, optoMLKL exhibits antitumor effects when activated in patient-derived pancreatic cancer organoids, highlighting its potential for clinical application. These findings will pave the way for innovative cancer therapies by leveraging optogenetic approaches to precisely control and enhance necroptosis.
