techniques-for-imaging-prometaphase-metaphase-meiosis-i-fixed

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed <em>Drosophila</em> Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Rutgers University

Taking Drosophila Rad51 for a SPiN.

Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates.

Increase in Ty1 cDNA recombination in yeast sir4 mutant strains at high temperature.

In most eukaryotes, crossovers are not independently distributed along the length of a chromosome. Instead, they appear to avoid close proximity to one another--a phenomenon known as crossover interference. Previously, for three of the five Arabidopsis chromosomes, we measured the strength of interference and suggested a model wherein some crossovers experience interference while others do not. Here we show, using the same model, that the fraction of interference-insensitive crossovers is significantly smaller on the remaining two chromosomes. Since these two chromosomes bear the Arabidopsis NOR domains, the possibility that these chromosomal regions influence interference is discussed.

Crossover interference on nucleolus organizing region-bearing chromosomes in Arabidopsis.

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.

Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers.

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2-7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.

REC, Drosophila MCM8, drives formation of meiotic crossovers.

Meiotic recombination gives rise to crossovers, which are required in most organisms for the faithful segregation of homologous chromosomes during meiotic cell division. Characterization of crossover-defective mutants has contributed much to our understanding of the molecular mechanism of crossover formation. We report here a molecular analysis of recombination in a Drosophila melanogaster crossover-defective mutant, mei-9. In the absence of mei-9 activity, postmeiotic segregation associated with noncrossovers occurs at the expense of crossover products, suggesting that the underlying meiotic function for MEI-9 is in crossover formation rather than mismatch repair. In support of this, analysis of the arrangement of heteroduplex DNA in the postmeiotic segregation products reveals different patterns from those observed in Drosophila Msh6 mutants, which are mismatch-repair defective. This analysis also provides evidence that the double-strand break repair model applies to meiotic recombination in Drosophila. Our results support a model in which MEI-9 nicks Holliday junctions to generate crossovers during meiotic recombination, and, in the absence of MEI-9 activity, the double Holliday junction intermediate instead undergoes dissolution to generate noncrossover products in which heteroduplex is unrepaired.

Heteroduplex DNA in meiotic recombination in Drosophila mei-9 mutants.

Crossovers (COs) generated through meiotic recombination are important for the correct segregation of homologous chromosomes during meiosis. Several models describing the molecular mechanism of meiotic recombination have been proposed. These models differ in the arrangement of heteroduplex DNA (hDNA) in recombination intermediates. Heterologies in hDNA are usually repaired prior to the recovery of recombination products, thereby obscuring information about the arrangement of hDNA. To examine hDNA in meiotic recombination in Drosophila melanogaster, we sought to block hDNA repair by conducting recombination assays in a mutant defective in mismatch repair (MMR). We generated mutations in the MMR gene Msh6 and analyzed recombination between highly polymorphic homologous chromosomes. We found that hDNA often goes unrepaired during meiotic recombination in an Msh6 mutant, leading to high levels of postmeiotic segregation; however, hDNA and gene conversion tracts are frequently discontinuous, with multiple transitions between gene conversion, restoration, and unrepaired hDNA. We suggest that these discontinuities reflect the activity of a short-patch repair system that operates when canonical MMR is defective.

Meiotic recombination in Drosophila Msh6 mutants yields discontinuous gene conversion tracts.

During meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes.

The chromosomal passenger complex is required for meiotic acentrosomal spindle assembly and chromosome biorientation.

During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation.

Microtubule-depolymerizing kinesin KLP10A restricts the length of the acentrosomal meiotic spindle in Drosophila females.

Abscission is the last step of cytokinesis that physically separates the cytoplasm of sister cells. As the final stage of cell division, abscission is poorly characterized during animal development. Here, we show that Aurora B and Survivin regulate the number of germ cells in each Drosophila egg chamber by inhibiting abscission during differentiation. This inhibition is mediated by an Aurora B-dependent phosphorylation of Cyclin B, as a phosphomimic form of Cyclin B rescues premature abscission caused by a loss of function of Aurora B. We show that Cyclin B localizes at the cytokinesis bridge, where it promotes abscission. We propose that mutual inhibitions between Aurora B and Cyclin B regulate the duration of abscission and thereby the number of sister cells in each cyst. Finally, we show that inhibitions of Aurora B and Cyclin-dependent kinase 1 activity in vertebrate cells also have opposite effects on the timing of abscission, suggesting a possible conservation of these mechanisms.

Aurora B and cyclin B have opposite effects on the timing of cytokinesis abscission in Drosophila germ cells and in vertebrate somatic cells.

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.

Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore-microtubule attachment in oocytes.

Sarah J. Radford

Waksman Institute

Rutgers University

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Sarah J. Radford

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Rutgers University

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Waksman Institute