RESUMEN
Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteómica/métodos , Algoritmos , Animales , Autofagia , Proteínas de Caenorhabditis elegans/análisis , Interpretación Estadística de Datos , Bases de Datos de Proteínas , Proteínas Asociadas a Microtúbulos/análisis , Unión Proteica , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). We establish here that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Galphai and Gbeta13F. Ric-8, Galphai and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis.
Asunto(s)
División Celular/fisiología , Drosophila/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Neuronas Aferentes/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Receptores Frizzled/fisiología , Subunidades beta de la Proteína de Unión al GTP/fisiología , Proteínas de Unión al GTP/genética , Huso Acromático/metabolismo , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
The orientation of cell division has a crucial role in early embryo body plan specification, axis determination and cell fate diversity generation, as well as in the morphogenesis of tissues and organs. In many instances, cell division orientation is regulated by the planar cell polarity (PCP) pathways: the Wnt/Frizzled non-canonical pathway or the Fat/Dachsous/Four-jointed pathway. Firstly, using asymmetric cell division in both Drosophila and C. elegans, we describe the central role of the Wnt/Frizzled pathway in the regulation of asymmetric cell division orientation, focusing on its cooperation with either the Src kinase pathway or the heterotrimeric G protein pathway. Secondly, we describe our present understanding of the mechanisms by which the planar cell polarity pathways drive tissue morphogenesis by regulating the orientation of symmetric cell division within a field of cells. Finally, we will discuss the important avenues that need to be explored in the future to better understand how planar cell polarity pathways control embryo body plan determination, cell fate specification or tissue morphogenesis by mitotic spindle orientation.
Asunto(s)
División Celular , Polaridad Celular , Transducción de Señal , Animales , Proteínas/metabolismo , Huso Acromático/metabolismoRESUMEN
Human and mouse oocytes' developmental potential can be predicted by their mechanical properties. Their development into blastocysts requires a specific stiffness window. In this study, we combine live-cell and computational imaging, laser ablation, and biophysical measurements to investigate how deregulation of cortex tension in the oocyte contributes to early developmental failure. We focus on extra-soft cells, the most common defect in a natural population. Using two independent tools to artificially decrease cortical tension, we show that chromosome alignment is impaired in extra-soft mouse oocytes, despite normal spindle morphogenesis and dynamics, inducing aneuploidy. The main cause is a cytoplasmic increase in myosin-II activity that could sterically hinder chromosome capture. We describe here an original mode of generation of aneuploidies that could be very common in oocytes and could contribute to the high aneuploidy rate observed during female meiosis, a leading cause of infertility and congenital disorders.
Asunto(s)
Aneuploidia , Proteínas del Citoesqueleto/metabolismo , Miosina Tipo II/metabolismo , Oocitos/patología , Animales , Segregación Cromosómica , Femenino , Infertilidad/etiología , Meiosis , Ratones , OogénesisRESUMEN
Laser ablation is a powerful tool to study forces within biological systems. This technique has been extensively used to study mitotic spindle formation and chromosome segregation. This chapter describes laser ablation of microtubule-chromosome attachments coupled to fluorescence live microscopy and quantitative analysis of individual chromosome movement after microtubule severing. This method allows to gain insight into the organization and dynamics of the meiotic spindle and chromosomes in metaphase I mouse oocytes.
Asunto(s)
Segregación Cromosómica , Terapia por Láser/métodos , Microscopía Fluorescente/métodos , Microtúbulos/metabolismo , Oocitos/fisiología , Animales , Femenino , Meiosis , Ratones , Oocitos/citología , Oocitos/efectos de la radiación , Huso Acromático/metabolismoRESUMEN
Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.
Asunto(s)
Centriolos/metabolismo , Células Germinativas/metabolismo , Oocitos/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Centriolos/efectos de los fármacos , Centrosoma/efectos de los fármacos , Centrosoma/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Metafase/efectos de los fármacos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Nocodazol/farmacología , Oocitos/citología , Oocitos/efectos de los fármacos , Oogonios/citología , Oogonios/efectos de los fármacos , Oogonios/metabolismo , Ovario/embriología , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Polos del Huso/efectos de los fármacos , Polos del Huso/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Edición Génica , Meiosis , Centro Organizador de los Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Femenino , Ratones , Ratones Transgénicos , Oocitos/citología , Huso Acromático/genéticaRESUMEN
Endocytosis and autophagy are key vesicular pathways involved in degradation and recycling of cellular material. Both degradative pathways finally fuse with lysosome but are indeed interconnected at several levels. In particular, the fusion between endosomes and autophagosomes can generate intermediate vesicles named amphisomes. We analyzed the physiological and developmental roles of the ESCRT machinery in a model organism, the nematode Caenorhabditis elegans and showed that the blockage of the endosomal maturation triggers the induction of autophagic activity. This chapter describes several methods for studying endocytosis, autophagy, and their interconnection in C. elegans. A series of genetic, biochemical, and microscopy analyses has been used to study at the cellular and developmental levels, the cross talks between autophagy and endocytosis.
Asunto(s)
Autofagia/genética , Caenorhabditis elegans/metabolismo , Endocitosis/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Fagosomas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Endosomas/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Vectores Genéticos , Lisosomas/ultraestructura , Microscopía Electrónica , Microscopía Fluorescente , Fagosomas/ultraestructura , Transducción de Señal , TransgenesRESUMEN
The formation of the autophagic vesicles requires the recruitment of ubiquitin-like Atg8 proteins to the membrane of nascent autophagosomes. Seven Atg8 homologs are present in mammals, split into the LC3 and the GABARAP/GATE-16 families, whose respective functions are unknown. Using Caenorhabditis elegans, we investigated the functions of the GABARAP and the LC3 homologs, LGG-1 and LGG-2, in autophagosome biogenesis. Both LGG-1 and LGG-2 localize to the autophagosomes but display partially overlapping patterns. During allophagy, a developmentally stereotyped autophagic flux, LGG-1 acts upstream of LGG-2 to allow its localization to autophagosomes. LGG-2 controls the maturation of LGG-1-positive autophagosomes and facilitates the tethering with the lysosomes through a direct interaction with the VPS-39 HOPS complex subunit. Genetic analyses sustain a sequential implication of LGG-1, LGG-2, RAB-7, and HOPS complex to generate autolysosomes. The duplications of Atg8 in metazoans thus allowed the acquisition of specialized functions for autophagosome maturation.
Asunto(s)
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación/genética , Fagosomas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Endosomas/metabolismo , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/metabolismoRESUMEN
Several reports in fly, nematode and mammalian cells have revealed that the inactivation of endosomal sorting complexes required for transport (ESCRT) blocks the endosomal maturation but also leads to the increased number of autophagosomal structures. In this review we compare these data and conclude that the way ESCRT mutations affect the relationships between autophagosomes and endosomes cannot be generalized but depends on the studied species. We propose that the effect of ESCRT mutations on autophagy is directly dependent of the level of interaction between autophagosomes and endosomes. In particular, the formation of amphisomes during autophagosomal maturation could be the key point to explain the differences observed between species. These observations highlight the importance of multiple model organisms to decipher the complexity of relationships between such dynamic vesicles.
RESUMEN
The Frizzled receptor and Dishevelled effector regulate mitotic spindle orientation in both vertebrates and invertebrates, but how Dishevelled orients the mitotic spindle is unknown. Using the Drosophila S2 cell "induced polarity" system, we find that Dishevelled cortical polarity is sufficient to orient the spindle and that Dishevelled's DEP domain mediates this function. This domain binds a C-terminal domain of Mud (the Drosophila NuMA ortholog), and Mud is required for Dishevelled-mediated spindle orientation. In Drosophila, Frizzled-Dishevelled planar cell polarity (PCP) orients the sensory organ precursor (pI) spindle along the anterior-posterior axis. We show that Dishevelled and Mud colocalize at the posterior cortex of pI, Mud localization at the posterior cortex requires Dsh, and Mud loss-of-function randomizes spindle orientation. During zebrafish gastrulation, the Wnt11-Frizzled-Dishevelled PCP pathway orients spindles along the animal-vegetal axis, and reducing NuMA levels disrupts spindle orientation. Overall, we describe a Frizzled-Dishevelled-NuMA pathway that orients division from Drosophila to vertebrates.