Genome integrity depends on the fidelity of chromosome segregation during mitosis and meiosis. Defects in these processes results in an abnormal number of chromosomes, or aneuploidy, which is usually catastrophic and is the leading cause of infertility in women and the cause of disorders such ‘as Down’s syndrome and is usually associated with tumor cell development. Research in the McKim laboratory is directed at understanding the mechanism of chromosome segregation during meiosis in the fruit fly Drosophila melanogaster. Our experiments include several innovative approaches that combine sophisticated genetics and high-resolution immunofluorescence microscopy to image the structure and behavior of meiotic proteins, chromosomes and spindles in oocytes.
Using molecular genetics, cell biology and genomic approaches, the McKim lab identifies and characterizes genes that are important for all aspects of the meiotic pathway including: i) the assembly of meiotic chromatin, ii) the pairing of homologous chromosomes, iii) recombination between chromosomes, iv) the assembly of a meiotic spindle and v) the orientation and segregation of homologous chromosome. Many of the important genes in this process are also involved in maintaining genome integrity and the fidelity of chromosome division in other cell types. Therefore, these studies also provide insights into the factors affecting genome stability in mitotic cells and the development of cancer cells.
Complete list of publications: [Pubmed]
Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila.
Joyce EF, Paul A, Chen KE, Tanneti N, McKim KS. 2012. Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila. Genetics. 191(3):739-46.
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.
Microtubule-depolymerizing kinesin KLP10A restricts the length of the acentrosomal meiotic spindle in Drosophila females
Radford SJ, Harrison AM, McKim KS. 2012. Microtubule-depolymerizing kinesin KLP10A restricts the length of the acentrosomal meiotic spindle in Drosophila females. Genetics. 192(2):431-40
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.
The chromosomal passenger complex is required for meiotic acentrosomal spindle assembly and chromosome biorientation.
Radford SJ, Jang JK, McKim KS. 2012. The chromosomal passenger complex is required for meiotic acentrosomal spindle assembly and chromosome biorientation. Genetics. 192(2):417-29.
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.
Aurora B and cyclin B have opposite effects on the timing of cytokinesis abscission in Drosophila germ cells and in vertebrate somatic cells.
Mathieu, J., Cauvin, C., Moch, C., Radford, S.J., Sampaio, P.C., Perdigoto, C.N., Schweisguth, F., Bardin, A.J., Sunkel, C.E., McKim, K.S., Echard, A., & Huynh, J. 2013. Aurora B and cyclin B have opposite effects on the timing of cytokinesis abscission in Drosophila germ cells and in vertebrate somatic cells. Developmental cell, 26:3, 250-65 .
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.
Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.
Radford SJ, Hoang TL, Głuszek AA, Ohkura H, McKim KS. 2015. Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes. PLoS genetics. 11(10):e1005605.
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.
The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.
Głuszek AA, Cullen CF, Li W, Battaglia RA, Radford SJ, Costa MF, McKim KS, Goshima G, Ohkura H. 2015. The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes. J Cell Biol. 211 (6) 1113-1120
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.
Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes.
Das A, Shah SJ, Fan B, Paik D, DiSanto DJ, Hinman AM, Cesario JM, Battaglia RA, Demos N, McKim KS. 2016. Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes. Genetics. 202(1):61-75.
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.
Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation.
Gyuricza MR, Manheimer KB, Apte V, Krishnan B, Joyce EF, McKee BD, McKim KS. 2016. Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation. Curr Biol. 26(13):1688-1698.
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.
Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes.
Radford SJ, McKim KS. 2016. Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes. J Vise Exp. (116).
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.
Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes.
Radford SJ, Go AM, McKim KS. 2017. Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes. Genetics. 205(2):517-527.
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.
The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes.
Radford SJ, Nguyen AL, Schindler K, McKim KS. 2017. The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes. Chromosoma. 126(3):351-364
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.
Kinesin 6 Regulation in Drosophila Female Meiosis by the Non-conserved N- and C- Terminal Domains.
Das A, Cesario J, Hinman AM, Jang JK, McKim KS. 2018. Kinesin 6 Regulation in Drosophila Female Meiosis by the Non-conserved N- and C- Terminal Domains. G3 (Bethesda, Md.). 8(5):1555-1569.
Bipolar spindle assembly occurs in the absence of centrosomes in the oocytes of most organisms. In the absence of centrosomes in Drosophila 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.
Kim McKim is a Principal Investigator at the Waksman Institute and Professor of Genetics in the School of Arts and Science. Professor McKim uses the fruit fly Drosophila melanogaster to study the fundamental mechanisms of heredity. His lab uses molecular genetics, cell biology and genomic approaches to identify and characterize genes that are important for reproductive fitness, including the segregation of chromosomes during meiosis and other processes that lead to the the generation of a healthy oocyte.
Complete list of publications here