Curated Optogenetic Publication Database

Abstract: The front of migratory cellular clusters during development, wound healing and cancer invasion is typically populated with highly protrusive cells that are called leader cells. Leader cells are thought to physically pull and direct their cohort of followers, but how leaders and followers are mechanically organized to migrate collectively remains controversial. One possibility is that the autonomous local action of a leader cell is sufficient to drive migration of the group. Yet another possibility is that a global mechanical organization is required for the group to move cohesively. Here we show that the effectiveness of leader-follower organization is proportional to the asymmetry of traction and tension within the cellular cluster. By combining hydrogel micropatterning and optogenetic activation of Rac1, we locally generate highly protrusive leaders at the edge of minimal cell groups. We find that the induced leader can robustly drag one follower but is generally unable to direct larger groups. By measuring traction forces and tension propagation in groups of increasing size, we establish a quantitative relationship between group velocity and the asymmetry of the traction and tension profiles. We propose a model of the motile cluster as an active polar fluid that explains this force-velocity relationship in terms of asymmetries in the distribution of active tractions. Our results challenge the notion of autonomous leader cells by showing that collective cell migration requires a global mechanical organization within the cluster.

Abstract: Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

Abstract: Mural cells are critical components of the cerebral vasculature. They are categorized into three primary subsets: arteriole smooth muscle cells (aSMCs), pericytes (PCs) and venule smooth muscle cells (vSMCs). It is well known that aSMCs can directly regulate cerebral blood flow (CBF) with their own contraction and dilation mechanisms. On the other hand, the direct involvement of PCs or vSMCs in CBF regulation is controversial. This ambiguity is largely due to the lack of specifically manipulable tools to isolate their function. To address this issue, we employed a set-subtraction approach by using a combination of tTA-mediated gene induction and Cre-mediated gene excision. We developed transgenic mice expressing optical actuators, channelrhodopsin-2 (ChR2) and photoactivated adenylyl cyclase (PAC) in smooth muscle actin (SMA)-negative mural cells that lack the machinery for SMA-mediated vasoregulation. Using these mouse models, we assessed CBF alterations in response to optical stimulation using laser Doppler techniques. Our results showed that optical stimulation induced notable CBF changes in both models. This study provides evidence for the potential regulatory role of PCs and vSMCs in cerebral hemodynamics and introduces powerful tools to specifically manipulate these cell types in vascular neurobiology.

Abstract: Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

Abstract: Microtubules filaments are assembled into higher-order structures and machines critical for cellular processes using microtubule-associated proteins (MAPs). However, the design of synthetic MAPs that direct the formation of new structures in cells is challenging, as nanoscale biochemical activities must be organized across micron length-scales. Here we develop synthetic MAP-IDR condensates (synMAPs) that provide tunable and regulatable assembly of higher-order microtubule structures in vitro and in mammalian cells. synMAPs harness a small microtubule-binding domain from oligodendrocytes (TPPP) whose activity can be synthetically rewired by interaction with condensate-forming IDR sequences. This combination allows synMAPs to self-organize multivalent structures that bind and bridge microtubules into synthetic architectures. Regulating the connection between the microtubule-binding and condensate-forming components allows synMAPs to act as nodes in more complex cytoskeletal circuits in which the formation and dynamics of the microtubule structure can be controlled by small molecules or cell-signaling inputs. By systematically testing a panel of synMAP circuit designs, we define a two-level control scheme for dynamic assembly of microtubule architectures at the nanoscale (via microtubule-binding) and microscale (via condensate formation). synMAPs provide a compact and rationally engineerable starting point for the design of more complex microtubule architectures and cellular machines.

Abstract: Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP’s influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.

Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to "lock" onto a particular direction, limiting the ability of cells to reorient. We use spatially defined optogenetic control of a leading edge organizer (PI3K) to probe how neutrophil-like HL-60 cells balance "decisiveness" needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibition process that destabilizes the leading edge to promote exploration. We show that this local inhibition enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.

Abstract: Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Abstract: General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Here, we report robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. We created topological variants of the DARPin scaffold by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following EGF-activation of EGFR, Ras is required for sustained EGFR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function.

Abstract: The ability of a single protein to trigger different functions is an assumed key feature of cell signaling, yet there are very few examples demonstrating it. Here, using an optogenetic tool to control membrane localization of RhoA nucleotide exchange factors (GEFs), we present a case where the same protein can trigger both protrusion and retraction when recruited to the plasma membrane, polarizing the cell in two opposite directions. We show that the basal concentration of the GEF prior to activation predicts the resulting phenotype. A low concentration leads to retraction, whereas a high concentration triggers protrusion. This unexpected protruding behavior arises from the simultaneous activation of Cdc42 by the GEF and inhibition of RhoA by the PH domain of the GEF at high concentrations. We propose a minimal model that recapitulates the phenotypic switch, and we use its predictions to control the two phenotypes within selected cells by adjusting the frequency of light pulses. Our work exemplifies a unique case of control of antagonist phenotypes by a single protein that switches its function based on its concentration or dynamics of activity. It raises numerous open questions about the link between signaling protein and function, particularly in contexts where proteins are highly overexpressed, as often observed in cancer.

Abstract: Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

Abstract: Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solidlike (less fluidlike) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solidlike properties than in wild-type embryos but less solidlike properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solidlike properties in tissues that are relatively isotropic, whereas high-tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

Abstract: The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

Abstract: Collective cell migration during embryonic development, wound healing, and cancer metastasis entails the emergence of leader cells at the migration front. These cells with conspicuous lamellipodial structures provide directional guidance to the collective. Despite their physiological relevance, the mechanisms underlying the emergence of leader cells remain elusive. Here we report that in diverse model systems for wound healing, including cultured epithelial monolayer, Drosophila embryo, and mouse embryonic skin, leader cells display a peripheral accumulation of lysosomes. This accumulation appears essential for leader cell emergence, involves lysosomal movement along microtubules, and depends on the actomyosin contractility-generated cellular forces. Peripheral lysosomes associate with inactive Rac1 molecules to remove them from the leading periphery, which increases local Rac1-activity, triggering actin polymerization and promoting lamellipodium formation. Taken together, we demonstrate that beyond their catabolic role, lysosomes act as the intracellular platform that links mechanical and biochemical signals to control the emergence of leader cells.

Abstract: RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

Abstract: In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging, and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition, and TGF-β signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish an hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

Abstract: An increasingly 24/7 connected and urbanised world has created a silent pandemic of noise-induced hearing loss. Ensuring survival to children born (extremely) preterm is crucial. The incubator is a closed medical device, modifying the internal climate, and thus providing an environment for the child, as safe, warm, and comfortable as possible. While sound outside the incubator is managed and has decreased over the years, managing the noise inside the incubator is still a challenge.

Abstract: Cells tend to divide along the direction in which they are longest, as famously stated by Oscar Hertwig in 1884 in his long axis rule. The orientation of the mitotic spindle determines the cell division axis, and the long axis rule is usually ensured by forces stemming from microtubules. Pulling on the spindle from the cell cortex can give rise to unstable behaviors, and we here set out to understand how the long axis rule is realized in early embryonic divisions where cortical pulling forces are prevalent. We focus on early C. elegans development, where we compressed embryos to reveal that cortical pulling forces favor an alignment of the spindle with the short axis of the cell. Strikingly, we find that this misalignment is corrected by an actomyosin-based mechanism that rotates the entire cell, including the mitotic spindle. We uncover that myosin-driven contractility in the cytokinetic ring generates inward forces that align it with the short axis, and thereby the spindle with the long axis. A theoretical model together with experiments using slightly compressed mouse zygotes suggest that a constricting cytokinetic ring can ensure the long axis rule in cells that are free to rotate inside a confining structure, thereby generalizing the underlying principle.

Abstract: Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Abstract: The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.

Abstract: Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.

Abstract: During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.

Abstract: An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

Abstract: Studying the generation of biomechanical force and how this force drives cell and tissue morphogenesis is challenging for understanding the mechanical mechanisms underlying embryogenesis. Actomyosin has been demonstrated to be the main source of intracellular force generation that drives membrane and cell contractility, thus playing a vital role in multi-organ formation in ascidian Ciona embryogenesis. However, manipulation of actomyosin at the subcellular level is impossible in Ciona because of the lack of technical tools and approaches. In this study, we designed and developed a myosin light chain phosphatase fused with a light-oxygen-voltage flavoprotein from Botrytis cinerea (MLCP-BcLOV4) as an optogenetics tool to control actomyosin contractility activity in the Ciona larva epidermis. We first validated the light-dependent membrane localization and regulatory efficiency on mechanical forces of the MLCP-BcLOV4 system as well as the optimum light intensity that activated the system in HeLa cells. Then, we applied the optimized MLCP-BcLOV4 system in Ciona larval epidermal cells to realize the regulation of membrane elongation at the subcellular level. Moreover, we successfully applied this system on the process of apical contraction during atrial siphon invagination in Ciona larvae. Our results showed that the activity of phosphorylated myosin on the apical surface of atrial siphon primordium cells was suppressed and apical contractility was disrupted, resulting in the failure of the invagination process. Thus, we established an effective technique and system that provide a powerful approach in the study of the biomechanical mechanisms driving morphogenesis in marine organisms.

Abstract: By acting both upstream of and downstream from biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTOR complex 2 (mTORC2) programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading-edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input phosphatidylinositol (3,4,5)-trisphosphate to robustly amplify mTORC2 activation. Our results suggest that different signaling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.