RESUMEN
The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.
Asunto(s)
Proteínas de Drosophila , Meiosis , Animales , Meiosis/genética , Segregación Cromosómica/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Profase/genética , Centrómero/genética , Centrómero/metabolismo , Drosophila/genética , Drosophila/metabolismo , Mitosis , Cinetocoros/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismoRESUMEN
The chromosome passenger complex (CPC) localizes to chromosomes and microtubules, sometimes simultaneously. The CPC also has multiple domains for interacting with chromatin and microtubules. Interactions between the CPC and both the chromatin and microtubules is important for spindle assembly and error correction. Such dual chromatin-microtubule interactions may increase the concentration of the CPC necessary for efficient kinase activity while also making it responsive to specific conditions or structures in the cell. CPC-microtubule dependent functions are considered in the context of the first meiotic division. Acentrosomal spindle assembly is a process that depends on transfer of the CPC from the chromosomes to the microtubules. Furthermore, transfer to the microtubules is not only to position the CPC for a later role in cytokinesis; metaphase I error correction and subsequent bi-orientation of bivalents may depend on microtubule associated CPC interacting with the kinetochores.
Asunto(s)
Cinetocoros , Microtúbulos , Cromatina , Segregación Cromosómica , Cromosomas , Meiosis , Huso AcromáticoRESUMEN
Meiosis in female oocytes lacks centrosomes, the microtubule-organizing centers. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, we examined the role of protein phosphatase 2A (PP2A) in Drosophila oocyte meiosis. We found that both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which has a B subunit encoded by two partially redundant paralogs, wdb and wrd, is also required for maintenance of sister chromatid cohesion, establishment of end-on microtubule attachments, and metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332 and kinetochore protein SPC105R. Although BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. We propose at least three populations of PP2A-B56 regulate meiosis, two of which depend on SPC105R and a third that is associated with the spindle.
Asunto(s)
Proteínas de Drosophila , Cinetocoros , Proteína Fosfatasa 2 , Huso Acromático , Animales , Proteínas de Ciclo Celular/genética , Segregación Cromosómica , Drosophila , Proteínas de Drosophila/genética , Femenino , Meiosis , Microtúbulos , Oocitos , Proteína Fosfatasa 2/genéticaRESUMEN
Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.
Asunto(s)
Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiosis , Microtúbulos/metabolismo , Proteína Fosfatasa 1/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Centrómero/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cromosomas de Insectos/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Regulación de la Expresión Génica , Microtúbulos/ultraestructura , Oocitos/citología , Oocitos/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Separasa/genética , Separasa/metabolismo , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Complejo Sinaptonémico/metabolismo , Complejo Sinaptonémico/ultraestructura , CohesinasRESUMEN
Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over.
Asunto(s)
Intercambio Genético , Recombinación Homóloga/genética , Meiosis/genética , Dominios RING Finger/genética , Animales , Roturas del ADN de Doble Cadena , Drosophila melanogaster/genética , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Dominios Proteicos/genética , Interferencia de ARN , Técnicas del Sistema de Dos Híbridos , Cromosoma X/genéticaRESUMEN
Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.
Asunto(s)
Cromosomas/genética , Meiosis , Oocitos/citología , Huso Acromático/metabolismo , Animales , Cromosomas/metabolismo , Femenino , Humanos , Masculino , Mitosis , Oocitos/metabolismo , Huso Acromático/genéticaRESUMEN
In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Cinetocoros , Proteínas Asociadas a Microtúbulos/genética , Animales , Centrosoma/metabolismo , Segregación Cromosómica/genética , Cromosomas/genética , Drosophila melanogaster/genética , Meiosis/genética , Microtúbulos/genética , Oocitos/crecimiento & desarrollo , Huso Acromático/genéticaRESUMEN
Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. In this study, we aimed to develop a screening pipeline for identifying novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. More than 240 genes with no known function were silenced using RNA interference (RNAi) and the effects on meiosis and fertility were assessed. We identified 94 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction.
Asunto(s)
Proteínas de Drosophila , Infertilidad , Humanos , Animales , Ratones , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transcriptoma , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Meiosis/genética , Segregación Cromosómica , Fertilidad/genética , Infertilidad/metabolismo , Oocitos/metabolismoRESUMEN
Crossovers mediate the accurate segregation of homologous chromosomes during meiosis. The widely conserved pch2 gene of Drosophila melanogaster is required for a pachytene checkpoint that delays prophase progression when genes necessary for DSB repair and crossover formation are defective. However, the underlying process that the pachytene checkpoint is monitoring remains unclear. Here we have investigated the relationship between chromosome structure and the pachytene checkpoint and show that disruptions in chromosome axis formation, caused by mutations in axis components or chromosome rearrangements, trigger a pch2-dependent delay. Accordingly, the global increase in crossovers caused by chromosome rearrangements, known as the "interchromosomal effect of crossing over," is also dependent on pch2. Checkpoint-mediated effects require the histone deacetylase Sir2, revealing a conserved functional connection between PCH2 and Sir2 in monitoring meiotic events from Saccharomyces cerevisiae to a metazoan. These findings suggest a model in which the pachytene checkpoint monitors the structure of chromosome axes and may function to promote an optimal number of crossovers.
Asunto(s)
Segregación Cromosómica , Cromosomas/genética , Intercambio Genético , Drosophila/genética , Meiosis , Animales , Drosophila/citología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Masculino , Fase PaquitenoRESUMEN
The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. It is not known, however, if these two functions require the same CENP-C molecules. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. Consistent with studies in mammals, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. In contrast to CID, maintaining high levels of CENP-C requires expression during prophase. Confirming the importance of this loading, we found CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.
RESUMEN
The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.
Asunto(s)
Quimera/genética , Drosophila/genética , Drosophila/fisiología , Infertilidad/genética , Retroelementos/genética , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/genética , Femenino , Haploidia , Masculino , Meiosis , Oocitos/citología , Oocitos/metabolismo , CigotoRESUMEN
Drosophila has been a model system for meiosis since the discovery of nondisjunction. Subsequent studies have determined that crossing over is required for chromosome segregation, and identified proteins required for the pairing of chromosomes, initiating meiotic recombination, producing crossover events, and building a spindle to segregate the chromosomes. With a variety of genetic and cytological tools, Drosophila remains a model organism for the study of meiosis. This review focusses on meiosis in females because in male meiosis, the use of chiasmata to link homologous chromosomes has been replaced by a recombination-independent mechanism. Drosophila oocytes are also a good model for mammalian meiosis because of biological similarities such as long pauses between meiotic stages and the absence of centrosomes during the meiotic divisions.
Asunto(s)
Drosophila , Meiosis , Animales , Segregación Cromosómica/genética , Cromosomas , Drosophila/genética , Femenino , Masculino , Mamíferos/genética , Meiosis/genética , No Disyunción GenéticaRESUMEN
The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, we developed a strategy to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. We found that an interaction between Borealin and the chromatin is crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. We propose that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/genética , Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Mitosis , Oocitos/fisiología , Animales , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Cinetocoros , Masculino , Microtúbulos/genética , Oocitos/citología , FosforilaciónRESUMEN
During meiosis, programmed DNA double-strand breaks (DSBs) are repaired to create at least one crossover per chromosome arm. Crossovers mature into chiasmata, which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. This process is usually monitored by one or more checkpoints that ensure that DSBs are repaired prior to the meiotic divisions. We show here that mutations in Drosophila genes required to process DSBs into crossovers delay two important steps in meiotic progression: a chromatin-remodeling process associated with DSB formation and the final steps of oocyte selection. Consistent with the hypothesis that a checkpoint has been activated, the delays in meiotic progression are suppressed by a mutation in the Drosophila homolog of pch2. The PCH2-dependent delays also require proteins thought to regulate the number and distribution of crossovers, suggesting that this checkpoint monitors events leading to crossover formation. Surprisingly, two lines of evidence suggest that the PCH2-dependent checkpoint does not reflect the accumulation of unprocessed recombination intermediates: the delays in meiotic progression do not depend on DSB formation or on mei-41, the Drosophila ATR homolog, which is required for the checkpoint response to unrepaired DSBs. We propose that the sites and/or conditions required to promote crossovers are established independently of DSB formation early in meiotic prophase. Furthermore, the PCH2-dependent checkpoint is activated by these events and pachytene progression is delayed until the DSB repair complexes required to generate crossovers are assembled. Interestingly, PCH2-dependent delays in prophase may allow additional crossovers to form.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Intercambio Genético , Roturas del ADN de Doble Cadena , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Fase Paquiteno , Animales , Ensamble y Desensamble de Cromatina/efectos de la radiación , Intercambio Genético/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , Drosophila melanogaster/efectos de la radiación , Femenino , Genes de Insecto , Modelos Genéticos , Mutación/genética , Oocitos/citología , Oocitos/metabolismo , Oocitos/efectos de la radiación , Fase Paquiteno/efectos de la radiación , Fenotipo , Intercambio de Cromátides Hermanas/efectos de la radiación , Coloración y Etiquetado , Complejo Sinaptonémico/metabolismo , Complejo Sinaptonémico/efectos de la radiación , Rayos XRESUMEN
Three Drosophila proteins, ERCC1, MUS312, and MEI-9, function in a complex proposed to resolve double-Holliday-junction intermediates into crossovers during meiosis. We report here the characterization of hold'em (hdm), whose protein product belongs to a single-strand-DNA-binding superfamily of proteins. Mutations in hdm result in reduced meiotic crossover formation and sensitivity to the DNA-damaging agent methyl methanesulfonate. Furthermore, HDM physically interacts with both MEI-9 and ERCC1 in a yeast two-hybrid assay. We conclude that HDM, MEI-9, MUS312, and ERCC1 form a complex that resolves meiotic recombination intermediates into crossovers.
Asunto(s)
Intercambio Genético , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Meiosis , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Intercambio Genético/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Drosophila melanogaster/citología , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Metilmetanosulfonato/toxicidad , Datos de Secuencia Molecular , Mutágenos/toxicidad , Mutación/genética , No Disyunción Genética/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Cinetocoros/metabolismo , Meiosis , Animales , Centrómero/metabolismo , Drosophila , Células HeLa , Humanos , RatonesRESUMEN
Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Óvulo/crecimiento & desarrollo , Anafase/genética , Animales , Proteínas de Unión al Calcio , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Ciclina B/metabolismo , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Femenino , Fertilidad/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Meiosis/fisiología , Modelos Biológicos , Mutación , Óvulo/citología , Óvulo/metabolismo , Poliadenilación , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Espermatozoides/citología , Espermatozoides/ultraestructura , Membrana Vitelina/metabolismoRESUMEN
Meiotic chromosome segregation occurs in Drosophila oocytes on an acentrosomal spindle, which raises interesting questions regarding spindle assembly and function. One is how to organize a bipolar spindle without microtubule organizing centers at the poles. Another question is how to orient the chromosomes without kinetochore capture of microtubules that grow from the poles. We have characterized the mei-38 gene in Drosophila and found it may be required for chromosome organization within the karyosome. Nondisjunction of homologous chromosomes occurs in mei-38 mutants primarily at the first meiotic division in females but not in males where centrosomes are present. Most meiotic spindles in mei-38 oocytes are bipolar but poorly organized, and the chromosomes appear disorganized at metaphase. mei-38 encodes a novel protein that is conserved in the Diptera and may be a member of a multigene family. Mei-38 was previously identified (as ssp1) due to a role in mitotic spindle assembly in a Drosophila cell line. MEI-38 protein localizes to a specific population of spindle microtubules, appearing to be excluded from the overlap of interpolar microtubules in the central spindle. We suggest MEI-38 is required for the stability of parallel microtubules, including the kinetochore microtubules.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Segregación Cromosómica , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila/genética , Animales , Proteínas de Ciclo Celular/genética , Cromosomas/ultraestructura , Cruzamientos Genéticos , Intercambio Genético , Femenino , Genotipo , Cinetocoros/metabolismo , Masculino , Metafase , Microtúbulos/metabolismo , Huso Acromático , TransgenesRESUMEN
Methods are described to analyze two different parts of the Drosophila ovary, which correspond to early stages (pachytene) and late stages (metaphase I and beyond) of meiosis. In addition to taking into account morphology, the techniques differ by fixation conditions and the method to isolate the tissue. Most of these methods are whole mounts, which preserve the three-dimensional structure.
Asunto(s)
Técnicas Citológicas/métodos , Drosophila/citología , Meiosis/fisiología , Ovario/citología , Animales , Análisis Citogenético/métodos , Drosophila/genética , Embrión no Mamífero/citología , Femenino , Meiosis/genética , Modelos Biológicos , Ovario/metabolismo , Fijación del Tejido/métodosRESUMEN
Bipolar spindles assemble in the absence of centrosomes in the oocytes of many species. In Drosophila melanogaster oocytes, the chromosomes have been proposed to initiate spindle assembly by nucleating or capturing microtubules, although the mechanism is not understood. An important contributor to this process is Subito, which is a kinesin-6 protein that is required for bundling interpolar microtubules located within the central spindle at metaphase I. We have characterized the domains of Subito that regulate its activity and its specificity for antiparallel microtubules. This analysis has revealed that the C-terminal domain may interact independently with microtubules while the motor domain is required for maintaining the interaction with the antiparallel microtubules. Surprisingly, deletion of the N-terminal domain resulted in a Subito protein capable of promoting the assembly of bipolar spindles that do not include centrosomes or chromosomes. Bipolar acentrosomal spindle formation during meiosis in oocytes may be driven by the bundling of antiparallel microtubules. Furthermore, these experiments have revealed evidence of a nuclear- or chromosome-based signal that acts at a distance to activate Subito. Instead of the chromosomes directly capturing microtubules, signals released upon nuclear envelope breakdown may activate proteins like Subito, which in turn bundles together microtubules.