Publications by authors named "Hiroyuki Ohkura"

44 Publications

SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56.

J Cell Biol 2021 Feb;220(2)

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK.

Meiosis creates genetic diversity by recombination and segregation of chromosomes. The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous chromosomes, but how its assembly/disassembly is regulated remains to be understood. Here, we report how two major posttranslational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. We found that the ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in Drosophila female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly and maintenance.
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http://dx.doi.org/10.1083/jcb.202009167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780726PMC
February 2021

Identification of 15 New Bypassable Essential Genes of Fission Yeast.

Cell Struct Funct 2019 Sep 31;44(2):113-119. Epub 2019 Aug 31.

Division of Biological Science, Graduate School of Science, Nagoya University.

Every organism has a different set of genes essential for its viability. This indicates that an organism can become tolerant to the loss of an essential gene under certain circumstances during evolution, via the manifestation of 'masked' alternative mechanisms. In our quest to systematically uncover masked mechanisms in eukaryotic cells, we developed an extragenic suppressor screening method using haploid spores deleted of an essential gene in the fission yeast Schizosaccharomyces pombe. We screened for the 'bypass' suppressors of lethality of 92 randomly selected genes that are essential for viability in standard laboratory culture conditions. Remarkably, extragenic mutations bypassed the essentiality of as many as 20 genes (22%), 15 of which have not been previously reported. Half of the bypass-suppressible genes were involved in mitochondria function; we also identified multiple genes regulating RNA processing. 18 suppressible genes were conserved in the budding yeast Saccharomyces cerevisiae, but 13 of them were non-essential in that species. These trends suggest that essentiality bypass is not a rare event and that each organism may be endowed with secondary or backup mechanisms that can substitute for primary mechanisms in various biological processes. Furthermore, the robustness of our simple spore-based methodology paves the way for genome-scale screening.Key words: Schizosaccharomyces pombe, extragenic suppressor screening, bypass of essentiality (BOE), cut7 (kinesin-5), hul5 (E3 ubiquitin ligase).
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http://dx.doi.org/10.1247/csf.19025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877344PMC
September 2019

The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes.

J Cell Biol 2019 09 5;218(9):2854-2864. Epub 2019 Jul 5.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

Before fertilization, oocytes of most species undergo a long, natural arrest in metaphase. Before this, prometaphase I is also prolonged, due to late stable kinetochore-microtubule attachment. How oocytes stably maintain the dynamic spindle for hours during these periods is poorly understood. Here we report that the bipolar spindle changes its molecular architecture during the long prometaphase/metaphase I in oocytes. By generating transgenic flies expressing GFP-tagged spindle proteins, we found that 14 of 25 spindle proteins change their distribution in the bipolar spindle. Among them, microtubule cross-linking kinesins, MKlp1/Pavarotti and kinesin-5/Klp61F, accumulate to the spindle equator in late metaphase. We found that the late equator accumulation of MKlp1/Pavarotti is regulated by a mechanism distinct from that in mitosis. While MKlp1/Pavarotti contributes to the control of spindle length, kinesin-5/Klp61F is crucial for maintaining a bipolar spindle during metaphase I arrest. Our study provides novel insight into how oocytes maintain a bipolar spindle during metaphase arrest.
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http://dx.doi.org/10.1083/jcb.201902110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719438PMC
September 2019

A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes.

J Cell Biol 2018 10 7;217(10):3431-3445. Epub 2018 Aug 7.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Scotland, UK

The meiotic spindle in oocytes is assembled in the absence of centrosomes, the major microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved question in meiosis is how spindle microtubules are generated without centrosomes and only around chromosomes in the exceptionally large volume of oocytes. Here we report a novel oocyte-specific microtubule nucleation pathway that is essential for assembling most spindle microtubules complementarily with the Augmin pathway. This pathway is mediated by the kinesin-6 Subito/MKlp2, which recruits the γ-tubulin complex to the spindle equator to nucleate microtubules in oocytes. Away from chromosomes, Subito interaction with the γ-tubulin complex is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. We further demonstrate in vitro that the Subito complex from ovaries can nucleate microtubules from pure tubulin dimers. Collectively, microtubule nucleation regulated by Subito drives spatially restricted spindle assembly in oocytes.
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http://dx.doi.org/10.1083/jcb.201803172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168254PMC
October 2018

Combining microscopy and biochemistry to study meiotic spindle assembly in Drosophila oocytes.

Methods Cell Biol 2018 11;145:237-248. Epub 2018 Apr 11.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom. Electronic address:

Studies using Drosophila have played pivotal roles in advancing our understanding of molecular mechanisms of mitosis throughout the past decades, due to the short generation time and advanced genetic research of this organism. Drosophila is also an excellent model to study female meiosis in oocytes. Pathways such as the acentrosomal assembly of the meiotic spindle in oocytes are conserved from fly to humans. Collecting and manipulating large Drosophila oocytes for microscopy and biochemistry are both time and cost efficient, offering advantages over mouse or human oocytes. Therefore, Drosophila oocytes serve as an excellent platform for molecular studies of female meiosis using a combination of genetics, microscopy, and biochemistry. Here we describe key methods to observe the formation of the meiotic spindle either in fixed or in live oocytes. Moreover, biochemical methods are described to identify protein-protein interactions in vivo.
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http://dx.doi.org/10.1016/bs.mcb.2018.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031290PMC
December 2018

14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes.

J Cell Biol 2017 10 31;216(10):3029-3039. Epub 2017 Aug 31.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, UK

The meiotic spindle is formed without centrosomes in a large volume of oocytes. Local activation of crucial spindle proteins around chromosomes is important for formation and maintenance of a bipolar spindle in oocytes. We found that phosphodocking 14-3-3 proteins stabilize spindle bipolarity in oocytes. A critical 14-3-3 target is the minus end-directed motor Ncd (human HSET; kinesin-14), which has well-documented roles in stabilizing a bipolar spindle in oocytes. Phospho docking by 14-3-3 inhibits the microtubule binding activity of the nonmotor Ncd tail. Further phosphorylation by Aurora B kinase can release Ncd from this inhibitory effect of 14-3-3. As Aurora B localizes to chromosomes and spindles, 14-3-3 facilitates specific association of Ncd with spindle microtubules by preventing Ncd from binding to nonspindle microtubules in oocytes. Therefore, 14-3-3 translates a spatial cue provided by Aurora B to target Ncd selectively to the spindle within the large volume of oocytes.
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http://dx.doi.org/10.1083/jcb.201704120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626551PMC
October 2017

Kdm5/Lid Regulates Chromosome Architecture in Meiotic Prophase I Independently of Its Histone Demethylase Activity.

PLoS Genet 2016 08 5;12(8):e1006241. Epub 2016 Aug 5.

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

During prophase of the first meiotic division (prophase I), chromatin dynamically reorganises to recombine and prepare for chromosome segregation. Histone modifying enzymes are major regulators of chromatin structure, but our knowledge of their roles in prophase I is still limited. Here we report on crucial roles of Kdm5/Lid, one of two histone demethylases in Drosophila that remove one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). In the absence of Kdm5/Lid, the synaptonemal complex was only partially formed and failed to be maintained along chromosome arms, while localisation of its components at centromeres was unaffected. Kdm5/Lid was also required for karyosome formation and homologous centromere pairing in prophase I. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid can rescue the above cytological defects. Therefore Kdm5/Lid controls chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity.
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http://dx.doi.org/10.1371/journal.pgen.1006241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975413PMC
August 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

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

A negative loop within the nuclear pore complex controls global chromatin organization.

Genes Dev 2015 Sep;29(17):1789-94

The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.

The nuclear pore complex (NPC) tethers chromatin to create an environment for gene regulation, but little is known about how this activity is regulated to avoid excessive tethering of the genome. Here we propose a negative regulatory loop within the NPC controlling the chromatin attachment state, in which Nup155 and Nup93 recruit Nup62 to suppress chromatin tethering by Nup155. Depletion of Nup62 severely disrupts chromatin distribution in the nuclei of female germlines and somatic cells, which can be reversed by codepleting Nup155. Thus, this universal regulatory system within the NPC is crucial to control large-scale chromatin organization in the nucleus.
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http://dx.doi.org/10.1101/gad.264341.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573852PMC
September 2015

Accurate chromosome segregation by probabilistic self-organisation.

BMC Biol 2015 Aug 12;13:65. Epub 2015 Aug 12.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh, EH9 3BF, UK.

Background: For faithful chromosome segregation during cell division, correct attachments must be established between sister chromosomes and microtubules from opposite spindle poles through kinetochores (chromosome bi-orientation). Incorrect attachments of kinetochore microtubules (kMTs) lead to chromosome mis-segregation and aneuploidy, which is often associated with developmental abnormalities such as Down syndrome and diseases including cancer. The interaction between kinetochores and microtubules is highly dynamic with frequent attachments and detachments. However, it remains unclear how chromosome bi-orientation is achieved with such accuracy in such a dynamic process.

Results: To gain new insight into this essential process, we have developed a simple mathematical model of kinetochore-microtubule interactions during cell division in general, i.e. both mitosis and meiosis. Firstly, the model reveals that the balance between attachment and detachment probabilities of kMTs is crucial for correct chromosome bi-orientation. With the right balance, incorrect attachments are resolved spontaneously into correct bi-oriented conformations while an imbalance leads to persistent errors. In addition, the model explains why errors are more commonly found in the first meiotic division (meiosis I) than in mitosis and how a faulty conformation can evade the spindle assembly checkpoint, which may lead to a chromosome loss.

Conclusions: The proposed model, despite its simplicity, helps us understand one of the primary causes of chromosomal instability-aberrant kinetochore-microtubule interactions. The model reveals that chromosome bi-orientation is a probabilistic self-organisation, rather than a sophisticated process of error detection and correction.
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http://dx.doi.org/10.1186/s12915-015-0172-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4533937PMC
August 2015

Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system.

Mol Biol Cell 2015 Apr 18;26(8):1491-508. Epub 2015 Feb 18.

Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, United Kingdom

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end-tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain-containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin-dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.
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http://dx.doi.org/10.1091/mbc.E14-06-1083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395129PMC
April 2015

Meiosis: an overview of key differences from mitosis.

Authors:
Hiroyuki Ohkura

Cold Spring Harb Perspect Biol 2015 Jan 20;7(5). Epub 2015 Jan 20.

The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.

Meiosis is the specialized cell division that generates gametes. In contrast to mitosis, molecular mechanisms and regulation of meiosis are much less understood. Meiosis shares mechanisms and regulation with mitosis in many aspects, but also has critical differences from mitosis. This review highlights these differences between meiosis and mitosis. Recent studies using various model systems revealed differences in a surprisingly wide range of aspects, including cell-cycle regulation, recombination, postrecombination events, spindle assembly, chromosome-spindle interaction, and chromosome segregation. Although a great degree of diversity can be found among organisms, meiosis-specific processes, and regulation are generally conserved.
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http://dx.doi.org/10.1101/cshperspect.a015859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448623PMC
January 2015

The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes.

J Cell Sci 2015 Feb;128(3):566-75

Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. Here, we took advantage of Drosophilaoocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, we found severe undercondensation of chromosomes after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. We show that the main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. We further show that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes.
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http://dx.doi.org/10.1242/jcs.158477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311133PMC
February 2015

Kank Is an EB1 interacting protein that localises to muscle-tendon attachment sites in Drosophila.

PLoS One 2014 9;9(9):e106112. Epub 2014 Sep 9.

The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom.

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106112PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159139PMC
May 2015

Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics.

Mol Biol Cell 2014 Apr 29;25(7):1025-36. Epub 2014 Jan 29.

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

EB1 is a conserved protein that plays a central role in regulating microtubule dynamics and organization. It binds directly to microtubule plus ends and recruits other plus end-localizing proteins. Most EB1-binding proteins contain a Ser-any residue-Ile-Pro (SxIP) motif. Here we describe the isolation of peptide aptamers with optimized versions of this motif by screening for interaction with the Drosophila EB1 protein. The use of small peptide aptamers to competitively inhibit protein interaction and function is becoming increasingly recognized as a powerful technique. We show that SxIP aptamers can bind microtubule plus ends in cells and functionally act to displace interacting proteins by competitive binding. Their expression in developing flies can interfere with microtubules, altering their dynamics. We also identify aptamers binding to human EB1 and EB3, which have sequence requirements similar to but distinct from each other and from Drosophila EB1. This suggests that EB1 paralogues within one species may interact with overlapping but distinct sets of proteins in cells.
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http://dx.doi.org/10.1091/mbc.E13-08-0504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967968PMC
April 2014

In vivo siRNA delivery system for targeting to the liver by poly-l-glutamic acid-coated lipoplex.

Results Pharma Sci 2014 25;4:1-7. Epub 2014 Jan 25.

Institute of Medicinal Chemistry, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan.

In this study, we developed anionic polymer-coated liposome/siRNA complexes (lipoplexes) with chondroitin sulfate C (CS), poly-l-glutamic acid (PGA) and poly-aspartic acid (PAA) for siRNA delivery by intravenous injection, and evaluated the biodistribution and gene silencing effect in mice. The sizes of CS-, PGA- and PAA-coated lipoplexes were about 200?nm and their ?-potentials were negative. CS-, PGA- and PAA-coated lipoplexes did not induce agglutination after mixing with erythrocytes. In terms of biodistribution, siRNAs after intravenous administration of cationic lipoplexes were largely observed in the lungs, but those of CS-, PGA- and PAA-coated lipoplexes were in both the liver and the kidneys, indicating that siRNA might be partially released from the anionic polymer-coated lipoplexes in the blood circulation and accumulate in the kidney, although the lipoplexes can prevent the agglutination with blood components. To increase the association between siRNA and cationic liposome, we used cholesterol-modified siRNA (siRNA-Chol) for preparation of the lipoplexes. When CS-, PGA- and PAA-coated lipoplexes of siRNA-Chol were injected into mice, siRNA-Chol was mainly observed in the liver, not in the kidneys. In terms of the suppression of gene expression in vivo, apolipoprotein B (ApoB) mRNA in the liver was significantly reduced 48?h after single intravenous injection of PGA-coated lipoplex of ApoB siRNA-Chol (2.5?mg?siRNA/kg), but not cationic, CS- and PAA-coated lipoplexes. In terms of toxicity after intravenous injection, CS-, PGA- and PAA-coated lipoplexes did not increase GOT and GPT concentrations in blood. From these findings, PGA coatings for cationic lipoplex of siRNA-Chol might produce a systemic vector of siRNA to the liver.
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http://dx.doi.org/10.1016/j.rinphs.2014.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050376PMC
March 2015

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs.

J Cell Biol 2013 Aug;202(3):479-94

Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, England, UK.

Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin.
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http://dx.doi.org/10.1083/jcb.201211052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734092PMC
August 2013

Cell cycle regulation of microtubule interactomes: multi-layered regulation is critical for the interphase/mitosis transition.

Mol Cell Proteomics 2013 Nov 26;12(11):3135-47. Epub 2013 Jul 26.

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.

Microtubules dramatically change their dynamics and organization at the entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we used an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that the suppression of the microtubule affinity of this mitotic MAP in combination with nuclear localization is essential for microtubule organization in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.
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http://dx.doi.org/10.1074/mcp.M113.028563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820929PMC
November 2013

Meiosis-specific stable binding of augmin to acentrosomal spindle poles promotes biased microtubule assembly in oocytes.

PLoS Genet 2013 Jun 13;9(6):e1003562. Epub 2013 Jun 13.

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

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.
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http://dx.doi.org/10.1371/journal.pgen.1003562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681601PMC
June 2013

The conserved kinase SRPK regulates karyosome formation and spindle microtubule assembly in Drosophila oocytes.

J Cell Sci 2012 Oct 1;125(Pt 19):4457-62. Epub 2012 Aug 1.

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

In Drosophila oocytes, after the completion of recombination, meiotic chromosomes form a compact cluster called the karyosome within the nucleus, and later assemble spindle microtubules without centrosomes. Although these oocyte-specific phenomena are also observed in humans, their molecular basis is not well understood. Here, we report essential roles for the conserved kinase SRPK in both karyosome formation and spindle microtubule assembly in oocytes. We have identified a female-sterile srpk mutant through a cytological screen for karyosome defects. Unlike most karyosome mutants, the karyosome defect is independent of the meiotic recombination checkpoint. Heterochromatin clustering found within the wild-type karyosome is disrupted in the mutant. Strikingly, a loss of SRPK severely prevents microtubule assembly for acentrosomal spindles in mature oocytes. Subsequently, bi-orientation and segregation of meiotic chromosomes are also defective. Therefore, this study demonstrates new roles of this conserved kinase in two independent meiotic steps specific to oocytes.
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http://dx.doi.org/10.1242/jcs.107979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500864PMC
October 2012

Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis.

PLoS One 2011 2;6(9):e24174. Epub 2011 Sep 2.

The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom.

Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024174PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166307PMC
December 2011

A PAR-1-dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte.

J Cell Biol 2011 Jul;194(1):121-35

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, England, UK.

Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. In this paper, we use rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport. We found that all MTs in the oocyte were highly dynamic and were organized with a biased random polarity that increased toward the posterior. This organization originated through MT nucleation at the oocyte nucleus and cortex, except at the posterior end of the oocyte, where PAR-1 suppressed nucleation. Our findings explain the biased random posterior cargo movements in the oocyte that establish the germline and posterior.
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http://dx.doi.org/10.1083/jcb.201103160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135408PMC
July 2011

The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila.

PLoS Genet 2010 Oct 28;6(10):e1001179. Epub 2010 Oct 28.

The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.
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http://dx.doi.org/10.1371/journal.pgen.1001179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965759PMC
October 2010

Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis.

J Cell Biol 2009 Mar 16;184(6):777-84. Epub 2009 Mar 16.

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

The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex.
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http://dx.doi.org/10.1083/jcb.200811102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699157PMC
March 2009

Spatial control of branching within dendritic arbors by dynein-dependent transport of Rab5-endosomes.

Nat Cell Biol 2008 Oct 31;10(10):1164-71. Epub 2008 Aug 31.

Department of Biophysics, Graduate School of Science, Sakyo-ku, Kyoto 606-8507, Japan.

Dendrites allow neurons to integrate sensory or synaptic inputs, and the spatial disposition and local density of branches within the dendritic arbor limit the number and type of inputs. Drosophila melanogaster dendritic arborization (da) neurons provide a model system to study the genetic programs underlying such geometry in vivo. Here we report that mutations of motor-protein genes, including a dynein subunit gene (dlic) and kinesin heavy chain (khc), caused not only downsizing of the overall arbor, but also a marked shift of branching activity to the proximal area within the arbor. This phenotype was suppressed when dominant-negative Rab5 was expressed in the mutant neurons, which deposited early endosomes in the cell body. We also showed that 1) in dendritic branches of the wild-type neurons, Rab5-containing early endosomes were dynamically transported and 2) when Rab5 function alone was abrogated, terminal branches were almost totally deleted. These results reveal an important link between microtubule motors and endosomes in dendrite morphogenesis.
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http://dx.doi.org/10.1038/ncb1776DOI Listing
October 2008

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

A microtubule interactome: complexes with roles in cell cycle and mitosis.

PLoS Biol 2008 Apr;6(4):e98

Department of Zoology, University of Oxford, Oxford, United Kingdom.

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.
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http://dx.doi.org/10.1371/journal.pbio.0060098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323305PMC
April 2008

Regulation of gene expression during M-G1-phase in fission yeast through Plo1p and forkhead transcription factors.

J Cell Sci 2008 Jan 4;121(Pt 1):38-47. Epub 2007 Dec 4.

Division of Biochemistry and Molecular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.

In fission yeast the expression of several genes during M-G1 phase is controlled by binding of the PCB binding factor (PBF) transcription factor complex to Pombe cell cycle box (PCB) promoter motifs. Three components of PBF have been identified, including two forkhead-like proteins Sep1p and Fkh2p, and a MADS-box-like protein, Mbx1p. Here, we examine how PBF is controlled and reveal a role for the Polo kinase Plo1p. plo1(+) shows genetic interactions with sep1(+), fkh2(+) and mbx1(+), and overexpression of a kinase-domain mutant of plo1 abolishes M-G1-phase transcription. Plo1p binds to and directly phosphorylates Mbx1p, the first time a Polo kinase has been shown to phosphorylate a MADS box protein in any organism. Fkh2p and Sep1p interact in vivo and in vitro, and Fkh2p, Sep1p and Plo1p contact PCB promoters in vivo. However, strikingly, both Fkh2p and Plo1p bind to PCB promoters only when PCB-controlled genes are not expressed during S- and G2-phase, whereas by contrast Sep1p contacts PCBs coincident with M-G1-phase transcription. Thus, Plo1p, Fkh2p and Sep1p control M-G1-phase gene transcription through a combination of phosphorylation and cell-cycle-specific DNA binding to PCBs.
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http://dx.doi.org/10.1242/jcs.019489DOI Listing
January 2008

NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus.

J Cell Biol 2007 Dec 26;179(5):817-24. Epub 2007 Nov 26.

The Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. In this study, we report that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. We find that the noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. We propose that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms.
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http://dx.doi.org/10.1083/jcb.200706067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2099182PMC
December 2007