Qr: application:"Control of cytoskeleton / cell motility / cell shape"
Showing 1 - 25 of 235 results
1.
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.
2.
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.
3.
An intrinsic cytoskeletal oscillator establishes neuronal polarity.
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Lin, TC
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Coles, CH
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Alfadil, E
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Fäßler, F
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Husch, A
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Dupraz, S
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Pietralla, T
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Narita, A
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Schelski, M
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Flynn, KC
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Stern, S
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Möhl, C
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Hilton, BJ
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Vauti, F
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Arnold, HH
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Schur, FKM
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Bradke, F
Abstract:
Neurons acquire polarity by specifying one neurite as the axon, whereas the others become dendrites. But how this fundamental asymmetry is established remains unclear1. Neuronal polarization has been thought to rely primarily on growth cones that sense external cues2. Here we show that growth cones alone do not direct this process and that the soma acts as a central organizer of neuronal polarization. Using live imaging and genetic loss-of-function approaches in vivo, combined with optogenetic control and local cytoskeletal perturbations in cultured neurons, we uncover a soma-initiated oscillatory program that primes axon selection. Periodic actin branching that depends on the actin-related protein 2/3 (ARP2/3) complex at the soma remodels a global actomyosin network, thereby generating an actin wave that retracts neurites before propagating into a single neurite tip. Exposure to this wave relaxes local actomyosin contractility, which drives a transient microtubule-based protrusion and biases this neurite towards axon fate. As the cell exits this oscillatory stage, this neurite can overcome global inhibition and extend independently of ARP2/3, whereas actomyosin activity suppresses axon formation in the remaining neurites so that they subsequently become dendrites. This soma-driven mechanism ensures the emergence of a single axon independent of environmental cues and underpins the unidirectional information flow in neuronal circuits.
4.
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.
5.
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.
6.
GTPase-activating protein DLC1 spatio-temporally regulates Rho signaling.
Abstract:
Emerging evidence suggests that Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) bind to the cytoskeleton or focal adhesions (FAs), controlling spatio-temporal Rho GTPase activity through feedback mechanisms. We explore such feedback in the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1), which binds to FAs through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to globally increased Rho activity and contractility in fibroblasts. Although Rho activity appears macroscopically steady, individual molecules undergo 'signaling flux'-a dynamic cycle of activation and deactivation. To measure this flux, we built a genetic circuit that enables both optogenetic activation of Rho and simultaneous readout of Rho activity. In cells at mechanical steady state, this reveals that DLC1 globally controls the rate of Rho deactivation, both at FAs and at the plasma membrane. Transient induction of local contractility, however, shows DLC1 associating with and dissociating from FAs during their reinforcement and relaxation, which might provide local positive feedback on Rho activity for robust FA disassembly. Together, our results indicate that DLC1 regulates Rho activity both globally at steady state and locally at FAs under tension, highlighting the complexity of spatio-temporal Rho GTPase signaling.
7.
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.
8.
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.
9.
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.
10.
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.
11.
Myosin II-driven contractions of supporting cap cells promote sensory adaptation of Drosophila proprioceptors.
Abstract:
Mechanoreceptors can be motile and actively amplify their mechanical input.1,2,3,4 We here found that the responses of mechanoreceptor cells can also be shaped actively by contractile supporting cells. Drosophila larvae monitor body movements with pentascolopidial chordotonal (lch5) organs that are stretched out between cuticular attachment sites.5,6,7,8 These proprioceptive organs contain five stretch-receptor neurons each that receive mechanical stimuli from supporting cap cells. The elastic cap cells are surrounded by extracellular matrix and contain actin cables and non-muscle myosin II motors, suggesting that the cells might be motile.9,10 We show that the supporting cap cells are pre-strained at rest to about twice their relaxed length, and that the force they transmit is modulated by myosin II in the cap cells. Cap cells contracted upon optogenetic activation of myosin II. Cap cell-specific knockdown of the regulatory light chain of myosin II relieved tension and converted the spiking responses of the stretch receptors from phasic to more tonic, impairing adaptation to sustained stimuli. Our findings thus illustrate that mechanoreceptor responses can be actively tailored by contractile neighboring cells.
12.
Actin-membrane interface stress regulates Arp2/3-branched actin density during lamellipodial protrusion.
Abstract:
Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.
13.
The dynamic response of the bacterial flagellar motor to its direct intracellular input signal.
Abstract:
The bacterial flagellar motor drives bacterial swimming and chemotaxis by rotating helical flagellar filaments. When Escherichia coli navigates chemical gradients, the motor switches from counterclockwise (CCW) during forward swimming to clockwise (CW) during direction-changing tumbles. The motor responds indirectly to extracellular chemosensory input to membrane-bound chemoreceptors using an intervening intracellular signaling pathway. How the motor responds to its direct input signal-the diffusible messenger phosphorylated CheY (CheY-P)-remains poorly understood. Steady-state motor measurements have been modeled as an allosteric switch between CCW/CW states that depends on mean CheY-P levels. Allosteric models have suggested that as many as 20 CheY-P molecules can be bound to the motor when it switches rotational direction. But steady-state models cannot predict the sensitivity of the motor to dynamic changes in CheY-P that essentially modulate chemotactic behavior. We present an optogenetic reagent that precisely controls the direct dynamical input signal to the motor. We designed a "caged" molecule, Opto-CheY, that is transiently activated by photon absorption. We find that activation and binding of one to three additional CheY-P molecules is sufficient to switch the motor from the CCW to CW state. The sensitivity of the motor to small changes in CheY-P occupancy helps resolve a long-standing paradox about the high sensitivity of the chemotactic response to external sensory input. Optogenetic biochemistry by light-activated uncaging of signal molecules is a new strategy to dissect information-processing in the living cell.
14.
Optogenetic BlueGENEs engineered into a human safe harbor locus.
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Geidies, A
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Nieke, M
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Witte, N
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McLean, BJ
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Evangelopoulou, M
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Koh, CS
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Kuschel, S
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Stölting, F
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Zurbriggen, MD
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Beyer, HM
Abstract:
Crafting synthetic in vitro tissues with mammalian cells faces a shortage of methods to define spatial features. Optogenetic tissue engineering can provide the desired spatial and temporal control but requires stable genomic engineering to support long-term cultivation and high response resolution. Here, we developed BlueGENEs, a set of optimized optogenetic gene switches. BlueGENEs support rapid, stable cell line generation, including precision engineering into the human AAVS1 safe harbor locus. By combining a designer endonuclease and a phage integrase, the approach overcomes gene-disruptive effects of random gene delivery and enables reproducible cell line development. BlueGENEs comprise an optogenetic blue light-responsive gene switch, a synthetic response promoter, and selection strategies serving broad use scenarios. We generated various human cell lines for optical control of apoptotic cell fate, 3D tissue formation, and signals promoting cytoskeletal remodeling. Our results demonstrate the integration of optogenetic cells with bioprinting technologies, illustrating the potential of BlueGENEs in advancing the synthesis of de novo or patient-derived in vitro model systems.
15.
Closed-loop optogenetic control of cell biology enables outcome-driven microscopy.
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Passmore, JB
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Rates, A
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Schröder, J
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van Laarhoven, MTP
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Hellebrekers, VJW
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van Hoef, HG
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Geurts, AJM
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van Straaten, W
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Nijenhuis, W
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Berger, F
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Smith, CS
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Smal, I
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Kapitein, LC
Abstract:
Smart microscopy is transforming biological imaging by integrating real-time analysis with adaptive acquisition to enhance imaging efficiency. Whereas many emerging implementations are event-driven and focus on on-demand data acquisition to reduce phototoxicity, we here present 'outcome-driven' microscopy, a framework combining smart microscopy with optogenetics to control cell biological processes and achieve predefined outcomes. We validate this approach using light-based control of cell migration and nucleocytoplasmic transport, demonstrating robust spatiotemporal control of cellular behaviour in single cells and in cell populations.
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.
A Modular Platform for the Optogenetic Control of Small GTPase Activity in Living Cells Reveals Long-Range RhoA Signaling.
Abstract:
Small GTPases are critical regulators of cellular processes, such as cell migration, and comprise a family of over 167 proteins in the human genome. Importantly, the location-dependent regulation of small GTPase activity is integral to coordinating cellular signaling. Currently, there are no generalizable methods for directly controlling the activity of these signaling enzymes with subcellular precision. To address this issue, we introduce a modular, optogenetic platform for the spatial control of small GTPase activity within living cells, termed spLIT-small GTPases. This platform enabled spatially precise control of cytoskeletal dynamics such as filopodia formation (spLIT-Cdc42) and directed cell migration (spLIT-Rac1). Furthermore, a spLIT-RhoA system uncovered previously unreported long-range RhoA signaling in HeLa cells, resulting in bipolar membrane retraction. These results establish spLIT-small GTPases as a versatile platform for the direct, spatial control of small GTPase signaling and demonstrate the ability to uncover spatially defined aspects of small GTPase signaling.
18.
Why epithelial cells collectively move against a traveling signal wave.
Abstract:
The response of cell populations to external stimuli plays a central role in biological mechanical processes such as epithelial wound healing and developmental morphogenesis. Wave-like propagation of a signal of ERK MAP kinase has been shown to direct collective migration in one direction; however, the mechanism based on continuum mechanics under a traveling wave is not fully understood. To elucidate how the traveling wave of the ERK kinase signal directs collective migration, we constructed the mechanical model of the epithelial cell monolayer by considering the signal-dependent coordination of contractile stress and cellular orientation. The proposed model was studied by using an optogenetically controlled cell system where we found that local signal activation induces changes in cell density and orientation with the direction of propagation. The net motion of the cell population occurred relative to the wave, and the migration velocity showed a maximum in resonance with the velocity of the ERK signal wave. The presented mechanical model was further validated in an in vitro wound healing process.
19.
Optogenetic control of PLC-γ1 activity polarizes cell motility.
Abstract:
Phospholipase C-γ1 (PLC-γ1) signaling is required for mesenchymal chemotaxis, but is it sufficient to bias motility? PLC-γ1 enzyme activity is basally autoinhibited, and light-controlled membrane recruitment of wild-type (WT) PLC-γ1 (OptoPLC-γ1) in Plcg1-null fibroblasts does not trigger lipid hydrolysis, complicating efforts to isolate its contribution. Utilizing cancer-associated mutations to investigate the regulatory logic of PLC-γ1, we demonstrate that the canonical hallmark of enzyme activity, phosphorylated Tyr783 (pTyr783), is not a proxy for activity level, but is rather a marker of dysregulated autoinhibition. Accordingly, OptoPLC-γ1 with a deregulating mutation (P867R, S345F, or D1165H) exhibits elevated phosphorylation, and membrane localization of such is sufficient to activate substrate hydrolysis and concomitant motility responses. In particular, local recruitment of OptoPLC-γ1 S345F polarizes cell motility on demand. This response is spatially dose-sensitive and only partially reduced by blocking canonical PLC-γ1 signaling yet is lipase-dependent. Our findings reframe the interpretation of PLC-γ1 regulation and demonstrate that local activation of PLC-γ1 is sufficient to direct cell motility.
20.
Optogenetic actin network assembly on lipid bilayer uncovers the network density-dependent functions of actin-binding proteins.
Abstract:
The actin cytoskeleton forms a meshwork that drives cellular deformation. Network properties, determined by density and actin-binding proteins, are crucial, yet how density governs protein penetration and dynamics remains unclear. Here, we report an in vitro optogenetic system, named OptoVCA, enabling Arp2/3 complex-mediated actin assembly on lipid membranes. By tuning illumination power, duration, and pattern, OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine how network density affects two actin-binding proteins: myosin and ADF/cofilin. We find that even modest increases in density strictly inhibit myosin filament penetration by steric hindrance. Penetrated myosin filaments generate directional actin flow in networks with density gradients. In contrast, ADF/cofilin accesses networks regardless of density, yet network disassembly is markedly reduced by increased density. Thus, OptoVCA reveals that network density differentially regulates actin-binding protein penetration and activity. These findings advance understanding of cell mechanics through precise, light-based manipulation of cytoskeletal structure.
21.
Ras-mediated dynamic and biphasic regulation of cell migration.
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Lin, Y
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Parajón, E
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Yuan, Q
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Ye, S
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Qin, G
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Deng, Y
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Borleis, J
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Koyfman, A
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Iglesias, PA
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Konstantopoulos, K
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Robinson, DN
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Devreotes, PN
Abstract:
Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, nontranscriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling, whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells, whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that Ras activity is regulated by guanine nucleotide exchange factors generally increase cortical tension while Ras activity is regulated by GTPase-activating proteins decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.
22.
Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments.
Abstract:
Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but it reduced cell stiffness. Mitochondria and endoplasmic reticulum (ER) sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.
23.
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.
24.
Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics.
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Inaba, H
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Imasaki, T
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Aoyama, K
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Yoshihara, S
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Takazaki, H
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Kato, T
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Goto, H
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Mitsuoka, K
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Nitta, R
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Nakata, T
Abstract:
Lamellipodia are sheet-like protrusions essential for cell migration and endocytosis, but their ultrastructural dynamics remain poorly understood because conventional electron microscopy lacks temporal resolution. Here, we combined optogenetics with cryo-electron tomography (cryo-ET) to visualize the actin cytoskeleton and membrane structures during lamellipodia formation with temporal precision. Using photoactivatable-Rac1 (PA-Rac1) in COS-7 cells, we induced lamellipodia formation with a 2-min blue light irradiation, rapidly vitrified samples, and analyzed their ultrastructure with cryo-ET. We obtained 16 tomograms of lamellipodia at different degrees of extension from three cells. These revealed small protrusions with unbundled actin filaments, "mini filopodia" composed of short, bundled actin filaments at the leading edge, and actin bundles running nearly parallel to the leading edge within inner regions of lamellipodia, suggesting dynamic reorganizations of the actin cytoskeleton. This approach provides a powerful framework for elucidating the ultrastructural dynamics of cellular processes with precise temporal control.
25.
Optogenetic control of pheromone gradients and mating behavior in budding yeast.
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Banderas, A
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Hofmann, M
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Cordier, C
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Le Bec, M
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Elizondo-Cantú, MC
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Chiron, L
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Pouzet, S
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Lifschytz, Y
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Ji, W
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Amir, A
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Scolari, V
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Hersen, P
Abstract:
During mating in budding yeast, cells use pheromones to locate each other and fuse. This model system has shaped our current understanding of signal transduction and cell polarization in response to extracellular signals. The cell populations producing extracellular signal landscapes themselves are, however, less well understood, yet crucial for functionally testing quantitative models of cell polarization and for controlling cell behavior through bioengineering approaches. Here we engineered optogenetic control of pheromone landscapes in mating populations of budding yeast, hijacking the mating-pheromone pathway to achieve spatial control of growth, cell morphology, cell-cell fusion, and distance-dependent gene expression in response to light. Using our tool, we were able to spatially control and shape pheromone gradients, allowing the use of a biophysical model to infer the properties of large-scale gradients generated by mating populations in a single, quantitative experimental setup, predicting that the shape of such gradients depends quantitatively on population parameters. Spatial optogenetic control of diffusible signals and their degradation provides a controllable signaling environment for engineering artificial communication and cell-fate systems in gel-embedded cell populations without the need for physical manipulation.
