RESUMEN
Developing tissues form spatial patterns by establishing concentration gradients of diffusible signaling proteins called morphogens. The bone morphogenetic protein (BMP) morphogen pathway uses a family of extracellular modulators to reshape signaling gradients by actively "shuttling" ligands to different locations. It has remained unclear what circuits are sufficient to enable shuttling, what other patterns they can generate, and whether shuttling is evolutionarily conserved. Here, using a synthetic, bottom-up approach, we compared the spatiotemporal dynamics of different extracellular circuits. Three proteins-Chordin, Twsg, and the BMP-1 protease-successfully displaced gradients by shuttling ligands away from the site of production. A mathematical model explained the different spatial dynamics of this and other circuits. Last, combining mammalian and Drosophila components in the same system suggests that shuttling is a conserved capability. Together, these results reveal principles through which extracellular circuits control the spatiotemporal dynamics of morphogen signaling.
Asunto(s)
Drosophila , Endopeptidasas , Animales , Ligandos , Péptido Hidrolasas , Transducción de Señal , MamíferosRESUMEN
In developing tissues, cells estimate their spatial position by sensing graded concentrations of diffusible signaling proteins called morphogens. Morphogen-sensing pathways exhibit diverse molecular architectures, whose roles in controlling patterning dynamics and precision have been unclear. In this work, combining cell-based in vitro gradient reconstitution, genetic rewiring, and mathematical modeling, we systematically analyzed the distinctive architectural features of the Sonic Hedgehog pathway. We found that the combination of double-negative regulatory logic and negative feedback through the PTCH receptor accelerates gradient formation and improves robustness to variation in the morphogen production rate compared with alternative designs. The ability to isolate morphogen patterning from concurrent developmental processes and to compare the patterning behaviors of alternative, rewired pathway architectures offers a powerful way to understand and engineer multicellular patterning.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Animales , Tipificación del Cuerpo/genética , Retroalimentación Fisiológica , Redes y Vías Metabólicas , Ratones , Modelos Biológicos , Células 3T3 NIH , Receptor Patched-1/genética , Eliminación de Secuencia , Transducción de SeñalRESUMEN
The cyanobacterial circadian clock generates genome-wide transcriptional oscillations and regulates cell division, but the underlying mechanisms are not well understood. Here, we show that the response regulator RpaA serves as the master regulator of these clock outputs. Deletion of rpaA abrogates gene expression rhythms globally and arrests cells in a dawn-like expression state. Although rpaA deletion causes core oscillator failure by perturbing clock gene expression, rescuing oscillator function does not restore global expression rhythms. We show that phosphorylated RpaA regulates the expression of not only clock components, generating feedback on the core oscillator, but also a small set of circadian effectors that, in turn, orchestrate genome-wide transcriptional rhythms. Expression of constitutively active RpaA is sufficient to switch cells from a dawn-like to a dusk-like expression state as well as to block cell division. Hence, complex global circadian phenotypes can be generated by controlling the phosphorylation of a single transcription factor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ritmo Circadiano , Regulación Bacteriana de la Expresión Génica , Synechococcus/genética , Relojes Circadianos , Genoma Bacteriano , Fosforilación , Regiones Promotoras Genéticas , Synechococcus/fisiología , Transcripción GenéticaRESUMEN
Circadian clocks are ubiquitous biological oscillators that coordinate an organism's behavior with the daily cycling of the external environment. To ensure synchronization with the environment, the period of the clock must be maintained near 24 h even as amplitude and phase are altered by input signaling. We show that, in a reconstituted circadian system from cyanobacteria, these conflicting requirements are satisfied by distinct functions for two domains of the central clock protein KaiC: the C-terminal autokinase domain integrates input signals through the ATP/ADP ratio, and the slow N-terminal ATPase acts as an input-independent timer. We find that phosphorylation in the C-terminal domain followed by an ATPase cycle in the N-terminal domain is required to form the inhibitory KaiBâ¢KaiC complexes that drive the dynamics of the clock. We present a mathematical model in which this ATPase-mediated delay in negative feedback gives rise to a compensatory mechanism that allows a tunable phase and amplitude while ensuring a robust circadian period.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Ritmo Circadiano/fisiología , Cianobacterias/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Dominio Catalítico , Relojes Circadianos/genética , Relojes Circadianos/fisiología , Ritmo Circadiano/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Cianobacterias/genética , Cinética , Modelos Biológicos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942 is governed by a core oscillator consisting of the proteins KaiA, KaiB, and KaiC. Remarkably, circadian oscillations in the phosphorylation state of KaiC can be reconstituted in a test tube by mixing the three Kai proteins and adenosine triphosphate. The in vitro oscillator provides a well-defined system in which experiments can be combined with mathematical analysis to understand the mechanism of a highly robust biological oscillator. In this Review, we summarize the biochemistry of the Kai proteins and examine models that have been proposed to explain how oscillations emerge from the properties of the oscillator's constituents.
Asunto(s)
Proteínas Bacterianas/metabolismo , Relojes Biológicos , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Synechococcus/metabolismo , Proteínas Bacterianas/química , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Modelos Moleculares , Conformación ProteicaRESUMEN
The simple circadian oscillator found in cyanobacteria can be reconstituted in vitro using three proteins-KaiA, KaiB, and KaiC. The total phosphorylation level of KaiC oscillates with a circadian period, but the mechanism underlying its sustained oscillation remains unclear. We have shown that four forms of KaiC differing in their phosphorylation state appear in an ordered pattern arising from the intrinsic autokinase and autophosphatase rates of KaiC and their modulation by KaiA. Kinetic and biochemical data indicate that one of these phosphoforms inhibits the activity of KaiA through interaction with KaiB, providing the crucial feedback that sustains oscillation. A mathematical model constrained by experimental data quantitatively reproduces the circadian period and the distinctive dynamics of the four phosphoforms.