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- Chauhan, S., et al.
(author)
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Cdk2 catalytic activity is essential for meiotic cell division in vivo
- 2016
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In: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 473, s. 2783-2798
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Journal article (peer-reviewed)abstract
- Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two different knockin mouse strains in which Cdk2 point mutations ablated enzyme activity without altering protein expression levels. Mice homozygous for the mutations Cdk2(D145N/D145N) or Cdk2(T160A/T160A) expressed only 'kinase-dead' variants of Cdk2 under the control of the endogenous promoter, and despite exhibiting normal expression of cell cycle regulatory proteins and complexes, both mutations rendered mice sterile. Mouse cells that expressed only 'kinase-dead' variants of Cdk2 displayed normal mitotic cell cycle progression and proliferation both in vitro and in vivo, indicating that loss of Cdk2 kinase activity exerted little effect on this mode of cell division. In contrast, the reproductive organs of Cdk2 mutant mice exhibited abnormal morphology and impaired function associated with defective meiotic cell division and inability to produce gametes. Cdk2 mutant animals were therefore comparable to gene knockout mice, which completely lack the Cdk2 protein. Together, our data indicate that the essential meiotic functions of Cdk2 depend on its kinase activity, without which the generation of haploid cells is disrupted, resulting in sterility of otherwise healthy animals.
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| 4. |
- Gopinathan, L., et al.
(author)
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Emi2 Is Essential for Mouse Spermatogenesis
- 2017
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In: Cell Reports. - : Elsevier BV. - 2211-1247. ; 20:3, s. 697-708
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Journal article (peer-reviewed)abstract
- The meiotic functions of Emi2, an inhibitor of the APC/C complex, have been best characterized in oocytes where it mediates metaphase II arrest as a component of the cytostatic factor. We generated knockout mice to determine the in vivo functions of Emi2-in particular, its functions in the testis, where Emi2 is expressed at high levels. Male and female Emi2 knockout mice are viable but sterile, indicating that Emi2 is essential for meiosis but dispensable for embryonic development and mitotic cell divisions. We found that, besides regulating cell-cycle arrest in mouse eggs, Emi2 is essential for meiosis I progression in spermatocytes. In the absence of Emi2, spermatocytes arrest in early diplotene of prophase I. This arrest is associated with decreased Cdk1 activity and was partially rescued by a knockin mouse model of elevated Cdk1 activity. Additionally, we detected expression of Emi2 in spermatids and sperm, suggesting potential post-meiotic functions for Emi2.
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| 5. |
- Risal, Sanjiv, et al.
(author)
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MASTL is essential for anaphase entry of proliferating primordial germ cells and establishment of female germ cells in mice
- 2017
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In: Cell Discovery. - : Springer Science and Business Media LLC. - 2056-5968. ; 3
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Journal article (peer-reviewed)abstract
- In mammals, primordial germ cells (PGCs) are the embryonic cell population that serve as germ cell precursors in both females and males. During mouse embryonic development, the majority of PGCs are arrested at the G2 phase when they migrate into the hindgut at 7.75-8.75 dpc (days post coitum). It is after 9.5 dpc that the PGCs undergo proliferation with a doubling time of 12.6 h. The molecular mechanisms underlying PGC proliferation are however not well studied. In this work. Here we studied how MASTL (microtubule-associated serine/threonine kinase-like)/Greatwall kinase regulates the rapid proliferation of PGCs. We generated a mouse model where we specifically deleted Mastl in PGCs and found a significant loss of PGCs before the onset of meiosis in female PGCs. We further revealed that the deletion of Mastl in PGCs did not prevent mitotic entry, but led to a failure of the cells to proceed beyond metaphase-like stage, indicating that MASTL-mediated molecular events are indispensable for anaphase entry in PGCs. These mitotic defects further led to the death of Mastl-null PGCs by 12.5 dpc. Moreover, the defect in mitotic progression observed in the Mastl-null PGCs was rescued by simultaneous deletion of Ppp2r1a (a subunit of PP2A). Thus, our results demonstrate that MASTL, PP2A, and therefore regulated phosphatase activity have a fundamental role in establishing female germ cell population in gonads by controlling PGC proliferation during embryogenesis.
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| 6. |
- Tu, Zhaowei, et al.
(author)
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Speedy A-Cdk2 binding mediates initial telomere-nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation
- 2017
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:3, s. 592-597
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Journal article (peer-reviewed)abstract
- Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosomemovement duringmeiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere-NE attachment. Deletion of Spdya in mice disrupts telomere-NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2' s localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2-mediated telomere-NE attachment is independent of Cdk2 activation. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere-NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression.
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| 7. |
- Wang, L. N., et al.
(author)
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Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
- 2018
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In: Cell Death and Differentiation. - 1350-9047. ; 25:6, s. 1174-1188
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Journal article (peer-reviewed)abstract
- Telomeres integrity is indispensable for chromosomal stability by preventing chromosome erosion and end-to-end fusions. During meiosis, telomeres attach to the inner nuclear envelope and cluster into a highly crowded microenvironment at the bouquet stage, which requires specific mechanisms to protect the telomeres from fusion. Here, we demonstrate that germ cell-specific knockout of a shelterin complex subunit, Ttf1, results in arrest of spermatocytes at two different stages. The obliterated telomere-nuclear envelope attachment in Trf1/-deficient spermatocytes impairs homologue synapsis and recombination, resulting in a pachytene-like arrest, while the meiotic division arrest might stem from chromosome end-to end fusion due to the failure of recruiting meiosis specific telomere associated proteins. Further investigations uncovered that TRF1 could directly interact with Speedy A, and Speedy A might work as a scaffold protein to further recruit Cdk2, thus protecting telomeres from fusion at this stage. Together, our results reveal a novel mechanism of TRF1, Speedy A, and Cdk2 in protecting telomere from fusion in a highly crowded microenvironment during meiosis.
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| 8. |
- Xu, Jianliang, et al.
(author)
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Protective functions of ZO-2/Tjp2 expressed in hepatocytes and cholangiocytes against liver injury and cholestasis
- 2021
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In: Gastroenterology. - : Elsevier BV. - 1528-0012 .- 0016-5085. ; 160:6, s. 2103-2118
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Journal article (peer-reviewed)abstract
- BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Since the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2.METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver and bile and .by EM, histology and immunostaining. TJ barrier permeability was evaluated using FITC-Dextran (4kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury.RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density and aberrant Radixin and BSEP distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid (BA) transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA-diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. TCPOBOP ameliorated CA-induced injury by enhancing Cyp2b10 expression and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes only showed mild CA-induced liver injury.CONCLUSION: Tjp2 is required for normal cortical distribution of Radixin, canalicular volume regulation and microvilli density, Its inactivation deregulated expression of Cldn1 and key BA transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.
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