RESUMEN
Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas del Citoesqueleto/deficiencia , Situs Inversus/genética , Adolescente , Adulto , Animales , Astenozoospermia/patología , Axonema/ultraestructura , Sistemas CRISPR-Cas/genética , Cilios/metabolismo , Cilios/ultraestructura , Proteínas del Citoesqueleto/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Epidídimo/patología , Femenino , Flagelos/metabolismo , Flagelos/ultraestructura , Humanos , Mutación con Pérdida de Función , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Planarias/citología , Planarias/genética , Planarias/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/patología , Situs Inversus/diagnóstico por imagen , Situs Inversus/patología , Motilidad Espermática/genética , Tomografía Computarizada por Rayos X , Secuenciación del ExomaRESUMEN
In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3'-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3'-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.
Asunto(s)
Células Madre Adultas/fisiología , Planarias/genética , Planarias/fisiología , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Regeneración/genética , Regeneración/fisiología , Células Madre Adultas/citología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Autorrenovación de las Células/genética , Autorrenovación de las Células/fisiología , Modelos Biológicos , Conformación de Ácido Nucleico , Planarias/citología , Interferencia de ARN , Procesamiento Postranscripcional del ARN , Empalme del ARN , ARN de Helminto/química , Homología de Secuencia de Ácido NucleicoRESUMEN
Despite the identification of numerous regulators of regeneration in different animal models, a fundamental question remains: why do some wounds trigger the full regeneration of lost body parts, whereas others resolve by mere healing? By selectively inhibiting regeneration initiation, but not the formation of a wound epidermis, here we create headless planarians and finless zebrafish. Strikingly, in both missing-tissue contexts, injuries that normally do not trigger regeneration activate complete restoration of heads and fin rays. Our results demonstrate that generic wound signals have regeneration-inducing power. However, they are interpreted as regeneration triggers only in a permissive tissue context: when body parts are missing, or when tissue-resident polarity signals, such as Wnt activity in planarians, are modified. Hence, the ability to decode generic wound-induced signals as regeneration-initiating cues may be the crucial difference that distinguishes animals that regenerate from those that cannot.
Asunto(s)
Sistema de Señalización de MAP Quinasas/genética , Planarias/genética , Regeneración/genética , Vía de Señalización Wnt/genética , Cicatrización de Heridas/genética , Pez Cebra/genética , Aletas de Animales/fisiología , Animales , Cabeza/fisiología , Planarias/fisiología , Regeneración/fisiología , Transducción de Señal , Cicatrización de Heridas/fisiología , Heridas y Lesiones , Pez Cebra/fisiologíaRESUMEN
Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating ß1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.