Curated Optogenetic Publication Database

Abstract: Chemokine-guided cell migration is pivotal for many immunological and developmental processes. How chemokine receptor signaling persists to guarantee sustained directional migration despite receptor desensitization and internalization remains poorly understood. Here, we uncover a function for an intracellular pool of the chemokine receptor CCR7 present in human dendritic cells and cellular model systems. We find that CCR7 signaling, initiated at the plasma membrane, is translocated by joint trafficking of β-arrestin and Src kinase to endomembrane-residing CCR7. There, Src tyrosine phosphorylates CCR7, required for the recruitment of Vav1 to form an endomembrane-residing multi-protein signaling complex comprising CCR7, the RhoGEF Vav1, and its effector, Rac1. Interfering with vesicular trafficking affects CCR7-driven cell migration, whereas CCR7:Vav1 interaction at endomembranes is essential for local Rac1 recruitment to CCR7. Photoactivation of Rac1 at endomembranes leads to lamellipodia formation at the cell's leading edge, supporting the role of sustained endomembrane signaling in guiding cell migration.

Abstract: Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodelling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behaviour under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviours, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodelling and continuous strain relaxation. First, a critical strain threshold for tension remodelling triggers irreversible junction length changes for sufficiently strong contractions, making the system robust to small fluctuations in contractile activity. Second, continuous strain relaxation allows for mechanical memory removal, enabling frequency-dependent modulation of cell shape changes via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodelling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.

Abstract: Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.

Abstract: During symmetry breaking, the highly conserved Rho GTPase Cdc42 becomes stabilized at a defined site via an amplification process. However, little is known about how a new polarity site is established in an already asymmetric cell-a critical process in a changing environment. The human fungal pathogen Candida albicans switches from budding to filamentous growth in response to external cues, a transition controlled by Cdc42. Here, we have used optogenetic manipulation of cell polarity to reset growth in asymmetric filamentous C. albicans cells. We show that increasing the level of active Cdc42 on the plasma membrane results in disruption of the exocyst subunit Sec3 localization and a striking de novo clustering of secretory vesicles. This new cluster of secretory vesicles is highly dynamic, moving by hops and jumps, until a new growth site is established. Our results reveal that secretory vesicle clustering can occur in the absence of directional growth.

Abstract: The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.

Abstract: Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While non-muscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin co-localize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo. Movie S1 Movie S1 Live imaging of border cell specification and delamination from anterior epithelium From Figure 1D-I. Slbo promoter driving Lifeact-GFP (green) marks border cells, Upd-Gal4, UAS-DsRed.nls (red) mark polar cell nuclei. Hoechst 33342 (blue) marks DNA. Time resolution is 4 min. Movie S2 Movie S2 Representative Z-projected and registered live imaging of Sqh-mCherry accumulating in cortical junctions (flashing arrows) during border cell migration. From Figure 3J-K. Time resolution is 25 sec. Movie S3 Movie S3 Representative Z-projected and registered live imaging of E-cad-GFP during border cell migration. From Figure 3M-N. Time resolution is 60 sec. Movie S4 Movie S4 Representative Z-projection of control flpout cells from hs-Flp;, Slbo>Lifeact-GFP; AyGal4, UAS-RFP. Clonal cells are marked by magenta nuclei (nls-RFP). Time resolution is 2.5 min. From Supp. Figure 3 A-D. Movie S5 Movie S5 Representative Z-projection of Sqh-RNAi flpout cells from hs-Flp;, Slbo>Lifeact-GFP; AyGal4, UAS-RFP, UAS-sqh-RNAi. Clonal cells are marked by magenta nuclei (nls-RFP). Time resolution is 2.5 min. From Supp. Figure 3 E-H. Movie S6 Movie S6 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-white RNAi. Time resolution is 2 min. From Supp. Figure 4 A-D. Movie S7 Movie S7 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-sqh-RNAi showing frequent side protrusions. Time resolution is 2 min. From Supp. Figure 4 E-H. White arrows indicate ectopic side and rear protrusions. Movie S8 Movie S8 Representative Z-projected c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-sqh-RNAi showing long lived side protrusions. Time resolution is 2 min. From Supp. Figure 4 I-L. Movie S9 Movie S9 Representative Z-projected live imaging of c306-Gal4 driving UAS-white-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during periods of protrusive and round migration phases. From Figure 6A-D. 25 min corresponds to 6A and B and 1hr:25 min corresponds to 6C and D. Time resolution is 2.5 min. Movie S10 Movie S10 Sqh-mCherry (magenta) channel from Supplementary Movie 9. From Figure 6A-D. 25 min corresponds to 6A and B and 1hr:25 min corresponds to 6C and D. Time resolution is 2.5 min. Movie S11 Movie S11 Representative Z-projected live imaging of c306-Gal4 driving UAS-Ecad-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during a protrusive phase of migration. From Figure 6E-F. Time resolution is 2.5 min. Movie S12 Movie S12 Sqh-mCherry (magenta) channel from Supplementary Movie 11. From Figure 6E-F. Time resolution is 2.5 min. Movie S13 Movie S13 Representative Z-projected live imaging of c306-Gal4 driving UAS-Ecad-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during a rounded phase of migration. From Figure 6G-H. Time resolution is 2.5 min. Movie S14 Movie S14 Sqh-mCherry (magenta) channel from Supplementary Movie 13. From Figure 6G-H. Time resolution is 2.5 min. Movie S15 Movie S15 Example segmentation analysis from a representative Z-projected time lapse of a cluster expressing c306-Gal4 driving UAS-white-RNAi in clusters co-expressing Lifeact-GFP under the control of the slbo enhancer and Sqh-mCherry from its endogenous promoter during migration. Time lapse analyzed in Imaris by 1. segmentation of the cluster using Lifeact-GFP, 2. Rendering of Sqh-mCherry by masking the inside of the Life-act surface, 3. performing a distance transformation using the masked Sqh-mCherry that is color coded for distance from membrane (dark colors are short distances and bright/white colors are more distant), 4. combining the distance transformation with the Sqh-mCherry mask to only include the cortical 2 μm of the original Sqh-mCherry signal for quantification in Figure 6I. Movie S16 Movie S16 Representative Z-projected time lapse of Lifeact-GFP and Sqh-mCherry expressing clusters used for quantification of Figure 7B-C during protrusion/retractions cycles. Time resolution is 2 min. Movie S17 Movie S17 Sqh-mCherry channel from Supplementary movie 16. Time resolution is 2 min. Movie S18 Movie S18 Representative Z-projections of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-Sqh-E20E21 migrating border cells clusters that split. Time resolution is 2 min. Movie S19 Movie S19 Representative Z-projections of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-LifeactGFP and UAS-Sqh-E20E21 migrating border cells clusters during protrusive phase. Time resolution is 2 min. Movie S20 Movie S20 Representative Z-projection of Lifeact-GFP (green) in c306-Gal4; tub-GAL80ts driving UAS-Lifeact-GFP and UAS-Sqh-E20E21 border cells cluster at the oocyte border during a blebbing phase. Time resolution is 2 min. Movie S21 Movie S21 Representative Z-projection of control cluster expressing slbo-Gal4; UAS-PLCδ1-PH-GFP. Time resolution is 2 min. Movie S22 Movie S22 Representative Z-projection of cluster expressing slbo-Gal4; UAS-PLCδ1-PH-GFP, UAS-Rho1V14. Blebs are marked by white arrows. Time resolution is 2 min.

Abstract: We developed VIEW-MOD (Versatile Illumination Engine with a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.

Abstract: Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. Here we analyze the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. We show that early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which we show regulates actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.

Abstract: Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Abstract: The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

Abstract: Mitochondria play an essential role in regulating insulin secretion from beta cells by providing ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine if the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria 2-fold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely important for the generation of Ca2+ microdomains required for efficient insulin granule release.

Abstract: Cell morphogenesis is critical for embryonic development, tissue formation, and wound healing. Our ability to manipulate endogenous mechanisms to control cell shape, however, remains limited. Here we combined surface micropatterning of adhesion molecules with optogenetic activation of intracellular signaling pathways to control the nature and morphology of cellular protrusions. We employed geometry-dependent pre-organization of cytoskeletal structures together with acute activation of signaling pathways that control actin assembly to create a tool capable of generating membrane protrusions at defined cellular locations. Further, we find that the size of microfabricated patterns of adhesion molecules influences the molecular mechanism of cell protrusion: larger patterns enable cells to create actin-filled lamellipodia while smaller patterns promote formation of spherical blebs. Optogenetic perturbation of signaling pathways in these cells changes the size of blebs and convert them into lamellipodia. Our results demonstrate how the coordinated manipulation of adhesion geometry and cytoskeletal dynamics can be used to control membrane protrusion and cell morphogenesis.

Abstract: Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.

Abstract: Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.

Abstract: During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, β2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.

Abstract: Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. Here we use optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. We show that while basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process.

Abstract: The Ras-ERK signaling pathway regulates diverse cellular processes in response to environmental stimuli and contains important therapeutic targets for cancer. Recent single cell studies revealed stochastic pulses of ERK activation, the frequency of which determines functional outcomes such as cell proliferation. Here we show that ERK pulses are initiated by localized protrusive activities. Chemically and optogenetically induced protrusions trigger ERK activation through various entry points into the feedback loop involving Ras, PI3K, the cytoskeleton, and cellular adhesion. The excitability of the protrusive signaling network drives stochastic ERK activation in unstimulated cells and oscillations upon growth factor stimulation. Importantly, protrusions allow cells to sense combined signals from substrate stiffness and the growth factor. Thus, by uncovering the basis of ERK pulse generation we demonstrate how signals involved in cell growth and differentiation are regulated by dynamic protrusions that integrate chemical and mechanical inputs from the environment.

Abstract: Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information of membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2–CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

Abstract: The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anti-cancer strategies.

Abstract: Synthetic hydrogels are widely used as biomimetic in vitro model systems to understand how cells respond to complex microenvironments. The mechanical properties of hydrogels are deterministic for many cellular behaviors, including cell migration, spreading, and differentiation. However, it remains a major challenge to engineer hydrogels that recapture the dynamic mechanical properties of native extracellular matrices. Here, we provide a new hydrogel platform with spatiotemporally tunable mechanical properties to assay and define cellular behaviors under light. The change in the mechanical properties of the hydrogel is effected by a photo-induced switch of the cross-linker fluorescent protein, Dronpa145N, between the tetrameric and monomeric states, which causes minimal changes to the chemical properties of the hydrogel. The mechanical properties can be rapidly and reversibly tuned for multiple cycles using visible light, as confirmed by rheological measurements and atomic force microscopybased nano-indentation. We further demonstrated real-time and reversible modulation of cell migration behaviors on the hydrogels through photo-induced stiffness switching, with minimal invasion to the cultured cells. Hydrogels with a programmable mechanical history and a spatially defined mechanical hierarchy might serve as an ideal model system to better understand complex cellular functions.

Abstract: Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

Abstract: Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the initiation and promotion of cancer cell metastasis. The phosphoinositide 3-kinase (PI3K) signaling pathway has been demonstrated to be involved in TGF-β induced EMT, but the complicated TGF-β signaling network makes it challenging to dissect the important role of PI3K on regulation of EMT process. Here, we applied optogenetic controlled PI3K module (named 'Opto-PI3K'), which based on CRY2 and the N-terminal of CIB1 (CIBN), to rapidly and reversibly control the endogenous PI3K activity in cancer cells with light. By precisely modulating the kinetics of PI3K activation, we found that E-cadherin is an important downstream target of PI3K signaling. Compared with TGF-β treatment, Opto-PI3K had more potent effect in down-regulation of E-cadherin expression, which was demonstrated to be regulated in a light dose-dependent manner. Surprisingly, sustained PI3K activation induced partial EMT state in A549 cells that is highly reversible. Furthermore, we demonstrated that Opto-PI3K only partially mimicked TGF-β effects on promotion of cell migration in vitro. These results reveal the importance of PI3K signaling in TGF-β induced EMT, suggesting other TGF-β regulated signaling pathways are necessary for the full and irreversible promotion of EMT in cancer cells. In addition, our study implicates the great promise of optogenetics in cancer research for mapping input-output relationships in oncogenic pathways.

Abstract: The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα-GDP, Gα-GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

Abstract: Cells migrate by applying rearward forces against extracellular media. It is unclear how this is achieved in amoeboid migration, which lacks adhesions typical of lamellipodia-driven mesenchymal migration. To address this question, we developed optogenetically controlled models of lamellipodia-driven and amoeboid migration. On a two-dimensional surface, migration speeds in both modes were similar. However, when suspended in liquid, only amoeboid cells exhibited rapid migration accompanied by rearward membrane flow. These cells exhibited increased endocytosis at the back and membrane trafficking from back to front. Genetic or pharmacological perturbation of this polarized trafficking inhibited migration. The ratio of cell migration and membrane flow speeds matched the predicted value from a model where viscous forces tangential to the cell-liquid interface propel the cell forward. Since this mechanism does not require specific molecular interactions with the surrounding medium, it can facilitate amoeboid migration observed in diverse microenvironments during immune function and cancer metastasis.

Abstract: To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.