30 research outputs found
Casein kinase 1Ī³ acts as a molecular switch for cell polarization through phosphorylation of the polarity factor Tea1 in fission yeast
Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1Ī³, Cki3 is a critical determinant of NETO onset. Not only did cki3ā cells undergo NETO during G1ā or Sāphase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinaseādead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3ā cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues are critical for proteināprotein interaction and NETO onset. Our findings provide significant insight into the mechanism by which cell polarization is regulated in a spatiotemporal manner.T.K. was the recipient of a JSPS fellowship (PD) and was partly supported by āStrategic Young Researcher Overseas Visits Program for Accelerating Brain Circulationā from JSPS. This work was supported by Cancer Research UK (T.T. and A.P.S) and the Ministry of Education, Culture, Sports, Science and Technology (D.H.)
Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice
Noda, T., Lu, Y., Fujihara, Y., Oura, S., Koyano, T., Kobayashi, S., . . . Ikawa, M. (2020). Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice. Proceedings of the National Academy of Sciences of the United States of America, 117(21) doi:10.1073/pnas.192265011
Two-stage sinus floor augmentation using carbonate apatite
Purpose: The purpose of this study was to elucidate the efficacy and safety of carbonate apatite (CO3Ap) granules in 2-stage sinus floor augmentation through the radiographic and histomorphometric assessment of bone biopsy specimens.
Methods: Two-stage sinus floor augmentation was performed on 13 patients with a total of 17 implants. Radiographic assessment using panoramic radiographs was performed immediately after augmentation and was also performed 2 additional times, at 7Ā±2 months and 18Ā±2 months post-augmentation, respectively. Bone biopsy specimens taken from planned implant placement sites underwent micro-computed tomography, after which histological sections were prepared.
Results: Postoperative healing of the sinus floor augmentation was uneventful in all cases. The mean preoperative residual bone height was 3.5Ā±1.3 mm, and this was increased to 13.3Ā±1.7 mm by augmentation with the CO3Ap granules. The mean height of the augmented site had decreased to 10.7Ā±1.9 mm by 7Ā±2 months after augmentation; however, implants with lengths in the range of 6.5 to 11.5 mm could still be placed. The mean height of the augmented site had decreased to 9.6Ā±1.4 mm by 18Ā±2 months post-augmentation. No implant failure or complications were observed. Few inflammatory cells or foreign body giant cells were observed in the bone biopsy specimens. Although there were individual differences in the amount of new bone detected, new bone was observed to be in direct contact with the CO3Ap granules in all cases, without an intermediate layer of fibrous tissue. The amounts of bone and residual CO3Ap were 33.8%Ā±15.1% and 15.3%Ā±11.9%, respectively.
Conclusions: In this first demonstration, low-crystalline CO3Ap granules showed excellent biocompatibility, and bone biopsy showed them to be replaced with bone in humans. CO3Ap granules are a useful and safe bone substitute for two-stage sinus floor augmentation
The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment
Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPĪ²-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor Ī± (TNF-Ī±) and fibrotic marker collagen I in injured kidneys. C/EBPĪ², upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-Ī±, suggesting that C/EBPĪ² plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to No. Y)
Determination of Solidifying Shell Thickness of Continuously Cast Slab by Rivet Pin Shooting
Casein kinase 1Ī³ acts as a molecular switch for cell polarization through phosphorylation of the polarity factor T
Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1Ī³, Cki3 is a critical determinant of NETO onset. Not only did cki3ā cells undergo NETO during G1ā or Sāphase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinaseādead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3ā cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues are critical for proteināprotein interaction and NETO onset. Our findings provide significant insight into the mechanism by which cell polarization is regulated in a spatiotemporal manner
Successful production of genome-edited rats by the rGONAD method
Abstract Background Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as āimproved GONADā (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. Results Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. Conclusions We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals
KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice.
Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice