RESUMO
Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.
Assuntos
Núcleo Celular , Drosophila , Animais , Difusão , Embrião de Mamíferos , OptogenéticaRESUMO
A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Células Germinativas/fisiologia , Animais , Blastoderma , Padronização Corporal , Diferenciação Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Feminino , Células Germinativas/metabolismo , Masculino , Transdução de SinaisRESUMO
Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.
Assuntos
Quimiotaxia , Fibronectinas/farmacologia , Pseudópodes/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Quimiotaxia/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrina beta1/metabolismo , Camundongos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
Filopodia are thin, fingerlike structures that contain bundled actin filaments and project from the cell periphery. These structures are dogmatically endowed with the ability to sense cues in the microenvironment, implying that filopodia foster local signal transduction, yet their small diameter hampers the imaging of dynamic processes therein. To overcome this challenge, we analyzed total internal reflection fluorescence images of migrating fibroblasts coexpressing either a plasma membrane marker or tagged AktPH domain, a translocation biosensor for signaling through the phosphoinositide 3-kinase pathway, along with a cytosolic volume marker. We devised a scheme to estimate the radii of filopodia using either the membrane marker or volume marker data, and we used that information to account for geometry effects in the biosensor data. With conservative estimates of relative target molecule abundance, it is revealed that filopodia typically harbor higher densities of 3' phosphoinositides than adjacent regions at the cell periphery. In this context at least, the analysis supports the filopodial signaling hypothesis.
Assuntos
Movimento Celular , Pseudópodes/metabolismo , Transdução de Sinais , Animais , Camundongos , Células NIH 3T3 , Imagem ÓpticaRESUMO
The development of optogenetic control over signaling pathways has provided a unique opportunity to decode the role of signaling dynamics in cell fate programing. Here I present a protocol for decoding cell fates through systematic interrogation with optogenetics and visualization of signaling with live biosensors. Specifically, this is written for Erk control of cell fates using the optoSOS system in mammalian cells or Drosophila embryos, though it is intended to be adapted to apply generally for several optogenetic tools, pathways, and model systems. This guide focuses on calibrating these tools, tricks of their use, and using them to interrogate features which program cell fates.
Assuntos
Técnicas Biossensoriais , Optogenética , Animais , Optogenética/métodos , Transdução de Sinais , Diferenciação Celular , Drosophila , MamíferosRESUMO
Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn . We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be 'blurred' through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.
RESUMO
Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.
Assuntos
Padronização Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Luz , Morfogênese , Optogenética/métodos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/efeitos da radiação , Feminino , Sistema de Sinalização das MAP Quinases , Masculino , Transdução de SinaisRESUMO
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.
Assuntos
Ectoderma/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Drosophila/embriologia , Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Morfogênese/genéticaRESUMO
In developmental biology, localization is everything. The same stimulus-cell signaling event or expression of a gene-can have dramatically different effects depending on the time, spatial position, and cell types in which it is applied. Yet the field has long lacked the ability to deliver localized perturbations with high specificity in vivo. The advent of optogenetic tools, capable of delivering highly localized stimuli, is thus poised to profoundly expand our understanding of development. We describe the current state-of-the-art in cellular optogenetic tools, review the first wave of major studies showcasing their application in vivo, and discuss major obstacles that must be overcome if the promise of developmental optogenetics is to be fully realized.
Assuntos
Biologia do Desenvolvimento , Optogenética/métodos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Luz , Transdução de SinaisRESUMO
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.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Desenvolvimento Embrionário , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Luz , Optogenética , Fatores de Tempo , Transcrição Gênica/efeitos da radiação , Proteínas ras/metabolismoRESUMO
An optogenetic approach reveals how cells encode external information in complex patterns of protein activity.
Assuntos
Cálcio da Dieta , Cálcio , OptogenéticaRESUMO
Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator.
Assuntos
Actinas/fisiologia , Quimiotaxia/genética , Proteínas dos Microfilamentos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Pseudópodes/fisiologia , Receptores Odorantes/genética , Células 3T3 , Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Forma Celular , Quimiotaxia/fisiologia , Fibroblastos/citologia , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Camundongos , Proteínas dos Microfilamentos/biossíntese , Microscopia de Fluorescência , Dados de Sequência Molecular , Fator de Crescimento Derivado de Plaquetas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores Odorantes/biossínteseRESUMO
The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFß, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFß decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFß in vivo. Furthermore, depletion or overexpression of GMFß disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFß plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.
Assuntos
Actinas/biossíntese , Movimento Celular/fisiologia , Fator de Maturação da Glia/fisiologia , Pseudópodes/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Fibroblastos/fisiologia , Fibronectinas/farmacologia , Indóis/farmacologia , Camundongos , Isoformas de Proteínas/biossínteseRESUMO
Cells contain multiple F-actin assembly pathways, including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay and/or competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2(-/-) cells lack lamellipodia and migrate more slowly than WT cells but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2(-/-) cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. Antagonism of the Arp2/3 complex by profilin-1 in cells appears to maintain actin homeostasis by balancing Arp2/3 complex-dependent and -independent actin assembly pathways.
Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Profilinas/metabolismo , Animais , Feminino , Proteínas Fetais , Fibroblastos/citologia , Forminas , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Microscopia de Fluorescência , Proteínas Nucleares , Transdução de Sinais , Fibras de EstresseRESUMO
Chemotaxis, migration toward soluble chemical cues, is critical for processes such as wound healing and immune surveillance and is exhibited by various cell types, from rapidly migrating leukocytes to slow-moving mesenchymal cells. To study mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of platelet-derived growth factor (PDGF). Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mammalian target of rapamycin signaling, are dispensable for PDGF chemotaxis. Instead, we find that local inactivation of Myosin IIA, through a noncanonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of live imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge are required for mesenchymal chemotaxis.
Assuntos
Quimiotaxia/fisiologia , Mesoderma/fisiologia , Miosina Tipo II/metabolismo , Fosfolipase C gama/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Linhagem Celular , Movimento Celular , Diglicerídeos/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Transgênicos , Ésteres de Forbol , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de SinaisRESUMO
This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Processamento de Imagem Assistida por Computador , Camundongos , Células NIH 3T3 , Fosfoproteínas Fosfatases/genética , Ligação Proteica , Transporte Proteico , Receptor Cross-Talk , Proteínas Recombinantes de Fusão/genética , Transdução de SinaisRESUMO
Animal cell migration is a complex process characterized by the coupling of adhesion, cytoskeletal, and signaling dynamics. Here we model local protrusion of the cell edge as a function of the load-bearing properties of integrin-based adhesions, actin polymerization fostered by adhesion-mediated signaling, and mechanosensitive activation of RhoA that promotes myosin II-generated stress on the lamellipodial F-actin network. Analysis of stochastic model simulations illustrates how these pleiotropic functions of nascent adhesions may be integrated to govern temporal persistence and frequency of protrusions. The simulations give mechanistic insight into the documented effects of extracellular matrix density and myosin abundance, and they show characteristic, nonnormal distributions of protrusion duration times that are similar to those extracted from live-cell imaging experiments. Analysis of the model further predicts relationships between measurable quantities that reflect the partitioning of stress between tension on F-actin-bound adhesions, which act as a molecular clutch, and dissipation by retrograde F-actin flow.
Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Simulação por Computador , Adesões Focais/fisiologia , Integrinas/fisiologia , Citoesqueleto de Actina , Actinas/metabolismo , Actomiosina/fisiologia , Animais , Moléculas de Adesão Celular/genética , Matriz Extracelular/metabolismo , Integrinas/genética , Integrinas/metabolismo , Mecanotransdução Celular , Miosina Tipo II/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.