Showing 201 - 225 of 647 results
Optogenetic interrogation of integrin αVβ3 function in endothelial cells.
αVβ3 is reported to promote angiogenesis in some model systems but not in others. Here we used optogenetics to study effects of αVβ3 interaction with the intracellular adapter, kindlin-2, on endothelial cell functions potentially relevant to angiogenesis. Since interaction of kindlin-2 with αVβ3 requires the C-terminal three residues of the β3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to β3ΔRGT-GFP and mCherry-kindlin2, respectively, and expressed in β3-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVβ3 as assessed by immunofluorescence and TIRF microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVβ3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with β3 and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.
Photo-Activatable Akt Probe - A New Tool to Study the Akt-Dependent Physiopathology of Cancer Cells.
Akt is commonly overexpressed and activated in cancer cells, and plays a pivotal role in cell survival, protection andchemo-resistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anti-cancer drugs. Here, we examined whether a newly developed photo-activatable Akt (PA-Akt) probe, based on a light-inducible protein interaction module of plant cryptochrome2 (CRY2) and cryptochrome-interacting basic-helix-loop-helix (CIB1), can regulate Akt-associated cell functions. By illuminating blue light to the cells stably transfected with PA-Akt probe, CRY2-Akt (a fusion protein of CRY2 and Akt) underwent structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1) anchoring at cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probe-Akt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3 , one of the main downstream signals of Akt, were also phosphorylated, depending on light intensity. These facts indicate that photo-activation of probe-Akt can activate endogenous Akt and its downstream signals. The photoactivated Akt conferred protection against nutritional deprivation and H2O2 stresses to the cells significantly. Using the newly developed PA-Akt probe, endogenous Akt was activated easily, transiently and repeatedly. This probe will be a unique tool in studying Akt-associated specific cellular functions in cancer cells and developing anti-cancer drugs.
Optogenetic regulation of insulin secretion in pancreatic β-cells.
Pancreatic β-cell insulin production is orchestrated by a complex circuitry involving intracellular elements including cyclic AMP (cAMP). Tackling aberrations in glucose-stimulated insulin release such as in diabetes with pharmacological agents, which boost the secretory capacity of β-cells, is linked to adverse side effects. We hypothesized that a photoactivatable adenylyl cyclase (PAC) can be employed to modulate cAMP in β-cells with light thereby enhancing insulin secretion. To that end, the PAC gene from Beggiatoa (bPAC) was delivered to β-cells. A cAMP increase was noted within 5 minutes of photostimulation and a significant drop at 12 minutes post-illumination. The concomitant augmented insulin secretion was comparable to that from β-cells treated with secretagogues. Greater insulin release was also observed over repeated cycles of photoinduction without adverse effects on viability and proliferation. Furthermore, the expression and activation of bPAC increased cAMP and insulin secretion in murine islets and in β-cell pseudoislets, which displayed a more pronounced light-triggered hormone secretion compared to that of β-cell monolayers. Calcium channel blocking curtailed the enhanced insulin response due to bPAC activity. This optogenetic system with modulation of cAMP and insulin release can be employed for the study of β-cell function and for enabling new therapeutic modalities for diabetes.
Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.
Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.
Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.
Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene’s transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.
Blue Light Switchable Bacterial Adhesion as a Key Step toward the Design of Biofilms.
The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tunable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.
Recent advances in the development of light-inducible transgene expression systems have overcome many inherent drawbacks of conventional chemically regulated systems. The latest generation of those light-regulated systems that are specifically responsive to different wavelengths allows spatiotemporal control of gene expression in a so far unprecedented manner.In this chapter, we first describe the available light-inducible gene expression systems compatible with mammalian cells and explain their underlying mechanisms. Afterward, we give a detailed protocol for the implementation of a UVB light-inducible expression system in mammalian cells.
An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis.
The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic "on" switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells. Illumination of cells with blue light activated myogenic differentiation, including upregulation of myogenic markers and fusion into multinucleated myotubes. Cell differentiation was spatially patterned by illumination of cell cultures through a photomask. To demonstrate the application of the system to controlling in vivo tissue development, the light inducible switch was used to control the expression of VEGF and angiopoietin-1, which induced angiogenic sprouting in a mouse dorsal window chamber model. Live intravital microscopy showed illumination-dependent increases in blood-perfused microvasculature. This optogenetic switch is broadly useful for applications in which sustained and patterned gene expression is desired following transient induction, including tissue engineering, gene therapy, synthetic biology, and fundamental studies of morphogenesis.
Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) signaling is transient and spatially confined in live cells. How this pattern of signaling regulates transmitter release and hormone secretion has not been addressed. We devised an optogenetic approach to control PI(4,5)P2 levels in time and space in insulin-secreting cells. Combining this approach with total internal reflection fluorescence microscopy, we examined individual vesicle-trafficking steps. Unlike long-term PI(4,5)P2 perturbations, rapid and cell-wide PI(4,5)P2 reduction in the plasma membrane (PM) strongly inhibits secretion and intracellular Ca(2+) concentration ([Ca(2+)]i) responses, but not sytaxin1a clustering. Interestingly, local PI(4,5)P2 reduction selectively at vesicle docking sites causes remarkable vesicle undocking from the PM without affecting [Ca(2+)]i. These results highlight a key role of local PI(4,5)P2 in vesicle tethering and docking, coordinated with its role in priming and fusion. Thus, different spatiotemporal PI(4,5)P2 signaling regulates distinct steps of vesicle trafficking, and vesicle docking may be a key target of local PI(4,5)P2 signaling in vivo.
Photocontrolled reversible self-assembly of dodecamer nitrilase.
Naturally photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. In this study, a method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein-protein dimerization.
Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging.
cAMP is a common second messenger that is involved in various physiological processes. To expand the colour palette of available cAMP indicators, we developed a red cAMP indicator named "Pink Flamindo" (Pink Fluorescent cAMP indicator). The fluorescence intensity of Pink Flamindo increases 4.2-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 567 nm and 590 nm, respectively. Live-cell imaging revealed that Pink Flamindo is effective for monitoring the spatio-temporal dynamics of intracellular cAMP generated by photoactivated adenylyl cyclase in response to blue light, and in dual-colour imaging studies using a green Ca2+ indicator (G-GECO). Furthermore, we successfully monitored the elevation of cAMP levels in vivo in cerebral cortical astrocytes by two-photon imaging. We propose that Pink Flamindo will facilitate future in vivo, optogenetic studies of cell signalling and cAMP dynamics.
Cells lay their own tracks: optogenetic Cdc42 activation stimulates fibronectin deposition supporting directed migration.
Rho GTPase family members are known regulators of directed migration and therefore play key roles in processes including development, immune response and cancer metastasis. However, their individual contributions to these processes are complex. Here, we regulate the activity of two family members, Rac and Cdc42, by optogenetically recruiting specific GEF DH/PH domains to defined regions on the cell membrane. We find that the localized activation of both GTPases produce lamellipodia in cells plated on a fibronectin substrate. Using a novel optotaxis assay, we show that biased activation can drive directional migration. Interestingly, in the absence of exogenous fibronectin, Rac activation is insufficient to produce stable lamellipodia or directional migration while Cdc42 activation is sufficient. We find that a remarkably small amount of fibronectin (<10 puncta per protrusion) is necessary to support stable GTPase-driven lamellipodia. Cdc42 bypasses the need for exogenous fibronectin by stimulating cellular fibronectin deposition under the newly formed lamellipodia.
PhiReX: a programmable and red light-regulated protein expression switch for yeast.
Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h.
Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.
The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.
Interactions Between phyB and PIF Proteins Alter Thermal Reversion Reactions in vitro.
The dynamic behavior of the plant red/far-red light photoreceptor phytochrome B (phyB) has been elucidated in natural and synthetic systems. Red light switches phyB from the inactive Pr state to the active Pfr state, a process that is reversed by far-red light. Alongside light signals, phyB activity is constrained by thermal reversion (that is prominent in the dark) and protein-protein interactions between phyB, other phytochrome molecules, and, among others, PHYTOCHROME INTERACTING FACTORs (PIFs). Requirements for phyB-PIF association have been well studied and are central to light-regulated synthetic tools. However, it is unknown whether PIF interactions influence transitions of phyB between different conformers. Here, we show that the in vitro thermal reversion of phyB involves multiple reactions. Thermal reversion of phyB in vitro is inhibited by PIF6, and this effect is observed at all temperatures tested. We analyzed our experimental data using a mathematical model containing multiple Pfr conformers, in accordance with previous findings. Remarkably, each Pfr conformer is differentially regulated by PIF6 and temperature. As a result, we speculate that in vivo phytochrome signaling networks may require similar levels of complexity to fine-tune responses to the external environment.
Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System.
The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call "Opto-SOS." Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics.
An engineered photoswitchable mammalian pyruvate kinase.
Changes in allosteric regulation of glycolytic enzymes have been linked to metabolic reprogramming involved in cancer. Remarkably, allosteric mechanisms control enzyme function at significantly shorter time-scales compared to the long-term effects of metabolic reprogramming on cell proliferation. It remains unclear if and how the speed and reversibility afforded by rapid allosteric control of metabolic enzymes is important for cell proliferation. Tools that allow specific, dynamic modulation of enzymatic activities in mammalian cells would help address this question. Towards this goal, we have used molecular dynamics simulations to guide the design of PiL[D24], an engineered pyruvate kinase M2 (PKM2) variant that harbours an insertion of the light-sensing LOV2 domain from Avena Sativa within a region implicated in allosteric regulation by fructose 1,6-bisphosphate (FBP). The LOV2 photoreaction is preserved in the PiL[D24] chimera and causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for PEP resulting in increased pyruvate kinase activity after light exposure. Importantly, this change in activity is reversible upon light withdrawal. Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose. PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner and paves the way for studying the importance of rapid allosteric phenomena in the regulation of metabolism and enzyme control. This article is protected by copyright. All rights reserved.
Synthetic biological approaches to optogenetically control cell signaling.
Precise spatial and temporal control of cellular processes is in life sciences a highly sought-after capability. In the recent years, this goal has become progressively achievable through the field of optogenetics, which utilizes light as a non-invasive means to control genetically encoded light-responsive proteins. The latest optogenetic systems, such as those for control of subcellular localization or cellular decision-making and tissue morphogenesis provide us with insights to gain a deeper understanding of the cellular inner workings. Besides, they hold a potential for further development into biomedical applications, from in vitro optogenetics-assisted drug candidate screenings to light-controlled gene therapy and tissue engineering.
Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo.
Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of Bicoid decoding.
A module for Rac temporal signal integration revealed with optogenetics.
Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades.
Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization.
Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.
A calcium- and light-gated switch to induce gene expression in activated neurons.
Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. We present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, we show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, we temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.
A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.
Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.
Optogenetic protein clustering through fluorescent protein tagging and extension of CRY2.
Protein homo-oligomerization is an important molecular mechanism in many biological processes. Therefore, the ability to control protein homo-oligomerization allows the manipulation and interrogation of numerous cellular events. To achieve this, cryptochrome 2 (CRY2) from Arabidopsis thaliana has been recently utilized for blue light-dependent spatiotemporal control of protein homo-oligomerization. However, limited knowledge on molecular characteristics of CRY2 obscures its widespread applications. Here, we identify important determinants for efficient cryptochrome 2 clustering and introduce a new CRY2 module, named ''CRY2clust'', to induce rapid and efficient homo-oligomerization of target proteins by employing diverse fluorescent proteins and an extremely short peptide. Furthermore, we demonstrate advancement and versatility of CRY2clust by comparing against previously reported optogenetic tools. Our work not only expands the optogenetic clustering toolbox but also provides a guideline for designing CRY2-based new optogenetic modules.Cryptochrome 2 (CRY2) from A. thaliana can be used to control light-dependent protein homo-oligomerization, but the molecular mechanism of CRY2 clustering is not known, limiting its application. Here the authors identify determinants of CRY2 clustering and engineer fusion partners to modulate clustering efficiency.
Optogenetic activation of Plexin-B1 reveals contact repulsion between osteoclasts and osteoblasts.
During bone remodelling, osteoclasts induce chemotaxis of osteoblasts and yet maintain spatial segregation. We show that osteoclasts express the repulsive guidance factor Semaphorin 4D and induce contact inhibition of locomotion (CIL) in osteoblasts through its receptor Plexin-B1. To examine causality and elucidate how localized Plexin-B1 stimulation may spatiotemporally coordinate its downstream targets in guiding cell migration, we develop an optogenetic tool for Plexin-B1 designated optoPlexin. Precise optoPlexin activation at the leading edge of migrating osteoblasts readily induces local retraction and, unexpectedly, distal protrusions to steer cells away. These morphological changes are accompanied by reorganization of Myosin II, PIP3, adhesion and active Cdc42. We attribute the resultant repolarization to RhoA/ROCK-mediated redistribution of β-Pix, which activates Cdc42 and promotes protrusion. Thus, our data demonstrate a causal role of Plexin-B1 for CIL in osteoblasts and reveals a previously unknown effect of Semaphorin signalling on spatial distribution of an activator of cell migration.