RESUMEN
The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Humanos , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , Polaridad Celular , Endodermo/metabolismo , Hiperplasia/metabolismo , Intestinos , Embrión no Mamífero/metabolismoRESUMEN
The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption and host defense. Studies on mammalian cultured epithelial cells have uncovered some of the molecular players and physical constraints required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not yet been investigated in detail. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode Caenorhabditis elegans to build a high-resolution and dynamic localization map of known and new brush border markers. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation, but become highly stable once microvilli are built. This new toolbox will be instrumental for understanding the molecular processes of microvilli growth and maintenance in vivo, as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity.
Asunto(s)
Caenorhabditis elegans/metabolismo , Enterocitos/metabolismo , Microvellosidades/metabolismo , Animales , Caenorhabditis elegans/genética , Microvellosidades/genéticaRESUMEN
LA-related protein 1 (LARP-1) belongs to an RNA-binding protein family containing a LA motif. Here, we identify LARP-1 as a regulator of sex determination. In C. elegans hermaphrodites, a complex regulatory network regulates the switch from sperm to oocyte production. We find that simultaneous depletion of larp-1 and the Nanos homologue nos-3 results in germline masculinization. This phenotype is accompanied by a strong reduction of the levels of TRA-1, a GLI-family transcription factor that promotes oogenesis. TRA-1 levels are regulated by CBC(FEM-1), a ubiquitin ligase consisting of the FEM proteins, FEM-1, FEM-2 and FEM-3 and the cullin CUL-2. We show that both the masculinization phenotype and the reduction of TRA-1 levels observed in nos-3;larp-1 mutants require fem-3 activity, suggesting that nos-3 and larp-1 regulate the sperm-oocyte switch by inhibiting the fem genes. Consistently, fem-3 mRNA levels are increased in larp-1 mutants. By contrast, levels of fem-3 mRNA are not affected in nos-3 mutants. Therefore, our data indicate that LARP-1 and NOS-3 promote oogenesis by regulating fem-3 expression through distinct mechanisms.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Oogénesis/fisiología , Proteínas de Unión al ARN/metabolismo , Procesos de Determinación del Sexo/fisiología , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Femenino , Sistema de Señalización de MAP Quinasas , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación , Oocitos/fisiología , Oogénesis/genética , Filogenia , Espermatozoides/fisiologíaRESUMEN
Cadherin-mediated adhesion can be regulated at many levels, as demonstrated by detailed analysis in cell lines. We have investigated the requirements for Drosophila melanogaster epithelial (DE) cadherin regulation in vivo. Investigating D. melanogaster oogenesis as a model system allowed the dissection of DE-cadherin function in several types of adhesion: cell sorting, cell positioning, epithelial integrity, and the cadherin-dependent process of border cell migration. We generated multiple fusions between DE-cadherin and alpha-catenin as well as point-mutated beta-catenin and analyzed their ability to support these types of adhesion. We found that (1) although linking DE-cadherin to alpha-catenin is essential, regulation of the link is not required in any of these types of adhesion; (2) beta-catenin is required only to link DE-cadherin to alpha-catenin; and (3) the cytoplasmic domain of DE-cadherin has an additional specific function for the invasive migration of border cells, which is conserved to other cadherins. The nature of this additional function is discussed.
Asunto(s)
Cadherinas/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Secuencia de Aminoácidos , Animales , Proteínas del Dominio Armadillo , Cadherinas/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/fisiología , Fenotipo , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , alfa Catenina , beta CateninaRESUMEN
Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. beta-catenin links cadherin to the actin cytoskeleton via alpha-catenin. The role of p120/delta-catenin proteins in regulating cadherin function is less clear. Both beta-catenin and p120/delta-catenin are conserved in Drosophila. Here, we address the importance of cadherin-catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or beta-catenin-armadillo (DE-cadherin-Delta beta) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Fosfoproteínas/metabolismo , Animales , Proteínas del Dominio Armadillo , Sitios de Unión/genética , Tipificación del Cuerpo/genética , Células CHO , Cadherinas/genética , Cateninas , Adhesión Celular/genética , Moléculas de Adhesión Celular/genética , Agregación Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Movimiento Celular/fisiología , Anomalías Congénitas/genética , Cricetinae , Proteínas del Citoesqueleto/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Modelos Animales , Mutación/genética , Oogénesis/fisiología , Fosfoproteínas/genética , Estructura Terciaria de Proteína/genética , Catenina deltaRESUMEN
Coordinating mitotic spindle and cytokinetic furrow positioning is essential to ensure proper DNA segregation. Here, we present a novel mechanism, which corrects DNA segregation defects due to cytokinetic furrow mispositioning during the first division of C. elegans embryos. Correction of DNA segregation defects due to an abnormally anterior cytokinetic furrow relies on the concomitant and opposite displacements of the furrow and of the anterior nucleus toward the posterior and anterior poles of the embryo, respectively. It also coincides with cortical blebbing and an anteriorly directed cytoplasmic flow. Although microtubules contribute to nuclear displacement, relaxation of an excessive tension at the anterior cortex plays a central role in the correction process and simultaneously regulates cytoplasmic flow as well as nuclear and furrow displacements. This work thus reveals the existence of a so-far uncharacterized correction mechanism, which is critical to correct DNA segregation defects due to cytokinetic furrow mispositioning.
Asunto(s)
Segregación Cromosómica/fisiología , Citocinesis/fisiología , Animales , Fenómenos Biomecánicos/fisiología , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Núcleo Celular , ADN , Microtúbulos/fisiología , Mitosis/fisiología , Huso Acromático/fisiologíaRESUMEN
The PAR proteins play an essential role in establishing and maintaining cell polarity. While their function is conserved across species, little is known about their regulators and effectors. Here we report the identification of 13 potential components of the C. elegans PAR polarity pathway, identified in an RNAi-based, systematic screen to find suppressors of par-2(it5ts) lethality. Most of these genes are conserved in other species. Phenotypic analysis of double-mutant animals revealed that some of the suppressors can suppress lethality associated with the strong loss-of-function allele par-2(lw32), indicating that they might impinge on the PAR pathway independently of the PAR-2 protein. One of these is the gene nos-3, which encodes a homolog of Drosophila Nanos. We find that nos-3 suppresses most of the phenotypes associated with loss of par-2 function, including early cell division defects and maternal-effect sterility. Strikingly, while PAR-1 activity was essential in nos-3; par-2 double mutants, its asymmetric localization at the posterior cortex was not restored, suggesting that the function of PAR-1 is independent of its cortical localization. Taken together, our results identify conserved components that regulate PAR protein function and also suggest a role for NOS-3 in PAR protein-dependent cell polarity.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Polaridad Celular/genética , Mapeo Cromosómico/métodos , Genes Supresores , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Trastornos del Desarrollo Sexual , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Letales , Masculino , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN/fisiología , ARN Interferente Pequeño/fisiología , Proteínas de Unión al ARN/fisiología , Supresión Genética , Distribución TisularRESUMEN
The first division of the one-cell C. elegans embryo has been a fundamental model in deciphering the mechanisms underlying asymmetric cell division. Polarization of the one-cell zygote is induced by a signal from the sperm centrosome and results in the asymmetric distribution of PAR proteins. Multiple mechanisms then maintain PAR polarity until the end of the first division. Once asymmetrically localized, PAR proteins control several essential aspects of asymmetric division, including the position of the mitotic spindle along the polarity axis. Coordination of the spindle and cytokinetic furrow positions is the next essential step to ensure proper asymmetric division. In this chapter, I review the different mechanisms underlying these successive steps of asymmetric division. Work from the last 30 years has revealed the existence of multiple and redundant regulatory pathways which ensure division robustness. Besides the essential role of PAR proteins, this work also emphasizes the importance of both microtubules and actomyosin throughout the different steps of asymmetric division.
Asunto(s)
División Celular Asimétrica/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Polaridad Celular/fisiología , Animales , Embrión no MamíferoRESUMEN
During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis.