| 1. |
- Agrawal, Ritvija, et al.
(författare)
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The KASH5 protein involved in meiotic chromosomal movements is a novel dynein activating adaptor
- 2022
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Ingår i: eLife. - 2050-084X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.
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| 2. |
- Fujiwara, Yasuhiro, et al.
(författare)
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Meiotic cohesins mediate initial loading of HORMAD1 to the chromosomes and coordinate SC formation during meiotic prophase.
- 2020
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Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 16:9
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Tidskriftsartikel (refereegranskat)abstract
- During meiotic prophase, sister chromatids are organized into axial element (AE), which underlies the structural framework for the meiotic events such as meiotic recombination and homolog synapsis. HORMA domain-containing proteins (HORMADs) localize along AE and play critical roles in the regulation of those meiotic events. Organization of AE is attributed to two groups of proteins: meiotic cohesins REC8 and RAD21L; and AE components SYCP2 and SYCP3. It has been elusive how these chromosome structural proteins contribute to the chromatin loading of HORMADs prior to AE formation. Here we newly generated Sycp2 null mice and showed that initial chromatin loading of HORMAD1 was mediated by meiotic cohesins prior to AE formation. HORMAD1 interacted not only with the AE components SYCP2 and SYCP3 but also with meiotic cohesins. Notably, HORMAD1 interacted with meiotic cohesins even in Sycp2-KO, and localized along cohesin axial cores independently of the AE components SYCP2 and SYCP3. Hormad1/Rad21L-double knockout (dKO) showed more severe defects in the formation of synaptonemal complex (SC) compared to Hormad1-KO or Rad21L-KO. Intriguingly, Hormad1/Rec8-dKO but not Hormad1/Rad21L-dKO showed precocious separation of sister chromatid axis. These findings suggest that meiotic cohesins REC8 and RAD21L mediate chromatin loading and the mode of action of HORMAD1 for synapsis during early meiotic prophase.
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| 3. |
- He, Shuwen, 1995, et al.
(författare)
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Distinct dynein complexes defined by DYNLRB1 and DYNLRB2 regulate mitotic and male meiotic spindle bipolarity.
- 2023
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target.
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| 4. |
- Lu, Yonggang, et al.
(författare)
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1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg interaction.
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 120:8
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Tidskriftsartikel (refereegranskat)abstract
- Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.
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| 5. |
- Morimoto, Akihiro, et al.
(författare)
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A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis
- 2012
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 198, s. 165-172
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Tidskriftsartikel (refereegranskat)abstract
- n yeasts and worms, KASH (Klarsicht/ANC-1/Syne/ homology) domain and SUN (Sad-1/UNC-84) domain nuclear envelope (NE) proteins play a crucial role in meiotic chromosome movement and homologue pair-ing. However, although the vertebrate SUN domain pro-tein SUN1 is involved in these processes, its partner has remained identified. Based on subcellular localiza-tion screening in mouse spermatocytes, we identified a novel germ cell-specific protein, KASH5, that localized exclusively at telomeres from the leptotene to diplotene stages in both spermatocytes and oocytes. KASH5 pos-sesses hitherto unknown KASH-related sequences that directly interacted with SUN1 and mediated telomere loca-lization. Thus, KASH5 is a mammalian meiosis-specific KASH domain protein. We show that meiotic chromo-some movement depended on microtubules and that KASH5 interacted with the microtubule-associated dynein-dynactin complex. These results suggest that KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force-generating mechanism involved in meiotic chromosome movement. Our study strongly sug-gests that the meiotic homologue-pairing mechanism mediated by the SUN-KASH NE bridge is highly con-served among eukaryotes. © 2012 Morimoto et al.
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| 6. |
- Padmanaban, Shilpa, et al.
(författare)
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Caenorhabditis elegans telomere- binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA
- 2024
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Ingår i: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - 0027-8424 .- 1091-6490. ; 121:16
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Tidskriftsartikel (refereegranskat)abstract
- Protecting chromosome ends from misrecognition as double- stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N- terminal homodimerization and C- terminal Myb- domain- mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb- containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X - ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA- binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X - ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.
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| 7. |
- Pendlebury, Devon F, et al.
(författare)
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Dissecting the telomere-inner nuclear membrane interface formed in meiosis.
- 2017
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Ingår i: Nature structural & molecular biology. - : Springer Science and Business Media LLC. - 1545-9985 .- 1545-9993. ; 24, s. 1064-1072
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Tidskriftsartikel (refereegranskat)abstract
- Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.
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| 8. |
- Pendlebury, Devon F, et al.
(författare)
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Structure of a meiosis-specific complex central to BRCA2 localization at recombination sites.
- 2021
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Ingår i: Nature structural & molecular biology. - : Springer Science and Business Media LLC. - 1545-9985 .- 1545-9993. ; 28:8, s. 671-680
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Tidskriftsartikel (refereegranskat)abstract
- Meiotic cells invoke breast cancer susceptibility gene 2 (BRCA2) to repair programmed double-stranded DNA breaks and accomplish homologous recombination. The meiosis-specific protein MEILB2 facilitates BRCA2 recruitment to meiotic recombination sites. Here, we combine crystallography, biochemical analysis and a mouse meiosis model to reveal a robust architecture that ensures meiotic BRCA2 recruitment. The crystal structure of the MEILB2-BRCA2 complex reveals how two MEILB2 homodimers sandwich two chains of BRCA2 to afford a 4:2 architecture. The sandwich lacks close contact between the two MEILB2 dimers or the two BRCA2 chains. Instead, the two halves of each BRCA2 chain bridge two MEILB2 subunits from different homodimers to form the MEILB2-BRCA2-MEILB2 sandwich. Several identical residues from the two MEILB2 subunits are employed to engage the BRCA2 halves, justifying their strict conservation. Mutational analysis of the interface reveals a synergistic mechanism for MEILB2-BRCA2 recruitment during meiosis. Overall, these studies demonstrate how BRCA2 efficiently localizes in the cell to facilitate meiosis.
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| 9. |
- Shibuya, Hiroki, 1985, et al.
(författare)
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Live-cell microscopy of meiosis in spermatocytes.
- 2018
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Ingår i: Methods in cell biology. - : Elsevier. - 0091-679X. ; 145, s. 269-277
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Tidskriftsartikel (refereegranskat)abstract
- For the analysis of the molecular mechanisms underlying mammalian meiosis, the establishment of a transient gene expression system for meiocytes has been long awaited. We have established an efficient in vivo electroporation method for live mouse testis and demonstrate short-term transgene expression in spermatocytes. By expressing specific marker proteins fused with GFP, this technique is applicable not only to fixed cell observations after transgene expression but also to live imaging to dissect dynamic cellular events in live spermatocytes. The protocol is also adapted to the dissection of mutant phenotypes with defective chromosome movement during meiotic prophase I, as well as a phenotype rescue assay by expressing functional cDNAs in mutant testes.
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| 10. |
- Stanzione, Marcello, et al.
(författare)
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Meiotic DNA break formation requires the unsynapsed chromosome axis-binding protein IHO1 (CCDC36) in mice
- 2016
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Ingår i: Nature Cell Biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 18, s. 1208-1220
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Tidskriftsartikel (refereegranskat)abstract
- © 2016 Macmillan Publishers Limited. part of Springer Nature All rights reserved. DNA double-strand breaks (DSBs) are induced by SPO11 during meiosis to initiate recombination-mediated pairing and synapsis of homologous chromosomes. Germline genome integrity requires spatiotemporal control of DSB formation, which involves the proteinaceous chromosome axis along the core of each meiotic chromosome. In particular, a component of unsynapsed axes, HORMAD1, promotes DSB formation in unsynapsed regions where DSB formation must occur to ensure completion of synapsis. Despite its importance, the underlying mechanism has remained elusive. We identify CCDC36 as a direct interactor of HORMAD1 (IHO1) that is essential for DSB formation. Underpinning this function, IHO1 and conserved SPO11-auxiliary proteins MEI4 and REC114 assemble chromatin-bound recombinosomes that are predicted activators of DSB formation. HORMAD1 is needed for robust recruitment of IHO1 to unsynapsed axes and efficient formation and/or stabilization of these recombinosomes. Thus, we propose that HORMAD1-IHO1 interaction provides a mechanism for the selective promotion of DSB formation along unsynapsed chromosome axes.
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