Showing 1 - 10 of 10 results
Engineered Illumination Devices for Optogenetic Control of Cellular Signaling Dynamics.
Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics affect cell behavior. Here, we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). By patterning human embryonic stem cell (hESC) cultures with varying light intensities, LAVA devices enabled dose-responsive control of optoWnt activation and Brachyury expression. Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models the embryonic presentation of Wnt signals in vitro. LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.
Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage.
Cell-autonomous changes in p53 expression govern the duration and outcome of cell-cycle arrest at the G2 checkpoint for DNA damage. Here, we report that mitogen-activated protein kinase (MAPK) signaling integrates extracellular cues with p53 dynamics to determine cell fate at the G2 checkpoint. Optogenetic tools and quantitative cell biochemistry reveal transient oscillations in MAPK activity dependent on ataxia-telangiectasia-mutated kinase after DNA damage. MAPK inhibition alters p53 dynamics and p53-dependent gene expression after checkpoint enforcement, prolonging G2 arrest. In contrast, sustained MAPK signaling induces the phosphorylation of CDC25C, and consequently, the accumulation of pro-mitotic kinases, thereby relaxing checkpoint stringency and permitting cells to evade prolonged G2 arrest and senescence induction. We propose a model in which this MAPK-mediated mechanism integrates extracellular cues with cell-autonomous p53-mediated signals, to safeguard genomic integrity during tissue proliferation. Early steps in oncogene-driven carcinogenesis may imbalance this tumor-suppressive mechanism to trigger genome instability.
Chemokine Receptor CCR7 Triggers an Endomembrane Signaling Complex for Spatial Rac Activation.
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.
Secretory Vesicle Clustering in Fungal Filamentous Cells Does Not Require Directional Growth.
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.
Light Control of the Tet Gene Expression System in Mammalian Cells.
Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.
Activation of EphB2 Forward Signaling Enhances Memory Consolidation.
EphB2 is involved in enhancing synaptic transmission and gene expression. To explore the roles of EphB2 in memory formation and enhancement, we used a photoactivatable EphB2 (optoEphB2) to activate EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning, but not minutes afterward, enhanced long-term, but not short-term, auditory fear conditioning. Photoactivation of optoEphB2 during fear conditioning led to activation of the cAMP/Ca2+ responsive element binding (CREB) protein. Application of light to a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long-term, but not short-term, auditory fear conditioning memory was impaired in mice lacking EphB2 forward signaling (EphB2lacZ/lacZ). Activation of optoEphB2 in LA of EphB2lacZ/lacZ mice enhanced long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.
Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling.
Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.
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.
Plasma Membrane Association but Not Midzone Recruitment of RhoGEF ECT2 Is Essential for Cytokinesis.
Cytokinesis, the final step of cell division, begins with the formation of a cleavage furrow. How the mitotic spindle specifies the furrow at the equator in animal cells remains unknown. Current models propose that the concentration of the RhoGEF ECT2 at the spindle midzone and the equatorial plasma membrane directs furrow formation. Using chemical genetic and optogenetic tools, we demonstrate that the association of ECT2 with the plasma membrane during anaphase is required and sufficient for cytokinesis. Local membrane targeting of ECT2 leads to unilateral furrowing, highlighting the importance of local ECT2 activity. ECT2 mutations that prevent centralspindlin binding compromise concentration of ECT2 at the midzone and equatorial membrane but sustain cytokinesis. While the association of ECT2 with the plasma membrane is essential for cytokinesis, our data suggest that ECT2 recruitment to the spindle midzone is insufficient to account for equatorial furrowing and may act redundantly with yet-uncharacterized signals.
Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation.
During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.