Publications by authors named "Kim S McKim"

39 Publications

Borealin directs recruitment of the CPC to oocyte chromosomes and movement to the microtubules.

J Cell Biol 2021 Jun;220(6)

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ.

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.
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http://dx.doi.org/10.1083/jcb.202006018DOI Listing
June 2021

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space.

Essays Biochem 2020 09;64(2):251-261

Waksman Institute and Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ, United States of America.

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.
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http://dx.doi.org/10.1042/EBC20190080DOI Listing
September 2020

Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments.

PLoS Genet 2019 05 31;15(5):e1008072. Epub 2019 May 31.

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1008072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581285PMC
May 2019

Narya, a RING finger domain-containing protein, is required for meiotic DNA double-strand break formation and crossover maturation in Drosophila melanogaster.

PLoS Genet 2019 01 7;15(1):e1007886. Epub 2019 Jan 7.

Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336347PMC
January 2019

Kinesin 6 Regulation in Female Meiosis by the Non-conserved N- and C- Terminal Domains.

G3 (Bethesda) 2018 05 4;8(5):1555-1569. Epub 2018 May 4.

Waksman Institute, Rutgers, the State University of New Jersey, NJ-08854

Bipolar spindle assembly occurs in the absence of centrosomes in the oocytes of most organisms. In the absence of centrosomes in oocytes, we have proposed that the kinesin 6 Subito, a MKLP-2 homolog, is required for establishing spindle bipolarity and chromosome biorientation by assembling a robust central spindle during prometaphase I. Although the functions of the conserved motor domains of kinesins is well studied, less is known about the contribution of the poorly conserved N- and C- terminal domains to motor function. In this study, we have investigated the contribution of these domains to kinesin 6 functions in meiosis and early embryonic development. We found that the N-terminal domain has antagonistic elements that regulate localization of the motor to microtubules. Other parts of the N- and C-terminal domains are not required for microtubule localization but are required for motor function. Some of these elements of Subito are more important for either mitosis or meiosis, as revealed by separation-of-function mutants. One of the functions for both the N- and C-terminals domains is to restrict the CPC to the central spindle in a ring around the chromosomes. We also provide evidence that CDK1 phosphorylation of Subito regulates its activity associated with homolog bi-orientation. These results suggest the N- and C-terminal domains of Subito, while not required for localization to the central spindle microtubules, have important roles regulating Subito, by interacting with other spindle proteins and promoting activities such as bipolar spindle formation and homologous chromosome bi-orientation during meiosis.
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http://dx.doi.org/10.1534/g3.117.300571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940148PMC
May 2018

Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes.

Genetics 2017 02 7;205(2):517-527. Epub 2016 Dec 7.

Waksman Institute, Rutgers University, Piscataway, New Jersey 08854

The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD, and the plus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. We have found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. We propose that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces, resulting in the capacity to accurately segregate chromosomes.
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http://dx.doi.org/10.1534/genetics.116.194647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289833PMC
February 2017

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

J Vis Exp 2016 10 31(116). Epub 2016 Oct 31.

Waksman Institute, Rutgers University; Department of Genetics, Rutgers University;

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.
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http://dx.doi.org/10.3791/54666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226183PMC
October 2016

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes.

Chromosoma 2017 06 11;126(3):351-364. Epub 2016 Nov 11.

Waksman Institute, 190 Frelinghuysen Rd, Piscataway, NJ, 08854, USA.

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.
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http://dx.doi.org/10.1007/s00412-016-0618-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426991PMC
June 2017

Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation.

Curr Biol 2016 07 9;26(13):1688-1698. Epub 2016 Jun 9.

Waksman Institute and Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA. Electronic address:

Assembly of the synaptonemal complex (SC) in Drosophila depends on two independent pathways defined by the chromosome axis proteins C(2)M and ORD. Because C(2)M encodes a Kleisin-like protein and ORD is required for sister-chromatid cohesion, we tested the hypothesis that these two SC assembly pathways depend on two cohesin complexes. Through single- and double-mutant analysis to study the mitotic cohesion proteins Stromalin (SA) and Nipped-B (SCC2) in meiosis, we provide evidence that there are at least two meiosis-specific cohesin complexes. One complex depends on C(2)M, SA, and Nipped-B. Despite the presence of mitotic cohesins SA and Nipped-B, this pathway has only a minor role in meiotic sister-centromere cohesion and is primarily required for homolog interactions. C(2)M is continuously incorporated into pachytene chromosomes even though SC assembly is complete. In contrast, the second complex, which depends on meiosis-specific proteins SOLO, SUNN, and ORD is required for sister-chromatid cohesion, localizes to the centromeres and is not incorporated during prophase. Our results show that the two cohesin complexes have unique functions and are regulated differently. Multiple cohesin complexes may provide the diversity of activities required by the meiotic cell. For example, a dynamic complex may allow the chromosomes to regulate meiotic recombination, and a stable complex may be required for sister-chromatid cohesion.
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http://dx.doi.org/10.1016/j.cub.2016.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942336PMC
July 2016

The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.

J Cell Biol 2015 Dec 14;211(6):1113-20. Epub 2015 Dec 14.

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK

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.
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http://dx.doi.org/10.1083/jcb.201507006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687879PMC
December 2015

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes.

Genetics 2016 Jan 12;202(1):61-75. Epub 2015 Nov 12.

Waksman Institute, Rutgers, The State University of New Jersey, New Jersey 08854 Department of Genetics, Rutgers, The State University of New Jersey, New Jersey 08854

Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.
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http://dx.doi.org/10.1534/genetics.115.181081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701103PMC
January 2016

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

PLoS Genet 2015 Oct 16;11(10):e1005605. Epub 2015 Oct 16.

Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America; Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1005605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608789PMC
October 2015

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

Genetics 2012 Oct 3;192(2):431-40. Epub 2012 Aug 3.

Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.

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.
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http://dx.doi.org/10.1534/genetics.112.143503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454874PMC
October 2012

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

Genetics 2012 Oct 3;192(2):417-29. Epub 2012 Aug 3.

Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.

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.
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http://dx.doi.org/10.1534/genetics.112.143495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454873PMC
October 2012

Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila.

Genetics 2012 Jul 27;191(3):739-46. Epub 2012 Apr 27.

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ 08854-8020, USA.

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.
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http://dx.doi.org/10.1534/genetics.112.140996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389970PMC
July 2012

A pathway for synapsis initiation during zygotene in Drosophila oocytes.

Curr Biol 2011 Nov 27;21(21):1852-7. Epub 2011 Oct 27.

Waksman Institute and Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.

Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation [1-4]. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. We investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In "early zygotene" oocytes, synapsis is only observed at the centromeres. We also found that nonhomologous centromeres are clustered during this process. In "mid-zygotene" oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In "late zygotene" oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. We propose that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers.
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http://dx.doi.org/10.1016/j.cub.2011.10.005DOI Listing
November 2011

Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair.

J Cell Biol 2011 Oct 24;195(3):359-67. Epub 2011 Oct 24.

Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA.

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis.
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http://dx.doi.org/10.1083/jcb.201104121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206348PMC
October 2011

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females.

Fly (Austin) 2011 Apr-Jun;5(2):134-40. Epub 2011 Apr 1.

Waksman Institute and Department of Genetics, Rutgers the State University of New Jersey, Piscataway, NJ, USA.

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127062PMC
http://dx.doi.org/10.4161/fly.5.2.14767DOI Listing
September 2011

Drosophila I-R hybrid dysgenesis is associated with catastrophic meiosis and abnormal zygote formation.

J Cell Sci 2010 Oct 14;123(Pt 20):3515-24. Epub 2010 Sep 14.

Centre de Génétique Moléculaire et Cellulaire, CNRS UMR5534, University of Lyon, UCBL Lyon1, Villeurbanne, F-69100, France.

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.
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http://dx.doi.org/10.1242/jcs.073890DOI Listing
October 2010

Chromosome axis defects induce a checkpoint-mediated delay and interchromosomal effect on crossing over during Drosophila meiosis.

PLoS Genet 2010 Aug 12;6(8). Epub 2010 Aug 12.

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, USA.

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.
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http://dx.doi.org/10.1371/journal.pgen.1001059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920846PMC
August 2010

Cytological analysis of meiosis in fixed Drosophila ovaries.

Methods Mol Biol 2009 ;558:197-216

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ, USA.

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.
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http://dx.doi.org/10.1007/978-1-60761-103-5_12DOI Listing
October 2009

Drosophila hold'em is required for a subset of meiotic crossovers and interacts with the dna repair endonuclease complex subunits MEI-9 and ERCC1.

Genetics 2009 Jan 28;181(1):335-40. Epub 2008 Oct 28.

Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854-8020, USA.

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.
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http://dx.doi.org/10.1534/genetics.108.093104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2621184PMC
January 2009

Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation.

Genetics 2009 Jan 28;181(1):39-51. Epub 2008 Oct 28.

Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854-8020, USA.

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.
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http://dx.doi.org/10.1534/genetics.108.093112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2621188PMC
January 2009

mei-38 is required for chromosome segregation during meiosis in Drosophila females.

Genetics 2008 Sep 30;180(1):61-72. Epub 2008 Aug 30.

Waksman Institute and Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA.

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.
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http://dx.doi.org/10.1534/genetics.108.091140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2535709PMC
September 2008

Spindle assembly in the oocytes of mouse and Drosophila--similar solutions to a problem.

Chromosome Res 2007 ;15(5):681-96

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ 08854-8020, USA.

In the oocytes of many organisms a bipolar spindle is assembled in the absence of centrosomes. In this article we review how this occurs in two model organisms, Drosophila melanogaster and Mus musculus. Common themes include an important role for the chromosomes but paradoxically, organization of a bipolar spindle may not involve kinetochore microtubules. Some comparisons are not yet possible, however, since the same genes have usually not been studied in both systems.
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http://dx.doi.org/10.1007/s10577-007-1148-8DOI Listing
October 2007

Misregulation of the kinesin-like protein Subito induces meiotic spindle formation in the absence of chromosomes and centrosomes.

Genetics 2007 Sep 29;177(1):267-80. Epub 2007 Jul 29.

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854-8020, USA.

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.
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http://dx.doi.org/10.1534/genetics.107.076091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2013708PMC
September 2007

When specialized sites are important for synapsis and the distribution of crossovers.

Bioessays 2007 Mar;29(3):217-26

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway NJ 08854, USA.

In C. elegans and D. melanogaster, specialized sites have an important role in meiotic recombination. Recent evidence has shown that these sites in C. elegans have a role in synapsis. Here we compare the initiation of synapsis in organisms with specialized sites and those without. We propose that, early in prophase, synapsis requires an initiator to overcome inhibitory factors that function to prevent synaptonemal complex (SC) formation between nonhomologous sequences. These initiators of SC formation can be stimulated by crossover sites, possibly other types of recombination sites and also specialized sites where recombination does not occur.
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http://dx.doi.org/10.1002/bies.20531DOI Listing
March 2007

Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators.

J Cell Sci 2006 Nov 31;119(Pt 22):4770-80. Epub 2006 Oct 31.

Waksman Institute and Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.

Drosophila Subito is a kinesin 6 family member and ortholog of mitotic kinesin-like protein (MKLP2) in mammalian cells. Based on the previously established requirement for Subito in meiotic spindle formation and for MKLP2 in cytokinesis, we investigated the function of Subito in mitosis. During metaphase, Subito localized to microtubules at the center of the mitotic spindle, probably interpolar microtubules that originate at the poles and overlap in antiparallel orientation. Consistent with this localization pattern, subito mutants improperly assembled microtubules at metaphase, causing activation of the spindle assembly checkpoint and lagging chromosomes at anaphase. These results are the first demonstration of a kinesin 6 family member with a function in mitotic spindle assembly, possibly involving the interpolar microtubules. However, the role of Subito during mitotic anaphase resembles other kinesin 6 family members. Subito localizes to the spindle midzone at anaphase and is required for the localization of Polo, Incenp and Aurora B. Genetic evidence suggested that the effects of subito mutants are attenuated as a result of redundant mechanisms for spindle assembly and cytokinesis. For example, subito double mutants with ncd, polo, Aurora B or Incenp mutations were synthetic lethal with severe defects in microtubule organization.
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http://dx.doi.org/10.1242/jcs.03235DOI Listing
November 2006

The Drosophila calcipressin sarah is required for several aspects of egg activation.

Curr Biol 2006 Jul;16(14):1441-6

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

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.
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http://dx.doi.org/10.1016/j.cub.2006.06.024DOI Listing
July 2006

When size does not matter: pairing sites during meiosis.

Authors:
Kim S McKim

Cell 2005 Dec;123(6):989-92

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.

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http://dx.doi.org/10.1016/j.cell.2005.11.031DOI Listing
December 2005