Qr: journal:"Cell"
Showing 1 - 25 of 253 results
1.
Delineation of signaling microdomains from live-cell movies.
Abstract:
Many molecular signals are known to be organized in subcellular regions termed microdomains. One example entails Rho GTPases, which control a range of cell behaviors through their functional patterning in spatiotemporal units. While fluorescent biosensors have allowed precise measurement of Rho GTPase activity in living cells, computational tools to delineate and track signaling microdomains in time-lapse image sequences of such biosensors do not exist. Here, we introduce a method that centers on the notion of activation time series coordination to identify signaling microdomains in space and time. After validating the algorithm with simulated microdomains, we show that our method identifies domains that support current hypotheses on the signaling architecture governing Rho GTPase organization and also respond to perturbation with optogenetics. A record of this paper's transparent peer review process is included in the supplemental information.
2.
GCL pruning of PIP3 establishes the soma-germline boundary.
Abstract:
Primordial germ cells (PGCs) are the first cells specified in the Drosophila embryo and are precursors to the germline. Their formation requires suppression of somatic fates, achieved by degrading the receptor tyrosine kinase Torso at the posterior pole through the ubiquitin ligase adaptor germ cell-less (GCL). Although Torso is known to antagonize PGC formation, the underlying mechanisms remained unclear. Here, we combine optogenetic Ras activation and Ras effector loop mutants to show that Ras suppresses PGC formation independently of the canonical Raf/MEK/ERK pathway. We identify an unexpected early role for Torso in activating phosphoinositide 3-kinase (PI3K), generating membrane domains enriched in phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Elevated PI3K activity disrupts PGC formation, while reduced PI3K activity creates ectopic PGCs. We demonstrate that GCL remodels the posterior pole membrane by suppressing Torso-dependent PI3K activation. Clearing PIP3 enables myosin II enrichment, allowing for PGC formation. Together, our findings reveal how antagonistic Torso and GCL activities establish the soma-germline boundary by organizing cortical lipids.
3.
A single-component optogenetic toolkit for reversible visualization and programmable control of microtubule dynamics.
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Lan, TH
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Ma, G
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Liu, X
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Duong, T
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Price, A
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Chiu, M
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Du, D
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Reiner, DJ
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Huang, Y
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Zhou, Y
Abstract:
Microtubules 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, single-component optogenetic platform for spatiotemporal control of microtubule organization and dynamics. By harnessing light-induced oligomerization to regulate microtubule association, this system supports reversible microtubule labeling and plus-end tracking, localized control of tubulin PTMs, optically regulated kinesin-driven cargo transport, and inducible microtubule severing within a unified design strategy. Using these tools, we reveal how local microtubule integrity governs lysosomal trafficking and endoplasmic reticulum (ER)-associated signaling dynamics. Collectively, this modular optogenetic toolkit bridges molecular design with cytoskeletal function, offering a versatile platform to dissect how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.
4.
An optogenetic smart microscopy platform reveals signaling dynamics-dependent control over collective cell migration.
Abstract:
In cell biology, optical techniques can measure cells' internal states (biosensors) and stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a predetermined stimulus pattern is applied to cells, biosensors are measured over time, and data are processed offline. Here, we develop PyCLM, a Python-based suite enabling closed-loop measurement, image segmentation, and optogenetic control of thousands of cells per experiment. We showcase PyCLM on diverse applications, including performing feedback control on single cells and delivering developmental signaling patterns to Drosophila embryos. We compare single-cell versus tissue-scale optogenetic control of epithelial migration, revealing that fast and slow waves of receptor tyrosine kinase activity determine the direction of tissue movement, matching prior in vivo observations in zebrafish and mouse. PyCLM enables simple setup of dynamic experiments to probe cell and tissue properties and provides a first step toward real-time control of single-cell states at the tissue scale.
5.
CD44 restricts EGFR mobility to polarize cytoskeletal signalling modules driving bleb-based migration.
Abstract:
Cells under high confinement migrate efficiently in low-adhesion environments by forming stable, polarized, hydrostatic pressure-driven leader blebs. Here we investigated the basis of polarized bleb morphology in metastatic melanoma cells migrating under low-adhesive and highly confined microenvironments. Using high-resolution live imaging, molecular perturbations and biosensors, we show that EGF signalling through PI3K stabilizes and maintains polarized leader blebs. EGFR and PI3K activities form a gradient within leader blebs that decreases from rear to front, promoting phosphatidylinositol 3,4,5-trisphosphate and Rac1-GTP accumulation at the bleb rear, whereas phosphatidylinositol 4,5-bisphosphate and RhoA-GTP concentrate at the bleb tip, the inverse of the organization observed in integrin-dependent mesenchymal migration. Optogenetic disruption of this gradient triggers bleb retraction, underscoring its functional importance. Mathematical modelling and experiments identified a mechanism whereby during bleb initiation, CD44 and ERM proteins restrict EGFR mobility within a membrane-apposed cortical actin meshwork at the bleb rear, establishing the EGFR-PI3K-Rac gradient. Together, these findings define the biophysical and molecular mechanisms that underlie polarity in bleb-based migration and highlight how alternative spatial organization of signalling modules supports distinct migration modes in different microenvironments.
6.
Long-range mutual activation establishes Rho and Rac polarity during cell migration.
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De Belly, H
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Gallén, AF
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Strickland, E
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Estrada, DC
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Godinez, DS
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Neiva, E
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Zager, PJ
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Nagy, TL
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Burkhardt, JK
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Turlier, H
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Weiner, OD
Abstract:
In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are positioned at opposite poles remains unclear. We leverage optogenetics, mechanical perturbations, and mathematical modelling to reveal a surprising mechanochemical long-range mutual activation between front and back polarity programmes that complements their well-known local mutual inhibition. Rac-based protrusions elevate membrane tension, stimulating an mTORC2-dependent activation of Rho at the opposite side of the cell. Conversely, Rho-mediated contractility induces cortical-flow-based regulation of phosphoinositide signalling that triggers Rac activation distally. We develop a minimal mechanochemical model to explain how long-range facilitation, together with local inhibition, enables robust Rho and Rac partitioning. Our findings demonstrate how the actin cortex and plasma membrane interact as an integrated mechanochemical system for long-range Rac-Rho patterning. This circuit is required for efficient polarity and migration in primary human T cells and is conserved in epithelial cells, highlighting the generality of this mechanism.
7.
Optogenetic control of plasma membrane O-GlcNAcylation regulates WNK1 condensates and cellular signaling.
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Zhu, Q
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Liu, Q
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Fan, Z
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Shi, Y
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Liu, X
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Guo, Y
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Luo, J
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Zhao, J
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Qin, W
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Wang, Y
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Wang, P
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Ye, H
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Yi, W
Abstract:
Glycosylation plays a pivotal role in regulating diverse biological processes. However, the lack of tools capable of controlling the spatiotemporal dynamics of glycosylation has largely hindered its functional elucidation. Here, we introduce an optogenetic approach that employs red/far-red light to dynamically and reversibly control the plasma membrane localization of O-linked N-acetylglucosamine transferase (OGT) in living systems. Red-light-induced translocation of OGT suppresses insulin signaling in both cells and mice. Glycoproteomic and phosphoproteomic analyses reveal a global impact of OGT-mediated glycosylation on signal transduction. Moreover, using protein semisynthesis, cell-based assays, and molecular dynamics simulations, we demonstrate that red-light-induced O-GlcNAcylation of WNK1 at S1949 inhibits downstream cell volume response signaling pathways by suppressing WNK1 biomolecular condensate formation. Together, our findings provide a valuable tool to modulate subcellular O-GlcNAcylation and control cellular signaling in living systems, with broad applicability to the study of glycosylation in cells.
8.
Tissue rigidity phase transition shapes morphogen gradients.
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Autorino, C
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Khoromskaia, D
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Harari, L
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Floris, E
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Booth, H
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Pallares-Cartes, C
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Petrasiunaite, V
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Dorrity, M
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Corominas-Murtra, B
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Hadjivasiliou, Z
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Petridou, NI
Abstract:
During development, local mechanochemical cues within the cell microenvironment are translated into signalling pathways that drive cell fate decisions. Yet, as cells differentiate collectively, how global tissue-level properties shape these instructive cues remains unclear. Here we show that a tissue-scale rigidity transition guides patterning by tuning the length scales and timescales of morphogen signalling. By combining rigidity percolation theory, reaction-diffusion modelling, quantitative imaging and optogenetics in zebrafish, we uncover dynamical global tissue rigidity patterns that actively shape the Nodal morphogen gradient by locally changing its concentration and accelerating its signalling activity. In this self-generated mechanism, Nodal, besides instructing meso-endoderm fate specification, increases cell-cell adhesion strength via regulating planar cell polarity genes. Once the adhesion strength reaches a critical point, it triggers a rigidity transition which, in turn, induces the collapse of tissue porosity. The abrupt tissue reorganization negatively feeds back on Nodal signalling, impacting both its length scales, by restricting Nodal diffusivity, and its timescales, by speeding up the expression of its antagonist Lefty, thereby ensuring timely signal termination and robust patterning. Overall, we uncover a multiscale regulatory mechanism by which positional information and tissue material properties dynamically tune one another.
9.
An extracellular, optogenetic antibody platform for stimulus-gated antigen recognition and modulation of cell behavior.
Abstract:
Here, we present extrabody, an activatable, modular antibody platform that enables optogenetic or chemical reassembly of split antibody fragments for inducible extracellular antigen recognition. We demonstrate compatibility across diverse targets, including GFP, mCherry, and the tumor-associated antigens EGFR and HER2, and show that both nanobody- and scFv-derived fragments support light-dependent reconstitution. Extrabody enables input-gated cell-cell interactions and antigen transfer, providing external control over intercellular communication. Integration with synNotch receptors and chimeric antigen receptors (CARs) further allows dual-input regulation of downstream responses, including gene expression, cytokine release, and cytotoxicity. Together, these results establish extrabody as a versatile and generalizable interface for externally controlled cellular communication and synthetic signaling.
10.
Approaches to visualize, quantify, and manipulate phosphoinositides in cells.
Abstract:
Phosphoinositides are low-abundance regulatory lipids that control a broad range of cellular processes, from membrane trafficking and cytoskeletal remodeling to transcriptional regulation and RNA processing. These lipids are distributed across distinct subcellular compartments, where they carry out compartment-specific regulatory functions. Dysregulation of phosphoinositide metabolism is associated with cancer, neurodegenerative diseases, and immune dysfunction. However, their roles remain difficult to investigate owing to technical limitations in lipid detection and manipulation. This review outlines current strategies for modulating, visualizing, and quantifying phosphoinositide pools, including genetic manipulation techniques such as RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches, and optogenetics. It also evaluates visualization tools such as fluorescent biosensors and live-cell imaging techniques, including superresolution microscopy. In parallel, quantitative methods such as thin-layer chromatography and mass spectrometry for profiling phosphoinositide species, including isomer- and acyl-specific variants, are discussed. By comparing the strengths and limitations of these approaches and highlighting how they can be combined, this review provides a practical framework for dissecting phosphoinositide function in defined subcellular contexts.
11.
Light-inducible FLPase reconstitution enables temporal control of gene expression in Drosophila melanogaster.
Abstract:
Precise temporal control of gene expression is a prerequisite for disentangling timing-specific effects of gene function within the life cycle of Drosophila melanogaster. Here, we implement light-inducible FLPase reconstitution (LIFR) as a conditional gene expression system in flies, which combines blue light-responsive Magnet photoswitches and split-FLPase to remove an FRT-flanked stop cassette and irreversibly switch on transgene expression in response to light. This system is highly efficient, has virtually no transgene leakage, and a single light pulse is sufficient to induce long-term transgene expression. We demonstrate that LIFR in adulthood overcomes the developmental lethality elicited by constitutive pan-neuronal overexpression of neurodegeneration-causing mutants TDP43G298S and HTTQ97. We also illustrate that LIFR can help trace specific cell-type fates across developmental stages. Thus, we demonstrate proof of principle that LIFR is a versatile platform to conditionally activate long-lasting gene expression without the side effects of existing systems, thereby extending the Drosophila melanogaster genetic toolbox.
12.
Dynamic heterogeneity in an E. coli stress response regulon mediates gene activation and antimicrobial peptide tolerance.
Abstract:
The bacterial stress response is an intricately regulated system that plays a critical role in cellular resistance to drug treatment. Stress response genes are organized into networks with transcriptional regulators controlling downstream genes, but how variability in these regulators affects the downstream response remains unclear. We investigate how heterogeneity in the virulence regulator PhoP propagates to its diverse regulon. Using optogenetic control of PhoP expression and information theory, we find that downstream genes differ in mean expression, sensitivity to PhoP levels, and signal transmission reliability. These response functions also vary between individual cells, increasing population heterogeneity. We tie these variations to cell survival upon exposure to a clinically relevant antimicrobial peptide, showing that while high expression of the PhoP-regulon gene pmrD provides protection, cell survival is best determined by the integrated PhoP and pmrD expression levels. Our findings demonstrate that heterogeneous expression of stress response regulators has clear consequences for bacterial stress survival.
13.
Optogenetic Control of the Integrated Stress Response Limits Glioblastoma Invasion.
Abstract:
The integrated stress response (ISR) is a highly conserved signaling network, allowing cells to adapt and respond to various stressors. With its aggressive spread and high recurrence rates, glioblastoma multiforme (GBM) is one of the toughest cancers to date, yet the role of the ISR is still to be well understood, whether activation may suppress or promote this disease, and drug-treatment of GBM has thus far shown inconclusive results. In this work, we use an optogenetic tool, opto-PKR, to specifically trigger ISR activation via light-induced oligomerizing PKR-kinases, offering high spatiotemporal and reversible control, while avoiding potential upstream damage or side effects from drugs. Using immunofluorescence and RNA-sequencing, we show that targeted ISR activation reaching levels where both adaptive (ATF4) and terminal responses (CHOP) are activated results in subsequent downregulation of genes associated with the extracellular environment and glial cell migration, further supported by ECM-stain and scratch assays. Next, we show inhibition of aggressive spread for ISR-activated GBM spheroids in collagen 3D culture. Photopatterning of ISR activation in spheroids demonstrates a cell-intrinsic effect at the tissue scale, and recovery studies indicate a tunable, non-ablative intervention space. These findings suggest a route to containment and motivate ISR-activating small molecule screening in GBM models.
14.
Optogenetic mediated contractility enables reversible control of microglial morphology and migration in vivo.
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Biermeier, CM
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Albert, M
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Pal, AA
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Gallo, E
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Wittmann, J
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Hartmann, J
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Bill, RT
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Julmi, J
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Gilmour, D
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Peri, F
Abstract:
Directed migration and rapid process extension-retraction allow microglia to continuously survey the brain and efficiently identify and phagocytose apoptotic neurons. Defining how cytoskeletal regulators coordinate these behaviors could guide strategies for targeted modulation of microglial activity. To this aim, using in vivo imaging in zebrafish, we identified a mechanistic framework in which myosin II-dependent contractility governs transitions between surveillance, migratory, and phagocytic states. Building on this, we engineered an optogenetic RhoA actuator (opto-ArhGEF25) that enables reversible, spatiotemporally precise manipulation of microglial behavior in vivo. We show that patterned RhoA activation can modulate process dynamics and induce front-rear polarity that drives rapid repulsive migration away from the applied light source, overriding injury-evoked cues and preventing migration toward lesions. Together, these results establish optogenetic control of the cytoskeleton as a powerful approach to probe and ultimately modulate microglial function in the living brain.
15.
Local RhoA activation induces anillin-independent septin recruitment in interphase cells.
Abstract:
The regulation of the actin cytoskeleton is key to controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here, we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together, these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton.
16.
OptoTAT reveals microtubule acetylation as a rapid trigger for GEF-H1-mediated cell migration.
Abstract:
Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise induction of microtubule acetylation within minutes in live cells. Implementing optoTAT in migration assays, we observed striking and rapid responses at both molecular and cellular levels. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation, enabling a dynamic crosstalk between the actin and microtubule cytoskeletal networks in the coordination of cellular motility.
17.
Light-directed evolution of dynamic, multi-state, and computational protein functionalities.
Abstract:
Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.
18.
OptoLoop - an optogenetic tool to probe the functional role of genome organization.
Abstract:
The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. If and how this genome organization influences the regulation of gene expression remains only partially understood. The structure-function relationship of genomes has traditionally been probed by population-wide measurements after mutation of crucial DNA elements or by perturbation of chromatin-associated proteins. To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion. OptoLoop is based on the fusion between a nuclease-dead SpCas9 protein and the light-inducible oligomerizing protein CRY2. We demonstrate that OptoLoop can bring together genomically distant, repetitive DNA loci. As a proof-of-principle application of OptoLoop, we probed the functional role of DNA looping in the regulation of the human telomerase gene TERT. By analyzing the extent of chromatin looping and nascent RNA production at individual alleles, we find evidence for looping-mediated repression of TERT. In sum, OptoLoop represents a novel means for the interrogation of structure-function relationships in the genome.
19.
An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair.
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Zhao, J
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Wang, Z
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Lu, L
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Bu, G
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Miao, Z
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Zhang, Y
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Guo, Y
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Yang, Z
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Ma, J
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Jiao, J
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Ma, X
Abstract:
CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: ORECC regulated by doxycycline (Dox) and ORECo controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC's therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.
20.
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.
21.
Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.
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Guo, L
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Mann, JR
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Mauna, JC
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Copley, KE
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Wang, H
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Rubien, JD
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Bergmann, CA
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Carey, JL
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Merjane, J
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Ngo, M
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Xu, J
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Odeh, HM
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Lin, J
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Lee, BL
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Ganser, L
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Robinson, E
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Kim, KM
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Murthy, AC
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Paul, T
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Portz, B
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Gleixner, AM
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Diaz, Z
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Smirnov, A
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Padilla, G
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Lavorando, E
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Espy, C
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Shang, Y
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Huang, EJ
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Chesi, A
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Fawzi, NL
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Myong, S
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Donnelly, CJ
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Shorter, J
Abstract:
RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs that prevent FUS fibrillization by promoting liquid phases and distinct short RNAs that prevent and reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. We define a short RNA that dissolves cytoplasmic FUS aggregates, restores nuclear FUS, and mitigates FUS toxicity in optogenetic models and ALS patient-derived motor neurons. Another short RNA dissolves cytoplasmic TDP-43 aggregates, restores nuclear TDP-43, and mitigates TDP-43 toxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have utility for ALS/FTD and related disorders.
22.
The cell biologist's guide to detecting and modulating membrane phospholipids.
Abstract:
Molecular biology has benefited enormously from repurposed tools-many enzymes and antibodies evolved for other functions but are now essential for interrogating biological function by manipulating proteins or nucleic acids. In contrast, lipids have remained technically difficult to visualize or manipulate in cells. This review introduces tools that bring lipid biology into reach for molecular cell biologists, using familiar experimental approaches. We first describe adaptations of immunofluorescence and live-cell imaging of fluorescent molecules to track lipids. Then, we discuss tools for manipulating lipid levels, including pharmacologic inhibitors, synthetic biology platforms for inducible lipid generation or degradation, and optogenetic systems for precise temporal control. While some methods remain technically demanding, most tools are now broadly accessible. Our goal is to offer a practical framework for integrating lipid biology into mainstream cell biology experiments.
23.
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.
24.
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.
25.
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.
