Showing 1 - 23 of 23 results
Actuation of single downstream nodes in growth factor network steers immune cell migration.
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.
Optogenetic manipulation identifies the roles of ERK and AKT dynamics in controlling mouse embryonic stem cell exit from pluripotency.
ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues.
Spatiotemporal control of ERK pulse frequency coordinates fate decisions during mammary acinar morphogenesis.
The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility and proliferation. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated multicellular ERK waves emerge, correlating with high and low ERK pulse frequencies in outer surviving and inner dying cells, respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation display increased ERK pulse frequency and inner cell survival. Thus, fate decisions during acinar morphogenesis are coordinated by different spatiotemporal modalities of ERK pulse frequency.
Compartmentalization of telomeres through DNA-scaffolded phase separation.
Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.
Rapid and robust optogenetic control of gene expression in Drosophila.
Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. Here we describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. We show that it allows efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, we illustrate how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions.
Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos.
Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY's efficiency and generated variants with different recovery times. iLEXY enables rapid (t1/2 < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.
Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival.
Cell death events continuously challenge epithelial barrier function yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of extracellular-signal-regulated kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.
Robustness of epithelial sealing is an emerging property of local ERK feedback driven by cell elimination.
What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.
Long-Range Optogenetic Control of Axon Guidance Overcomes Developmental Boundaries and Defects.
Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.
ERK-Mediated Mechanochemical Waves Direct Collective Cell Polarization.
During collective migration of epithelial cells, the migration direction is aligned over a tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here, we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and ERK activation triggers cell contraction. The contraction of the activated cell pulls neighboring cells, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear polarization guarantees unidirectional propagation of ERK activation, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration.
Tissue-Scale Mechanical Coupling Reduces Morphogenetic Noise to Ensure Precision during Epithelial Folding.
Morphological constancy is universal in developing systems. It is unclear whether precise morphogenesis stems from faithful mechanical interpretation of gene expression patterns. We investigate the formation of the cephalic furrow, an epithelial fold that is precisely positioned with a linear morphology. Fold initiation is specified by a precise genetic code with single-cell row resolution. This positional code activates and spatially confines lateral myosin contractility to induce folding. However, 20% of initiating cells are mis-specified because of fluctuating myosin intensities at the cellular level. Nevertheless, the furrow remains linearly aligned. We find that lateral myosin is planar polarized, integrating contractile membrane interfaces into supracellular "ribbons." Local reduction of mechanical coupling at the "ribbons" using optogenetics decreases furrow linearity. Furthermore, 3D vertex modeling indicates that polarized, interconnected contractility confers morphological robustness against noise. Thus, tissue-scale mechanical coupling functions as a denoising mechanism to ensure morphogenetic precision despite noisy decoding of positional information.
Rapid Dynamics of Signal-Dependent Transcriptional Repression by Capicua.
Optogenetic perturbations, live imaging, and time-resolved ChIP-seq assays in Drosophila embryos were used to dissect the ERK-dependent control of the HMG-box repressor Capicua (Cic), which plays critical roles in development and is deregulated in human spinocerebellar ataxia and cancers. We established that Cic target genes are activated before significant downregulation of nuclear localization of Cic and demonstrated that their activation is preceded by fast dissociation of Cic from the regulatory DNA. We discovered that both Cic-DNA binding and repression are rapidly reinstated in the absence of ERK activation, revealing that inductive signaling must be sufficiently sustained to ensure robust transcriptional response. Our work provides a quantitative framework for the mechanistic analysis of dynamics and control of transcriptional repression in development.
A Nudge or a Shove: Altering Actomyosin Pulse Profiles In Vivo.
Pulsed actomyosin contractions drive morphogenetic processes, but how cyclic frequencies and amplitudes of contractions are tuned to achieve processive shrinking of cell surfaces remains unclear. In this issue of Developmental Cell, Cavanaugh et al. (2020) use optogenetics and biophysical modeling to demonstrate how cells respond to different oscillatory force profiles.
RhoA Mediates Epithelial Cell Shape Changes via Mechanosensitive Endocytosis.
Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.
Primary Cilia Signaling Promotes Axonal Tract Development and Is Disrupted in Joubert Syndrome-Related Disorders Models.
Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.
Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.
The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.
Developmental Erk Signaling Illuminated.
How a small number of signaling pathways can be re-used in distinct embryonic contexts to control different fates remains unclear. In this issue of Developmental Cell, Johnson and Toettcher (2019) use optogenetic approaches to explore how different dynamic ERK signaling states control specific developmental fates in the Drosophila embryo.
"Rho"ing a Cellular Boat with Rearward Membrane Flow.
The physicist Edward Purcell wrote in 1977 about mechanisms that cells could use to propel themselves in a low Reynolds number environment. Reporting in Developmental Cell, O'Neill et al. (2018) provide direct evidence for one of these mechanisms by optogenetically driving the migration of cells suspended in liquid through RhoA activation.
Membrane Flow Drives an Adhesion-Independent Amoeboid Cell Migration Mode.
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.
Propagating Wave of ERK Activation Orients Collective Cell Migration.
The biophysical framework of collective cell migration has been extensively investigated in recent years; however, it remains elusive how chemical inputs from neighboring cells are integrated to coordinate the collective movement. Here, we provide evidence that propagation waves of extracellular signal-related kinase (ERK) mitogen-activated protein kinase activation determine the direction of the collective cell migration. A wound-healing assay of Mardin-Darby canine kidney (MDCK) epithelial cells revealed two distinct types of ERK activation wave, a "tidal wave" from the wound, and a self-organized "spontaneous wave" in regions distant from the wound. In both cases, MDCK cells collectively migrated against the direction of the ERK activation wave. The inhibition of ERK activation propagation suppressed collective cell migration. An ERK activation wave spatiotemporally controlled actomyosin contraction and cell density. Furthermore, an optogenetic ERK activation wave reproduced the collective cell migration. These data provide new mechanistic insight into how cells sense the direction of collective cell migration.
The Spatiotemporal Limits of Developmental Erk Signaling.
Animal development is characterized by signaling events that occur at precise locations and times within the embryo, but determining when and where such precision is needed for proper embryogenesis has been a long-standing challenge. Here we address this question for extracellular signal regulated kinase (Erk) signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hr post-fertilization, when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part, by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Ras/Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.
Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.
We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.
An Optogenetic Method to Modulate Cell Contractility during Tissue Morphogenesis.
Morphogenesis of multicellular organisms is driven by localized cell shape changes. How, and to what extent, changes in behavior in single cells or groups of cells influence neighboring cells and large-scale tissue remodeling remains an open question. Indeed, our understanding of multicellular dynamics is limited by the lack of methods allowing the modulation of cell behavior with high spatiotemporal precision. Here, we developed an optogenetic approach to achieve local modulation of cell contractility and used it to control morphogenetic movements during Drosophila embryogenesis. We show that local inhibition of apical constriction is sufficient to cause a global arrest of mesoderm invagination. By varying the spatial pattern of inhibition during invagination, we further demonstrate that coordinated contractile behavior responds to local tissue geometrical constraints. Together, these results show the efficacy of this optogenetic approach to dissect the interplay between cell-cell interaction, force transmission, and tissue geometry during complex morphogenetic processes.