Publications by authors named "Janet K Jang"

14 Publications

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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

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

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

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

Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis.

Development 2008 Oct 28;135(19):3239-46. Epub 2008 Aug 28.

Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh, UK.

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not in microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.
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http://dx.doi.org/10.1242/dev.022624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2536564PMC
October 2008

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

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

Meiotic recombination in Drosophila females depends on chromosome continuity between genetically defined boundaries.

Genetics 2005 Feb 15;169(2):767-81. Epub 2004 Nov 15.

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

In the pairing-site model, specialized regions on each chromosome function to establish meiotic homolog pairing. Analysis of these sites could provide insights into the mechanism used by Drosophila females to form a synaptonemal complex (SC) in the absence of meiotic recombination. These specialized sites were first established on the X chromosome by noting that there were barriers to crossover suppression caused by translocation heterozygotes. These sites were genetically mapped and proposed to be pairing sites. By comparing the cytological breakpoints of third chromosome translocations to their patterns of crossover suppression, we have mapped two sites on chromosome 3R. We have performed experiments to determine if these sites have a role in meiotic homolog pairing and the initiation of recombination. Translocation heterozygotes exhibit reduced gene conversion within the crossover-suppressed region, consistent with an effect on the initiation of meiotic recombination. To determine if homolog pairing is disrupted in translocation heterozygotes, we used fluorescent in situ hybridization to measure the extent of homolog pairing. In wild-type oocytes, homologs are paired along their entire lengths prior to accumulation of the SC protein C(3)G. Surprisingly, translocation heterozygotes exhibited homolog pairing similar to wild type within the crossover-suppressed regions. This result contrasted with our observations of c(3)G mutant females, which were found to be defective in pairing. We propose that each Drosophila chromosome is divided into several domains by specialized sites. These sites are not required for homolog pairing. Instead, the initiation of meiotic recombination requires continuity of the meiotic chromosome structure within each of these domains.
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http://dx.doi.org/10.1534/genetics.104.035824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449117PMC
February 2005

Relationship of DNA double-strand breaks to synapsis in Drosophila.

J Cell Sci 2003 Aug 10;116(Pt 15):3069-77. Epub 2003 Jun 10.

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, 190 Frelinghuysen RD, Piscataway, New Jersey 08854-8020, USA.

The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms. Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans. To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites, phosphorylation of HIS2AV (gamma-HIS2AV). Our results support the hypothesis that double-strand break formation occurs after synapsis. Interestingly, we detected a low (10-25% of wildtype) number of gamma-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks. Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants. There may also be an interaction between the recruitment of repair proteins and phosphorylation.
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http://dx.doi.org/10.1242/jcs.00614DOI Listing
August 2003

Meiotic recombination and chromosome segregation in Drosophila females.

Annu Rev Genet 2002 11;36:205-32. Epub 2002 Jun 11.

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

In this review, we describe the pathway for generating meiotic crossovers in Drosophila melanogaster females and how these events ensure the segregation of homologous chromosomes. As appears to be common to meiosis in most organisms, recombination is initiated with a double-strand break (DSB). The interesting differences between organisms appear to be associated with what chromosomal events are required for DSBs to form. In Drosophila females, the synaptonemal complex is required for most DSB formation. The repair of these breaks requires several DSB repair genes, some of which are meiosis-specific, and defects at this stage can have effects downstream on oocyte development. This has been suggested to result from a checkpoint-like signaling between the oocyte nucleus and gene products regulating oogenesis. Crossovers result from genetically controlled modifications to the DSB repair pathway. Finally, segregation of chromosomes joined by a chiasma requires a bipolar spindle. At least two kinesin motor proteins are required for the assembly of this bipolar spindle, and while the meiotic spindle lacks traditional centrosomes, some centrosome components are found at the spindle poles.
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http://dx.doi.org/10.1146/annurev.genet.36.041102.113929DOI Listing
July 2003

mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster.

Genetics 2002 Sep;162(1):245-58

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

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462256PMC
September 2002

subito encodes a kinesin-like protein required for meiotic spindle pole formation in Drosophila melanogaster.

Genetics 2002 Apr;160(4):1489-501

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

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462067PMC
April 2002

Cytoplasmic localization and evolutionary conservation of MEI-218, a protein required for meiotic crossing-over in Drosophila.

Mol Biol Cell 2002 Jan;13(1):84-95

Waksman Institute, Rutgers University, Piscataway, New Jersey 08854-8020.

During Drosophila oogenesis, the oocyte is formed within a 16-cell cyst immediately after four incomplete cell divisions. One of the primary events in oocyte development is meiotic recombination. Here, we report the intracellular localization of the MEI-218 protein that is specifically required for meiotic crossing-over. To understand the role of mei-218 in meiosis and to study the regulation of genes required for meiotic recombination, we characterized the expression pattern of its RNA and protein. Furthermore, we cloned and sequenced mei-218 from two other Drosophila species. The mei-218 RNA and protein have a similar expression pattern, appearing first in early meiotic prophase and then rapidly disappearing as prophase is completed. This pattern corresponds to a specific appearance of the mei-218 gene product in the region of the ovary where meiotic prophase occurs. Although mei-218 is required for 95% of all crossovers, the protein is found exclusively in the cytoplasm. Based on these results, we suggest that mei-218 does not have a direct role in recombination but rather regulates other factors required for the production of crossovers. We propose that mei-218 is a molecular link between oocyte differentiation and meiosis.
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http://dx.doi.org/10.1091/mbc.01-06-0318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC65074PMC
January 2002