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Wu L, Sun J, Wang L, Chen Z, Guan Z, Du L, Qu R, Liu C, Shao Y, Hua Y. Whole-transcriptome sequencing in neural and non-neural tissues of a mouse model identifies miR-34a as a key regulator in SMA pathogenesis. MOLECULAR THERAPY. NUCLEIC ACIDS 2025; 36:102490. [PMID: 40125274 PMCID: PMC11930137 DOI: 10.1016/j.omtn.2025.102490] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Accepted: 02/17/2025] [Indexed: 03/25/2025]
Abstract
Spinal muscular atrophy (SMA) is a severe neurodegenerative disorder caused by deficiency of survival of motor neuron (SMN). While significant progress has been made in SMA therapy by rescuing SMN expression, limited knowledge about SMN downstream genes has hindered the development of alternative therapies. Here, we conducted whole-transcriptome sequencing of spinal cord, heart, and liver tissues of a severe SMA mouse model at early postnatal ages to explore critical coding and non-coding RNAs (ncRNAs). A large number of differentially expressed RNAs (DE-RNAs) were obtained, including 2,771 mRNAs, 382 microRNAs (miRNAs), 1,633 long ncRNAs, and 1,519 circular RNAs. Through in-depth data mining, we unveiled deregulation of miR-34a in all tissues. Analysis of competitive endogenous RNA networks of DE-RNAs identified multiple novel targets of miR-34a including Spag5 mRNA, lncRNA00138536, and circRNA007386. Further in vitro studies using mouse myoblast and human cardiomyocyte cell lines showed that knockdown of SMN upregulated miR-34a-5p and overexpression of miR-34a-5p alone disrupted cell-cycle progression through regulating its targets, recapitulating gene expression patterns observed in cardiac tissue of SMA mice. Our results identified a critical miRNA involved in SMA pathology, which sheds insights into the molecular basis of widespread tissue abnormalities observed in severe forms of SMA.
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Affiliation(s)
- Liucheng Wu
- Department of Neurology and Suzhou Clinical Research Center of Neurological Disease, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China
- Laboratory Animal Center, Nantong University, Nantong 226001, China
- Institute of Neuroscience, Soochow University, 199 Renai Road, Suzhou, Jiangsu 215123, China
| | - Junjie Sun
- Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China
| | - Li Wang
- Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
| | - Zhiheng Chen
- Laboratory Animal Center, Nantong University, Nantong 226001, China
| | - Zeyuan Guan
- Department of Neurology and Suzhou Clinical Research Center of Neurological Disease, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China
- Institute of Neuroscience, Soochow University, 199 Renai Road, Suzhou, Jiangsu 215123, China
| | - Lili Du
- Laboratory Animal Center, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China
| | - Ruobing Qu
- Department of Neurology and Suzhou Clinical Research Center of Neurological Disease, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China
- Institute of Neuroscience, Soochow University, 199 Renai Road, Suzhou, Jiangsu 215123, China
| | - Chun Liu
- Laboratory Animal Center, Nantong University, Nantong 226001, China
| | - Yixiang Shao
- Laboratory Animal Center, Nantong University, Nantong 226001, China
| | - Yimin Hua
- Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
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Canu V, Vaccarella S, Sacconi A, Pulito C, Goeman F, Pallocca M, Rutigliano D, Lev S, Strano S, Blandino G. Targeting of mutant-p53 and MYC as a novel strategy to inhibit oncogenic SPAG5 activity in triple negative breast cancer. Cell Death Dis 2024; 15:603. [PMID: 39164278 PMCID: PMC11336084 DOI: 10.1038/s41419-024-06987-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2024] [Revised: 08/02/2024] [Accepted: 08/07/2024] [Indexed: 08/22/2024]
Abstract
Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient's outcome, and reliable biomarkers for effective anticancer therapies.
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Affiliation(s)
- Valeria Canu
- Translational Oncology Research Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Sebastiano Vaccarella
- Translational Oncology Research Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Andrea Sacconi
- Biostatistics, Bioinformatics and Clinical Trial Center, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Claudio Pulito
- Translational Oncology Research Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Frauke Goeman
- Department of Research, Diagnosis and Innovative Technologies, UOSD SAFU, Translational Research Area, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Matteo Pallocca
- Biostatistics, Bioinformatics and Clinical Trial Center, IRCCS Regina Elena National Cancer Institute, Rome, Italy
- Institute of Experimental Endocrinology and Oncology, National Research Council, Naples, Italy
| | - Daniela Rutigliano
- Translational Oncology Research Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Sima Lev
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
| | - Sabrina Strano
- Department of Research, Diagnosis and Innovative Technologies, UOSD SAFU, Translational Research Area, IRCCS Regina Elena National Cancer Institute, Rome, Italy.
| | - Giovanni Blandino
- Translational Oncology Research Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy.
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Jia X, Lin L, Guo S, Zhou L, Jin G, Dong J, Xiao J, Xie X, Li Y, He S, Wei Z, Yu C. CLASP-mediated competitive binding in protein condensates directs microtubule growth. Nat Commun 2024; 15:6509. [PMID: 39095354 PMCID: PMC11297316 DOI: 10.1038/s41467-024-50863-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2024] [Accepted: 07/23/2024] [Indexed: 08/04/2024] Open
Abstract
Microtubule organization in cells relies on targeting mechanisms. Cytoplasmic linker proteins (CLIPs) and CLIP-associated proteins (CLASPs) are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we reveal that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identify additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offer general mechanistic insights into intricate protein-protein interactions at the mesoscale level.
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Affiliation(s)
- Xuanyan Jia
- Shenzhen Key Laboratory of Biomolecular Assembling and Regulation, Shenzhen, Guangdong, 518055, China
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Leishu Lin
- Shenzhen Key Laboratory of Biomolecular Assembling and Regulation, Shenzhen, Guangdong, 518055, China
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Siqi Guo
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Lulu Zhou
- Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Gaowei Jin
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Jiayuan Dong
- Shenzhen Key Laboratory of Biomolecular Assembling and Regulation, Shenzhen, Guangdong, 518055, China
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Jinman Xiao
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Xingqiao Xie
- Shenzhen Key Laboratory of Biomolecular Assembling and Regulation, Shenzhen, Guangdong, 518055, China
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Yiming Li
- Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Sicong He
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China
| | - Zhiyi Wei
- Shenzhen Key Laboratory of Biomolecular Assembling and Regulation, Shenzhen, Guangdong, 518055, China.
- Brain Research Center, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China.
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China.
- Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, Guangdong, China.
| | - Cong Yu
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China.
- Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, Guangdong, China.
- Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, and Shenzhen Key Laboratory of Cell Microenvironment, Shenzhen, Guangdong, 518055, China.
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Guo L, Yuan H, Zhu H, Zhou J, Wan Z, Zhou Y. SPAG5 deficiency activates autophagy to reduce atherosclerotic plaque formation in ApoE -/- mice. BMC Cardiovasc Disord 2024; 24:275. [PMID: 38807081 PMCID: PMC11131316 DOI: 10.1186/s12872-024-03945-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Accepted: 05/16/2024] [Indexed: 05/30/2024] Open
Abstract
BACKGROUND Autophagy, as a regulator of cell survival, plays an important role in atherosclerosis (AS). Sperm associated antigen 5 (SPAG5) is closely associated with the classical autophagy pathway, PI3K/Akt/mTOR signaling pathway. This work attempted to investigate whether SPAG5 can affect AS development by regulating autophagy. METHODS Human umbilical vein endothelial cells (HUVECs) were treated with oxidized-low density lipoprotein (ox-LDL) to induce cell damage. ApoE-/- mice were fed a Western diet to establish an AS mouse model. Haematoxylin and eosin (H&E) staining and Oil Red O staining evaluated the pathological changes and in lipid deposition in aortic tissues. CCK-8 and flow cytometry detected cell proliferation and apoptosis. Immunohistochemistry, Enzyme linked immunosorbent assay, qRT-PCR and western blotting assessed the levels of mRNA and proteins. RESULTS Ox-LDL treatment elevated SPAG5 expression and the expression of autophagy-related proteins, LC3-I, LC3-II, Beclin-1, and p62, in HUVECs. GFP-LC3 dots were increased in ox-LDL-treated HUVECs and LPS-treated HUVECs. SPAG5 knockdown reversed both ox-LDL and LPS treatment-mediated inhibition of cell proliferation and promotion of apoptosis in HUVECs. SPAG5 silencing further elevated autophagy and repressed the expression of PI3K, p-Akt/Akt, and p-mTOR/mTOR in ox-LDL-treated HUVECs. 3-MA (autophagy inhibitor) treatment reversed SPAG5 silencing-mediated increase of cell proliferation and decrease of apoptosis in ox-LDL-treated HUVECs. In vivo, SPAG5 knockdown reduced atherosclerotic plaques in AS mice through activating autophagy and inhibiting PI3K/Akt/mTOR signaling pathway. CONCLUSION This work demonstrated that SPAG5 knockdown alleviated AS development through activating autophagy. Thus, SPAG5 may be a potential target for AS therapy.
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Affiliation(s)
- Liangyun Guo
- Department of Ultrasound, The Second Affiliated Hospital of Nanchang University, No.1, Minde Road, Nanchang, Jiangxi, 330006, China
| | - Huijing Yuan
- Department of Obstetrics, Jiangxi Maternal and Child Health Hospital, No. 318 Bayi avenue, Nanchang, Jiangxi, 330006, China
| | - Huayao Zhu
- Department of ICU, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road, Nanchang, Jiangxi, 330006, China
| | - Jie Zhou
- Department of ICU, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road, Nanchang, Jiangxi, 330006, China
| | - Zixin Wan
- Department of ICU, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road, Nanchang, Jiangxi, 330006, China
| | - Yihua Zhou
- Department of ICU, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road, Nanchang, Jiangxi, 330006, China.
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Bergeron JJM. Proteomics Impact on Cell Biology to Resolve Cell Structure and Function. Mol Cell Proteomics 2024; 23:100758. [PMID: 38574860 PMCID: PMC11070594 DOI: 10.1016/j.mcpro.2024.100758] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2024] [Revised: 03/23/2024] [Accepted: 03/26/2024] [Indexed: 04/06/2024] Open
Abstract
The acceleration of advances in proteomics has enabled integration with imaging at the EM and light microscopy levels, cryo-EM of protein structures, and artificial intelligence with proteins comprehensively and accurately resolved for cell structures at nanometer to subnanometer resolution. Proteomics continues to outpace experimentally based structural imaging, but their ultimate integration is a path toward the goal of a compendium of all proteins to understand mechanistically cell structure and function.
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Affiliation(s)
- John J M Bergeron
- Department of Medicine, McGill University Hospital Research Institute, Montreal, Quebec, Canada.
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He J, Li J, Liu Y, Li Y. Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells. Cancers (Basel) 2024; 16:1269. [PMID: 38610947 PMCID: PMC11010853 DOI: 10.3390/cancers16071269] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2024] [Revised: 03/18/2024] [Accepted: 03/21/2024] [Indexed: 04/14/2024] Open
Abstract
Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, the SPAG5 knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines. The knockout of SPAG5 was confirmed on both gene and protein levels using genomic PCR, DNA sequencing and western blotting. The functional loss of SPAG5 was determined by colony-formation assay. SPAG5-regulated doxorubicin- and docetaxel-resistance was assessed by MTT and apoptosis assays. The results indicated that all the SPAG5 knockout MDA-MB-231 and BT549 clones were biallelic, where one allele was replaced by the donor template, and the other allele had the same "T" insertion (indel) adjacent to the cutting sites of gRNAs at the exon 1 boundary, irrespective of the gRNAs and cell lines. The locus of indel interrupted the SPAG5 transcription by damaging the GT-AG mRNA processing rule. Deletion of SPAG5 decreased clonogenicity in both MDA-MB-231 and BT549 cells. SPAG5 was able to regulate the resistance and the drug-induced apoptosis of both doxorubicin and docetaxel. In conclusion, recombinant plasmid-based CRISPR-Cas9 technology can be used to delete the SPAG5 gene in the TNBC cell lines. SPAG5 has an important role in regulating cell proliferation and doxorubicin- and docetaxel-resistance in MDA-MB-231 and BT549 cells.
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Affiliation(s)
- Ji He
- School of Science, Auckland University of Technology, Auckland 1010, New Zealand; (J.H.); (J.L.); (Y.L.)
- Department of Food and Agriculture Technology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, China
| | - Jiawei Li
- School of Science, Auckland University of Technology, Auckland 1010, New Zealand; (J.H.); (J.L.); (Y.L.)
- General Medicine Department, Shenzhen Longhua District Central Hospital, Shenzhen 518110, China
| | - Yanbiao Liu
- School of Science, Auckland University of Technology, Auckland 1010, New Zealand; (J.H.); (J.L.); (Y.L.)
| | - Yan Li
- School of Science, Auckland University of Technology, Auckland 1010, New Zealand; (J.H.); (J.L.); (Y.L.)
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Gao X, Bu H, Gao X, Wang Y, Wang L, Zhang Z. Pan-cancer analysis: SPAG5 is an immunological and prognostic biomarker for multiple cancers. FASEB J 2023; 37:e23159. [PMID: 37650687 DOI: 10.1096/fj.202300626r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2023] [Revised: 06/30/2023] [Accepted: 08/10/2023] [Indexed: 09/01/2023]
Abstract
Sperm-associated antigen 5 (SPAG5) is a mitotic spindle protein that regulates the separation of sister chromatids into daughter cells. Recent studies have discovered its overexpression in various cancers, suggesting its oncogenic characteristics and functions. However, a comprehensive analysis of SPAG5 regarding its diagnostic, prognostic, and immune-related effects across different cancer types is lacking. In this study, we employed bioinformatics methods and integrated multiple public databases to explore the potential oncogenic role of SPAG5. We analyzed its expression, prognosis, related chemicals, enriched pathways, immune infiltration, and its impact on different tumor genetic alterations. The results revealed that SPAG5 is highly expressed in most cancers and significantly correlates with poor patient prognosis. Additionally, SPAG5 expression showed potential for early cancer diagnosis in 15 different cancer types. In terms of tumor immunity, high expression of SPAG5 was associated with an immunosuppressive tumor microenvironment and immune therapy efficacy indicators. SPAG5 expression exhibited a negative correlation with most immune cell infiltrates but demonstrated a significant positive correlation with Th2 cells and MDSC cells. Multicolor fluorescence immunohistochemistry demonstrated that SPAG5 activates immune cell populations within tumors, indicating its significant role in the tumor microenvironment. Enrichment analysis indicated that SPAG5-related genes are mainly involved in cell cycle, cellular senescence, P53 signaling pathway, and FoxO signaling pathway. Furthermore, we confirmed the high expression of SPAG5 in cancer cells and observed that its knockdown upregulated the expression of the p53 protein. In conclusion, SPAG5 holds value as a diagnostic, prognostic, and immune biomarker in various cancers and may provide a novel target for tumor immunotherapy.
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Affiliation(s)
- Xiaofeng Gao
- Medicine Research Institute/Hubei Key Laboratory of Diabetes and Angiopathy, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
- School of Basic Medical Sciences, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
| | - Huitong Bu
- College of Biology, Hunan University, Changsha, People's Republic of China
| | - Xuzheng Gao
- Medicine Research Institute/Hubei Key Laboratory of Diabetes and Angiopathy, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
| | - Ying Wang
- Medicine Research Institute/Hubei Key Laboratory of Diabetes and Angiopathy, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
| | - Long Wang
- Medicine Research Institute/Hubei Key Laboratory of Diabetes and Angiopathy, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
- School of Basic Medical Sciences, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
- School of Stomatology and Ophthalmology, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
| | - Zhenwang Zhang
- Medicine Research Institute/Hubei Key Laboratory of Diabetes and Angiopathy, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
- School of Basic Medical Sciences, Xianning Medical College, Hubei University of Science and Technology, Xianning, People's Republic of China
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Bunning AR, Gupta Jr. ML. The importance of microtubule-dependent tension in accurate chromosome segregation. Front Cell Dev Biol 2023; 11:1096333. [PMID: 36755973 PMCID: PMC9899852 DOI: 10.3389/fcell.2023.1096333] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Accepted: 01/11/2023] [Indexed: 01/24/2023] Open
Abstract
Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
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Ying Z, Wang K, Wu J, Wang M, Yang J, Wang X, Zhou G, Chen H, Xu H, Sze SCW, Gao F, Li C, Sha O. CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72. BMC Biol 2022; 20:240. [PMID: 36280838 PMCID: PMC9590400 DOI: 10.1186/s12915-022-01437-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Accepted: 10/14/2022] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND The centrosome is one of the most important non-membranous organelles regulating microtubule organization and progression of cell mitosis. The coiled-coil alpha-helical rod protein 1 (CCHCR1, also known as HCR) gene is considered to be a psoriasis susceptibility gene, and the protein is suggested to be localized to the P-bodies and centrosomes in mammalian cells. However, the exact cellular function of HCR and its potential regulatory role in the centrosomes remain unexplored. RESULTS We found that HCR interacts directly with astrin, a key factor in centrosome maturation and mitosis. Immunoprecipitation assays showed that the coiled-coil region present in the C-terminus of HCR and astrin respectively mediated the interaction between them. Astrin not only recruits HCR to the centrosome, but also protects HCR from ubiquitin-proteasome-mediated degradation. In addition, depletion of either HCR or astrin significantly reduced centrosome localization of CEP72 and subsequent MCPH proteins, including CEP152, CDK5RAP2, and CEP63. The absence of HCR also caused centriole duplication defects and mitotic errors, resulting in multipolar spindle formation, genomic instability, and DNA damage. CONCLUSION We conclude that HCR is localized and stabilized at the centrosome by directly binding to astrin. HCR are required for the centrosomal recruitment of MCPH proteins and centriolar duplication. Both HCR and astrin play key roles in keeping normal microtubule assembly and maintaining genomic stability.
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Affiliation(s)
- Zhenguang Ying
- Department of Anatomy, Histology and Developmental Biology, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Kaifang Wang
- Department of Anatomy, Histology and Developmental Biology, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Junfeng Wu
- Department of Anatomy, Histology and Developmental Biology, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Mingyu Wang
- Medical AI Laboratory, School of Biomedical Engineering, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Jing Yang
- Department of Anatomy, Histology and Developmental Biology, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Xia Wang
- Department of Anatomy, Histology and Developmental Biology, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Guowei Zhou
- Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Haibin Chen
- Department of Histology and Embryology, Shantou University Medical College, Shantou, 515000, China
| | - Hongwu Xu
- Department of Neurosurgery, The First Affiliated Hospital of Shantou University Medical College, Shantou, 515000, China
- Department of Clinically Oriented Anatomy, Shantou University Medical College, Shantou, 515000, China
| | - Stephen Cho Wing Sze
- Department of Biology, Faculty of Science, Hong Kong Baptist University, Hongkong, 999077, China
- Golden Meditech Centre for NeuroRegeneration Sciences, Hong Kong Baptist University, Hongkong, 999077, China
| | - Feng Gao
- School of Dentistry, Shenzhen University Health Science Centre, Shenzhen, 518000, China
| | - Chunman Li
- Department of Anatomy, Shantou University Medical College, Shantou, 515000, China.
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, 515000, China.
| | - Ou Sha
- Department of Anatomy, Histology and Developmental Biology, Shenzhen University Health Science Centre, Shenzhen, 518000, China.
- School of Dentistry, Shenzhen University Health Science Centre, Shenzhen, 518000, China.
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10
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Mora-Bermúdez F, Kanis P, Macak D, Peters J, Naumann R, Xing L, Sarov M, Winkler S, Oegema CE, Haffner C, Wimberger P, Riesenberg S, Maricic T, Huttner WB, Pääbo S. Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development. SCIENCE ADVANCES 2022; 8:eabn7702. [PMID: 35905187 PMCID: PMC9337762 DOI: 10.1126/sciadv.abn7702] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2021] [Accepted: 06/15/2022] [Indexed: 06/15/2023]
Abstract
Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.
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Affiliation(s)
- Felipe Mora-Bermúdez
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
- Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Philipp Kanis
- Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Dominik Macak
- Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Jula Peters
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Ronald Naumann
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Lei Xing
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Mihail Sarov
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Sylke Winkler
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | | | - Christiane Haffner
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Pauline Wimberger
- Department of Gynecology and Obstetrics, Technische Universität Dresden, Dresden, Germany
| | | | - Tomislav Maricic
- Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Wieland B. Huttner
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Svante Pääbo
- Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
- Okinawa Institute of Science and Technology, Onna-son 904-0495, Japan
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11
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The splicing factor SF3B4 drives proliferation and invasion in cervical cancer by regulating SPAG5. Cell Death Discov 2022; 8:326. [PMID: 35853859 PMCID: PMC9296558 DOI: 10.1038/s41420-022-01120-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Revised: 07/03/2022] [Accepted: 07/05/2022] [Indexed: 12/24/2022] Open
Abstract
Regulation of alternative splicing (AS) by the splicing factor 3b (SF3B) family plays an essential role in cancer. However, the biological function of SF3B family members in cervical cancer (CC) needs to be further elucidated. In this study, we found that splicing factor 3b subunit 4 (SF3B4) was highly expressed in CC by bioinformatics analysis using cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) data from The Cancer Genome Atlas (TCGA). Then, we demonstrated that high expression of SF3B4 promoted proliferation and invasion abilities of CC cells in vitro and in vivo and that reduced expression of SF3B4 performed the opposite effect. Further RNA-seq and AS analysis showed that sperm-associated antigen 5 (SPAG5) was a downstream target gene of SF3B4. Interestingly, SPAG5 expression was decreased after SF3B4 knockdown because of retained introns (RIs) and reduced maturation of SPAG5 pre-mRNA. Importantly, SPAG5 deficiency impaired the oncogenic effects of SF3B4 overexpression on CC cells. In conclusion, SF3B4 promotes CC progression by regulating the effective splicing of SPAG5. SF3B4 could be a promising target for CC.
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12
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Peyrégne S, Kelso J, Peter BM, Pääbo S. The evolutionary history of human spindle genes includes back-and-forth gene flow with Neandertals. eLife 2022; 11:e75464. [PMID: 35816093 PMCID: PMC9273211 DOI: 10.7554/elife.75464] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 06/14/2022] [Indexed: 12/13/2022] Open
Abstract
Proteins associated with the spindle apparatus, a cytoskeletal structure that ensures the proper segregation of chromosomes during cell division, experienced an unusual number of amino acid substitutions in modern humans after the split from the ancestors of Neandertals and Denisovans. Here, we analyze the history of these substitutions and show that some of the genes in which they occur may have been targets of positive selection. We also find that the two changes in the kinetochore scaffold 1 (KNL1) protein, previously believed to be specific to modern humans, were present in some Neandertals. We show that the KNL1 gene of these Neandertals shared a common ancestor with present-day Africans about 200,000 years ago due to gene flow from the ancestors (or relatives) of modern humans into Neandertals. Subsequently, some non-Africans inherited this modern human-like gene variant from Neandertals, but none inherited the ancestral gene variants. These results add to the growing evidence of early contacts between modern humans and archaic groups in Eurasia and illustrate the intricate relationships among these groups.
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Affiliation(s)
- Stéphane Peyrégne
- Department of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyLeipzigGermany
| | - Janet Kelso
- Department of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyLeipzigGermany
| | - Benjamin M Peter
- Department of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyLeipzigGermany
| | - Svante Pääbo
- Department of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyLeipzigGermany
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13
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Zhang S, Gong X, Zhou Y, Ma Q, Cai Q, Yang G, Guo X, Chen Y, Xu M, Zhu Y, Zeng Y, Zeng F. Maternal Prkce expression in mature oocytes is critical for the first cleavage facilitating maternal-to-zygotic transition in mouse early embryos. Cell Prolif 2022; 55:e13231. [PMID: 35582855 PMCID: PMC9201378 DOI: 10.1111/cpr.13231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2022] [Revised: 03/17/2022] [Accepted: 03/23/2022] [Indexed: 11/27/2022] Open
Abstract
Objectives Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal‐to‐zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high‐ or low‐ competence mouse strains. Materials and Methods In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real‐time quantitative PCR (RT‐qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment. Results The fertilization and 2‐cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty‐nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT‐qPCR. Prkce knockout mice showed a reduced 2‐cell rate (Prkce+/+ 80.1% vs. Prkce−/− 32.4%) that could be rescued by Prkce cRNA injection (2‐cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation. Conclusions The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2‐cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high‐quality mature oocytes.
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Affiliation(s)
- Shaoqing Zhang
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Xiuli Gong
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Yiye Zhou
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Qingwen Ma
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Qin Cai
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Guanheng Yang
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Xinbing Guo
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Yanwen Chen
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Miao Xu
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Yiwen Zhu
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Yitao Zeng
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China
| | - Fanyi Zeng
- School of Life Sciences and Biotechnology & Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.,Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, China.,School of Pharmacy, Macau University of Science and Technonlogy, Taipa, Macau, China
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14
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Almeida AC, Soares-de-Oliveira J, Drpic D, Cheeseman LP, Damas J, Lewin HA, Larkin DM, Aguiar P, Pereira AJ, Maiato H. Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals. Cell Rep 2022; 39:110610. [PMID: 35385739 PMCID: PMC8994134 DOI: 10.1016/j.celrep.2022.110610] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Revised: 01/07/2022] [Accepted: 03/11/2022] [Indexed: 11/29/2022] Open
Abstract
Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.
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Affiliation(s)
- Ana C Almeida
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
| | - Joana Soares-de-Oliveira
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
| | - Danica Drpic
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
| | - Liam P Cheeseman
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
| | - Joana Damas
- Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London NW1 0TU, UK; Department of Evolution and Ecology, University of California, Davis, CA 95616, USA
| | - Harris A Lewin
- Department of Evolution and Ecology, University of California, Davis, CA 95616, USA
| | - Denis M Larkin
- Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London NW1 0TU, UK
| | - Paulo Aguiar
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto Nacional de Engenharia Biomédica (INEB), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
| | - António J Pereira
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
| | - Helder Maiato
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Cell Division Group, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal.
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15
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Song X, Conti D, Shrestha RL, Braun D, Draviam VM. Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation. Nat Commun 2021; 12:7010. [PMID: 34853300 PMCID: PMC8636589 DOI: 10.1038/s41467-021-27131-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2020] [Accepted: 11/02/2021] [Indexed: 02/08/2023] Open
Abstract
Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation. Chromosome instability frequently occurs due to issues with chromosome-microtubule attachments. Here the authors show that the Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to protect chromosome-microtubule attachments independent of biorientation.
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Affiliation(s)
- Xinhong Song
- School of Biological and Chemical Sciences, Queen Mary, University of London, London, E1 4NS, UK
| | - Duccio Conti
- School of Biological and Chemical Sciences, Queen Mary, University of London, London, E1 4NS, UK.,Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany
| | - Roshan L Shrestha
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.,Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Dominique Braun
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK
| | - Viji M Draviam
- School of Biological and Chemical Sciences, Queen Mary, University of London, London, E1 4NS, UK. .,Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.
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16
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The Aurora B gradient sustains kinetochore stability in anaphase. Cell Rep 2021; 37:109818. [PMID: 34758321 PMCID: PMC8595645 DOI: 10.1016/j.celrep.2021.109818] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Revised: 08/20/2021] [Accepted: 09/17/2021] [Indexed: 12/02/2022] Open
Abstract
Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.
Central spindle Aurora B targets kinetochore substrates in anaphase Phosphorylation of Dsn1 by Aurora B stabilizes kinetochores in anaphase Dsn1 phosphorylation modulates chromosome movements in anaphase
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17
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Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse. Dev Cell 2021; 56:2273-2283.e3. [PMID: 34428397 DOI: 10.1016/j.devcel.2021.07.022] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 06/07/2021] [Accepted: 07/29/2021] [Indexed: 12/18/2022]
Abstract
Chromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. Lagging anaphase chromosomes are a common age-associated phenomenon in oocytes, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here, we show that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of chromosome motion that we refer to as "class-I" and "class-II" laggards. We use imaging approaches and mechanistic interventions to dissociate the two classes and find that whereas class-II laggards are largely benign, class-I laggards frequently directly lead to aneuploidy. Most notably, a controlled prolongation of meiosis I specifically lessens class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.
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18
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GSK3 as a Regulator of Cytoskeleton Architecture: Consequences for Health and Disease. Cells 2021; 10:cells10082092. [PMID: 34440861 PMCID: PMC8393567 DOI: 10.3390/cells10082092] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 08/06/2021] [Accepted: 08/12/2021] [Indexed: 12/17/2022] Open
Abstract
Glycogen synthase kinase 3 (GSK3) was initially isolated as a critical protein in energy metabolism. However, subsequent studies indicate that GSK-3 is a multi-tasking kinase that links numerous signaling pathways in a cell and plays a vital role in the regulation of many aspects of cellular physiology. As a regulator of actin and tubulin cytoskeleton, GSK3 influences processes of cell polarization, interaction with the extracellular matrix, and directional migration of cells and their organelles during the growth and development of an animal organism. In this review, the roles of GSK3–cytoskeleton interactions in brain development and pathology, migration of healthy and cancer cells, and in cellular trafficking of mitochondria will be discussed.
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19
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Canu V, Donzelli S, Sacconi A, Lo Sardo F, Pulito C, Bossel N, Di Benedetto A, Muti P, Botti C, Domany E, Bicciato S, Strano S, Yarden Y, Blandino G. Aberrant transcriptional and post-transcriptional regulation of SPAG5, a YAP-TAZ-TEAD downstream effector, fuels breast cancer cell proliferation. Cell Death Differ 2021; 28:1493-1511. [PMID: 33230261 PMCID: PMC8166963 DOI: 10.1038/s41418-020-00677-9] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 11/04/2020] [Accepted: 11/06/2020] [Indexed: 01/28/2023] Open
Abstract
Sperm-associated antigen 5 (SPAG5) is an important driver of the cell mitotic spindle required for chromosome segregation and progression into anaphase. SPAG5 has been identified as an important proliferation marker and chemotherapy-sensitivity predictor, especially in estrogen receptor-negative breast cancer subtypes. Here, we report that SPAG5 is a direct target of miR-10b-3p, and its aberrantly high expression associates with poor disease-free survival in two large cohorts of breast cancer patients. SPAG5 depletion strongly impaired cancer cell cycle progression, proliferation, and migration. Interestingly, high expression of SPAG5 pairs with a YAP/TAZ-activated signature in breast cancer patients. Reassuringly, the depletion of YAP, TAZ, and TEAD strongly reduced SPAG5 expression and diminished its oncogenic effects. YAP, TAZ coactivators, and TEAD transcription factors are key components of the Hippo signaling pathway involved in tumor initiation, progression, and metastasis. Furthermore, we report that SPAG5 is a direct transcriptional target of TEAD/YAP/TAZ, and pharmacological targeting of YAP and TAZ severely reduces SPAG5 expression. Collectively, our data uncover an oncogenic feedback loop, comprising miR-10b-3p, SPAG5, and YAP/TAZ/TEAD, which fuels the aberrant proliferation of breast cancer.
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Affiliation(s)
- Valeria Canu
- grid.417520.50000 0004 1760 5276Oncogenomic and Epigenetic Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Sara Donzelli
- grid.417520.50000 0004 1760 5276Oncogenomic and Epigenetic Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Andrea Sacconi
- grid.417520.50000 0004 1760 5276Clinical Trial Center, Biostatistics and Bioinformatics Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Federica Lo Sardo
- grid.417520.50000 0004 1760 5276Oncogenomic and Epigenetic Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Claudio Pulito
- grid.417520.50000 0004 1760 5276Oncogenomic and Epigenetic Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Noa Bossel
- grid.13992.300000 0004 0604 7563Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, 7610001 Israel
| | - Anna Di Benedetto
- grid.417520.50000 0004 1760 5276Department of Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Paola Muti
- grid.4708.b0000 0004 1757 2822Department of Biomedical Science and Oral Health, University of Milan, Milan, 20122 Italy
| | - Claudio Botti
- grid.417520.50000 0004 1760 5276Breast Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Eytan Domany
- grid.13992.300000 0004 0604 7563Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, 7610001 Israel
| | - Silvio Bicciato
- grid.7548.e0000000121697570Center for Genome Research, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Sabrina Strano
- grid.417520.50000 0004 1760 5276SAFU Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | - Yosef Yarden
- grid.13992.300000 0004 0604 7563Department of Biological Regulation, Weizmann Institute of Science, Rehovot, 7610001 Israel
| | - Giovanni Blandino
- grid.417520.50000 0004 1760 5276Oncogenomic and Epigenetic Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Rome, Italy
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20
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Lau EOC, Damiani D, Chehade G, Ruiz-Reig N, Saade R, Jossin Y, Aittaleb M, Schakman O, Tajeddine N, Gailly P, Tissir F. DIAPH3 deficiency links microtubules to mitotic errors, defective neurogenesis, and brain dysfunction. eLife 2021; 10:e61974. [PMID: 33899739 PMCID: PMC8102060 DOI: 10.7554/elife.61974] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Accepted: 04/23/2021] [Indexed: 12/13/2022] Open
Abstract
Diaphanous (DIAPH) three (DIAPH3) is a member of the formin proteins that have the capacity to nucleate and elongate actin filaments and, therefore, to remodel the cytoskeleton. DIAPH3 is essential for cytokinesis as its dysfunction impairs the contractile ring and produces multinucleated cells. Here, we report that DIAPH3 localizes at the centrosome during mitosis and regulates the assembly and bipolarity of the mitotic spindle. DIAPH3-deficient cells display disorganized cytoskeleton and multipolar spindles. DIAPH3 deficiency disrupts the expression and/or stability of several proteins including the kinetochore-associated protein SPAG5. DIAPH3 and SPAG5 have similar expression patterns in the developing brain and overlapping subcellular localization during mitosis. Knockdown of SPAG5 phenocopies DIAPH3 deficiency, whereas its overexpression rescues the DIAHP3 knockdown phenotype. Conditional inactivation of Diaph3 in mouse cerebral cortex profoundly disrupts neurogenesis, depleting cortical progenitors and neurons, leading to cortical malformation and autistic-like behavior. Our data uncover the uncharacterized functions of DIAPH3 and provide evidence that this protein belongs to a molecular toolbox that links microtubule dynamics during mitosis to aneuploidy, cell death, fate determination defects, and cortical malformation.
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Affiliation(s)
- Eva On-Chai Lau
- Université catholique de Louvain, Institute of Neuroscience, Developmental NeurobiologyBrusselsBelgium
| | - Devid Damiani
- Université catholique de Louvain, Institute of Neuroscience, Developmental NeurobiologyBrusselsBelgium
| | - Georges Chehade
- Université catholique de Louvain, Institute of Neuroscience, Developmental NeurobiologyBrusselsBelgium
| | - Nuria Ruiz-Reig
- Université catholique de Louvain, Institute of Neuroscience, Developmental NeurobiologyBrusselsBelgium
| | - Rana Saade
- Université catholique de Louvain, Institute of Neuroscience, Developmental NeurobiologyBrusselsBelgium
| | - Yves Jossin
- Université catholique de Louvain, Institute of Neuroscience, Mammalian Development and Cell BiologyBrusselsBelgium
| | | | - Olivier Schakman
- Université catholique de Louvain, Institute of Neuroscience, Cell PhysiologyBrusselsBelgium
| | - Nicolas Tajeddine
- Université catholique de Louvain, Institute of Neuroscience, Cell PhysiologyBrusselsBelgium
| | - Philippe Gailly
- Université catholique de Louvain, Institute of Neuroscience, Cell PhysiologyBrusselsBelgium
| | - Fadel Tissir
- Université catholique de Louvain, Institute of Neuroscience, Developmental NeurobiologyBrusselsBelgium
- College of Health and Life Sciences, HBKUDohaQatar
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21
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Navarro AP, Cheeseman IM. Kinetochore assembly throughout the cell cycle. Semin Cell Dev Biol 2021; 117:62-74. [PMID: 33753005 DOI: 10.1016/j.semcdb.2021.03.008] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 03/12/2021] [Accepted: 03/13/2021] [Indexed: 11/29/2022]
Abstract
The kinetochore plays an essential role in facilitating chromosome segregation during cell division. This massive protein complex assembles onto the centromere of chromosomes and enables their attachment to spindle microtubules during mitosis. The kinetochore also functions as a signaling hub to regulate cell cycle progression, and is crucial to ensuring the fidelity of chromosome segregation. Despite the fact that kinetochores are large and robust molecular assemblies, they are also highly dynamic structures that undergo structural and organizational changes throughout the cell cycle. This review will highlight our current understanding of kinetochore structure and function, focusing on the dynamic processes that underlie kinetochore assembly.
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Affiliation(s)
- Alexandra P Navarro
- Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Iain M Cheeseman
- Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
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22
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High expression of sperm-associated antigen 5 correlates with poor survival in ovarian cancer. Biosci Rep 2021; 40:221952. [PMID: 31985007 PMCID: PMC7007403 DOI: 10.1042/bsr20193087] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2019] [Revised: 01/07/2020] [Accepted: 01/24/2020] [Indexed: 12/31/2022] Open
Abstract
OBJECTIVES Sperm-associated antigen 5 (SPAG5), a spindle-binding protein, regulates the process of mitosis. The present study focused on the relationship between SPAG5 expression and the clinicopathological characteristics and prognosis of ovarian cancer. METHODS First, we used the Gene Expression Omnibus (GEO) database to analyze SPAG5 expression in ovarian cancer and its clinical relevance. Subsequently, qPCR test was used to detect SPAG5 mRNA expression in 20 cases of ovarian cancer. The expression of SPAG5 protein in a tissue microarray containing 102 cases of ovarian cancer was detected by immunohistochemistry. Cox regression and Kaplan-Meier survival analyses were performed to identify the prognostic factors for the 102 ovarian cancer patients. RESULTS In the GEO datasets, SPAG5 mRNA expression was significantly higher in ovarian cancer tissues than that in normal ovarian tissues (P < 0.001). qPCR and immunohistochemistry showed that SPAG5 expression in ovarian cancer tissues was significantly higher than that in paracancerous tissues (P = 0.002, P < 0.001). The high expression of SPAG5 in ovarian cancer was correlated with histological type (P = 0.009), lymph node metastasis (P = 0.001), distant metastasis (P = 0.001), TNM stage (P = 0.001), and prognosis (P = 0.001). The Kaplan-Meier curve indicated that rates of disease-free survival (DFS) and overall survival (OS) were even lower in patients with high SPAG5 expression. Multivariate analysis showed that SPAG5 expression (P = 0.001) and TNM staging (P = 0.002) were independent prognostic factors for the DFS of ovarian cancer. CONCLUSIONS These results suggest that high SPAG5 expression was correlated with multiple clinicopathological features of ovarian cancer and can be used as an evaluation indicator for a poor ovarian cancer prognosis.
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23
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Geraghty Z, Barnard C, Uluocak P, Gruneberg U. The association of Plk1 with the astrin-kinastrin complex promotes formation and maintenance of a metaphase plate. J Cell Sci 2021; 134:jcs251025. [PMID: 33288550 PMCID: PMC7803464 DOI: 10.1242/jcs.251025] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Accepted: 11/30/2020] [Indexed: 11/20/2022] Open
Abstract
Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin-kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which astrin-kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.
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Affiliation(s)
- Zoë Geraghty
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Christina Barnard
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Pelin Uluocak
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Ulrike Gruneberg
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
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24
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Liu Y, Yu W, Ren P, Zhang T. Upregulation of centromere protein M promotes tumorigenesis: A potential predictive target for cancer in humans. Mol Med Rep 2020; 22:3922-3934. [PMID: 33000180 PMCID: PMC7533490 DOI: 10.3892/mmr.2020.11461] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Accepted: 07/20/2020] [Indexed: 01/04/2023] Open
Abstract
Centromere protein M (CENPM), a protein required for chromosome separation, is involved in in mitosis. However, little has been reported about the roles of CENPM in various types of cancer. The present study identified that the mRNA expression levels of CENPM were significantly upregulated in 14 types of human cancer and identified a positive association between CENPM mRNA expression and patient mortality using the Oncomine, Gene Expression Profiling Interactive Analysis, Human Protein Atlas and Kaplan‑Meier Plotter databases. A protein interaction network constructed with CENPM‑interacting genes obtained from the cBioPortal demonstrated that nine genes participating in the cell cycle served key roles in the function of CENPM. Cell cycle analysis, reverse transcription‑quantitative polymerase chain reaction, a Cell Counting Kit‑8‑based proliferation assay and a terminal deoxynucleotidyl transferase dUTP nick end labelling assay further revealed the tumorigenic and carcinogenic roles of CENPM in vitro. In addition, it was identified that the mRNA expression levels of five of the nine identified genes were significantly associated with CENPM in MCF7 cells and that CENPM was rarely mutated among various types of human cancer. In conclusion, the data from the present study revealed that CENPM exerted its pro‑tumorigenic function by regulating cell cycle‑associated protein expression and suggested that CENPM could be used as a prognostic marker for breast cancer.
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Affiliation(s)
- Ying Liu
- Key Laboratory of Endemic and Ethnic Diseases of The Ministry of Education, Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China
| | - Wenfeng Yu
- Key Laboratory of Endemic and Ethnic Diseases of The Ministry of Education, Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China
| | - Peng Ren
- Department of Urology, The Second Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 556000, P.R. China
| | - Ting Zhang
- Key Laboratory of Endemic and Ethnic Diseases of The Ministry of Education, Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China
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25
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Ying Z, Yang J, Li W, Wang X, Zhu Z, Jiang W, Li C, Sha O. Astrin: A Key Player in Mitosis and Cancer. Front Cell Dev Biol 2020; 8:866. [PMID: 32984344 PMCID: PMC7484939 DOI: 10.3389/fcell.2020.00866] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Accepted: 08/11/2020] [Indexed: 12/04/2022] Open
Abstract
Astrin, which is a spindle-associated protein, was found to be closely related to mitotic spindle formation and maintenance. It interacts with other spindle-related proteins to play a key role in maintaining the attachment of the kinetochore-microtubule and integrity of centrosomes and promoting the centriole duplication. In addition, Astrin was quite recently found to be abnormally highly expressed in a variety of cancers. Astrin promotes the development of cancer by participating in various molecular pathways and is considered as a potential prognostic and survival predictor.
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Affiliation(s)
- Zhenguang Ying
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China
| | - Jing Yang
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China
| | - Wei Li
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China
| | - Xia Wang
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China
| | - Zeyao Zhu
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China
| | - Weipeng Jiang
- School of Dentistry, Shenzhen University Health Science Centre, Shenzhen, China
| | - Chunman Li
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China
| | - Ou Sha
- Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences, Shenzhen University Health Science Centre, Shenzhen, China.,School of Dentistry, Shenzhen University Health Science Centre, Shenzhen, China
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26
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SPAG5 is associated with unfavorable prognosis in patients with lung adenocarcinoma and promotes proliferation, motility and autophagy in A549 cells. Exp Ther Med 2020; 20:77. [PMID: 32968434 PMCID: PMC7500011 DOI: 10.3892/etm.2020.9205] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Accepted: 07/17/2020] [Indexed: 12/24/2022] Open
Abstract
Sperm-associated antigen 5 (SPAG5) is involved in the tumorigenesis of multiple cancer types. However, the role of SPAG5 during lung adenocarcinoma (LUAD) progression remains to be fully elucidated. In the present study, the expression of SPAG5 in tumor tissues of patients with LUAD from public cancer databases was analyzed using the online software Gene Expression Profiling Interactive Analysis and University of Alabama Cancer Database. The association of SPAG5 expression levels with the prognosis of patients with LUAD was analyzed using Kaplan-Meier Plotter. In addition, the role of SPAG5 in the LUAD cell line A549 was determined by knocking down its expression with specific small interfering RNA. The results demonstrated that SPAG5 expression was upregulated in LUAD tissues and its high expression was associated with unfavorable prognosis. Furthermore, in A549 cells, SPAG5 promoted proliferation, migration, invasion and autophagy, but inhibited apoptosis. The present results suggest that SPAG5 has an oncogenic role in LUAD and may be a potential prognostic predictor and therapeutic target for LUAD.
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27
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Mohamadalizadeh-Hanjani Z, Shahbazi S, Geranpayeh L. Investigation of the SPAG5 gene expression and amplification related to the NuMA mRNA levels in breast ductal carcinoma. World J Surg Oncol 2020; 18:225. [PMID: 32838814 PMCID: PMC7445913 DOI: 10.1186/s12957-020-02001-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Accepted: 08/12/2020] [Indexed: 12/26/2022] Open
Abstract
Background The cell proliferative markers are very important in breast cancer. Since SPAG5 and NuMA proteins play a significant role in the mitosis regulatory network and cell division, we aimed to study their mRNA levels as well as SPAG5 gene amplification correlated to clinicopathological status in ductal carcinoma of the breast. Methods SPAG5 and NuMA gene expressions were investigated in 40 breast cancer tissues and normal adjacent tissues via real-time PCR. PUM1 was selected as the reference gene. QMF PCR method was applied to study SPAG5 gene amplification and AGBL2, BOD1L, and POR were designated as internal control genes. Gene amplification was determined by calculating a dosage quotient for each DNA fragment. Results Increased SPAG5 mRNA expression was detected in breast cancer tissues (p = 0.005) and related to tumor size. No significant difference was observed between NuMA gene expression level in tumor tissue and the normal adjacent tissue (p = 0.56). However, we observed that NuMA expression was significantly increased in ER-positive tumor tissues. There was no clear correlation pattern between SPAG5 and NuMA mRNA levels (r = 0.33). Seventeen percent of tissues showed complete amplification in SPAG5 gene fragments. Conclusion Our results were consistent with the previous publications regarding SPAG5 gene expression and amplification in breast cancer with an emphasis on the prominent role of this protein in tumor pathogenesis. Our results failed to yield any correlation between SPAG5 and NuMA mRNA levels which implies independence of these genes in breast cancer pathogenesis.
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Affiliation(s)
| | - Shirin Shahbazi
- Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
| | - Loabat Geranpayeh
- Department of Surgery, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran
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28
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Mycoplasma hyopneumoniae J elicits an antioxidant response and decreases the expression of ciliary genes in infected swine epithelial cells. Sci Rep 2020; 10:13707. [PMID: 32792522 PMCID: PMC7426424 DOI: 10.1038/s41598-020-70040-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Accepted: 06/22/2020] [Indexed: 12/20/2022] Open
Abstract
Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.
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29
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Li Z, Li H, Chen J, Luo H, Zeng J, Yao Y, Duan M. SPAG5 promotes osteosarcoma metastasis via activation of FOXM1/MMP2 axis. Int J Biochem Cell Biol 2020; 126:105797. [PMID: 32668328 DOI: 10.1016/j.biocel.2020.105797] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 06/22/2020] [Accepted: 06/30/2020] [Indexed: 01/02/2023]
Abstract
Osteosarcoma (OS) is a primary malignancy of bone with a tendency to metastasize early. An understanding of the pathways that regulate OS metastasis is required for the design of novel treatment approaches. Sperm-associated antigen 5 (SPAG5) is upregulated and functions as a potential tumor promoter in diverse human cancers, but has yet to be investigated in the OS. In the present study, results showed that SPAG5 expression is upregulated in OS tissues, and SPAG5 overexpression is obviously associated with the malignant phenotype and poor survival in patients with OS. Multivariate analyses also revealed that SPAG5 overexpression is an independent prognostic factor for poor outcome of patients with OS. The functional assay indicated that SPAG5 silencing significantly inhibits the invasion and migration of OS cells in vitro. Additionally, knockdown of SPAG5 in OS cells suppresses lung metastasis in vivo. Further, we also found that SPAG5 silencing inhibits the epithelial-mesenchymal transition (EMT) process of OS cells. Moreover, our results indicated that SPAG5 promotes OS metastasis by increasing matrix metalloproteinase-2 (MMP2) expression, and demonstrated that MMP2 is crucial for the pro-metastasis role of SPAG5 in OS cells. Mechanistically, we identified that SPAG5 regulates MMP2 expression by modulating FOXM1 (Forkhead box M1) degradation to enhance the protein stability of FOXM1. Collectively, these findings describe the effects of SPAG5-FOXM1-MMP2 axis in the regulation of OS cell migration and metastasis formation. We provide a novel evidence that SPAG5 may serve as a prognostic indicator and potential therapeutic target for patients with osteosarcoma.
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Affiliation(s)
- Zhiyun Li
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Hu Li
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Jiangwei Chen
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Hao Luo
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Jin Zeng
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Yao Yao
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China
| | - Mansheng Duan
- Department of Orthopedic Surgery, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China.
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30
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Conti D, Gul P, Islam A, Martín-Durán JM, Pickersgill RW, Draviam VM. Kinetochores attached to microtubule-ends are stabilised by Astrin bound PP1 to ensure proper chromosome segregation. eLife 2019; 8:49325. [PMID: 31808746 PMCID: PMC6930079 DOI: 10.7554/elife.49325] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2019] [Accepted: 12/01/2019] [Indexed: 12/12/2022] Open
Abstract
Microtubules segregate chromosomes by attaching to macromolecular kinetochores. Only microtubule-end attached kinetochores can be pulled apart; how these end-on attachments are selectively recognised and stabilised is not known. Using the kinetochore and microtubule-associated protein, Astrin, as a molecular probe, we show that end-on attachments are rapidly stabilised by spatially-restricted delivery of PP1 near the C-terminus of Ndc80, a core kinetochore-microtubule linker. PP1 is delivered by the evolutionarily conserved tail of Astrin and this promotes Astrin’s own enrichment creating a highly-responsive positive feedback, independent of biorientation. Abrogating Astrin:PP1-delivery disrupts attachment stability, which is not rescued by inhibiting Aurora-B, an attachment destabiliser, but is reversed by artificially tethering PP1 near the C-terminus of Ndc80. Constitutive Astrin:PP1-delivery disrupts chromosome congression and segregation, revealing a dynamic mechanism for stabilising attachments. Thus, Astrin-PP1 mediates a dynamic ‘lock’ that selectively and rapidly stabilises end-on attachments, independent of biorientation, and ensures proper chromosome segregation.
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Affiliation(s)
- Duccio Conti
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom.,Department of Genetics, University of Cambridge, Cambridge, United Kingdom
| | - Parveen Gul
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
| | - Asifa Islam
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
| | - José M Martín-Durán
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
| | - Richard W Pickersgill
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
| | - Viji M Draviam
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom.,Department of Genetics, University of Cambridge, Cambridge, United Kingdom
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31
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Amin MA, Agarwal S, Varma D. Mapping the kinetochore MAP functions required for stabilizing microtubule attachments to chromosomes during metaphase. Cytoskeleton (Hoboken) 2019; 76:398-412. [PMID: 31454167 DOI: 10.1002/cm.21559] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2018] [Revised: 08/07/2019] [Accepted: 08/22/2019] [Indexed: 12/24/2022]
Abstract
In mitosis, faithful chromosome segregation is orchestrated by the dynamic interactions between the spindle microtubules (MTs) emanating from the opposite poles and the kinetochores of the chromosomes. However, the precise mechanism that coordinates the coupling of the kinetochore components to dynamic MTs has been a long-standing question. Microtubule-associated proteins (MAPs) regulate MT nucleation and dynamics, MT-mediated transport and MT cross-linking in cells. During mitosis, MAPs play an essential role not only in determining spindle length, position, and orientation but also in facilitating robust kinetochore-microtubule (kMT) attachments by linking the kinetochores to spindle MTs efficiently. The stability of MTs imparted by the MAPs is critical to ensure accurate chromosome segregation. This review primarily focuses on the specific function of nonmotor kinetochore MAPs, their recruitment to kinetochores and their MT-binding properties. We also attempt to synthesize and strengthen our understanding of how these MAPs work in coordination with the kinetochore-bound Ndc80 complex (the key component at the MT-binding interface in metaphase and anaphase) to establish stable kMT attachments and control accurate chromosome segregation during mitosis.
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Affiliation(s)
- Mohammed A Amin
- Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
| | - Shivangi Agarwal
- Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
| | - Dileep Varma
- Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
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32
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Akera T, Trimm E, Lampson MA. Molecular Strategies of Meiotic Cheating by Selfish Centromeres. Cell 2019; 178:1132-1144.e10. [PMID: 31402175 DOI: 10.1016/j.cell.2019.07.001] [Citation(s) in RCA: 87] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Revised: 01/11/2019] [Accepted: 06/25/2019] [Indexed: 10/26/2022]
Abstract
Asymmetric division in female meiosis creates selective pressure favoring selfish centromeres that bias their transmission to the egg. This centromere drive can explain the paradoxical rapid evolution of both centromere DNA and centromere-binding proteins despite conserved centromere function. Here, we define a molecular pathway linking expanded centromeres to histone phosphorylation and recruitment of microtubule destabilizing factors, leading to detachment of selfish centromeres from spindle microtubules that would direct them to the polar body. Exploiting centromere divergence between species, we show that selfish centromeres in two hybrid mouse models use the same molecular pathway but modulate it differently to enrich destabilizing factors. Our results indicate that increasing microtubule destabilizing activity is a general strategy for drive in both models, but centromeres have evolved distinct mechanisms to increase that activity. Furthermore, we show that drive depends on slowing meiotic progression, suggesting that selfish centromeres can be suppressed by regulating meiotic timing.
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Affiliation(s)
- Takashi Akera
- Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Emily Trimm
- Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Michael A Lampson
- Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA.
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33
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Wang T, Li K, Song H, Xu D, Liao Y, Jing B, Guo W, Hu M, Kuang Y, Sun B, Ling J, Zhang T, Xu J, Yao F, Deng J. p53 suppression is essential for oncogenic SPAG5 upregulation in lung adenocarcinoma. Biochem Biophys Res Commun 2019; 513:319-325. [PMID: 30955859 DOI: 10.1016/j.bbrc.2019.03.198] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2019] [Accepted: 03/29/2019] [Indexed: 01/18/2023]
Abstract
Aberrant expression of sperm-associated antigen 5 (SPAG5) is implicated to play oncogenic roles in several types of cancers. However, the functions of SPAG5 in lung adenocarcinoma remain unclear. In this study, we investigated the role of SPAG5 in lung adenocarcinoma. We found that SPAG5 was upregulated in most of the lung adenocarcinoma cell lines as compared to normal lung epithelial cells. SPAG5 knockdown suppressed proliferation, colony forming, and migration of lung adenocarcinoma A549 cells in vitro and inhibited tumor growth in vivo. These suggest that upregulated SPAG5 promotes lung tumor progression. Importantly, treatment with MDM2 inhibitor, Nutlin-3a, restored p53 and p21 expression and suppressed SPAG5 expression in wild-type p53 lung adenocarcinoma cells, A549 and H460, but not in p53-null lung cancer cells, H1299. This suggests that the p53 signal pathway is essential for SPAG5 suppression. In addition, knocking-down p53 or p21 in A549 and H460 cells attenuated Nutlin-3a-induced repression of SPAG5, which further supports that the p53-p21 axis is required for SPAG5 repression. Thus, SPAG5 can serve as a prognostic marker, and therapeutic strategy targeting the p53-p21-SPAG5 axis may have important clinical implications.
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Affiliation(s)
- Tong Wang
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Kaimi Li
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Hongyong Song
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Dongliang Xu
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yueling Liao
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Bo Jing
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Wenzheng Guo
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Min Hu
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yanbin Kuang
- Department of Respiratory Medicine, The Second Affiliated Hospital, Dalian Medical University, Dalian, China
| | - Beibei Sun
- Translational Medical Research Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Jing Ling
- Department of Oncology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Tuo Zhang
- Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Jianhua Xu
- Basic Medical School of Pathology and Pathophysiology, Kunming Medical University, Kunming, China
| | - Feng Yao
- Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Jiong Deng
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Translational Medical Research Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China.
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Zanchetta ME, Meroni G. Emerging Roles of the TRIM E3 Ubiquitin Ligases MID1 and MID2 in Cytokinesis. Front Physiol 2019; 10:274. [PMID: 30941058 PMCID: PMC6433704 DOI: 10.3389/fphys.2019.00274] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Accepted: 02/28/2019] [Indexed: 11/13/2022] Open
Abstract
Ubiquitination is a post-translational modification that consists of ubiquitin attachment to target proteins through sequential steps catalysed by activating (E1), conjugating (E2), and ligase (E3) enzymes. Protein ubiquitination is crucial for the regulation of many cellular processes not only by promoting proteasomal degradation of substrates but also re-localisation of cellular factors and modulation of protein activity. Great importance in orchestrating ubiquitination relies on E3 ligases as these proteins recognise the substrate that needs to be modified at the right time and place. Here we focus on two members of the TRIpartite Motif (TRIM) family of RING E3 ligases, MID1, and MID2. We discuss the recent findings on these developmental disease-related proteins analysing the link between their activity on essential factors and the regulation of cytokinesis highlighting the possible consequence of alteration of this process in pathological conditions.
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Affiliation(s)
| | - Germana Meroni
- Department of Life Sciences, University of Trieste, Trieste, Italy
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35
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Liu H, Hu J, Wei R, Zhou L, Pan H, Zhu H, Huang M, Luo J, Xu W. SPAG5 promotes hepatocellular carcinoma progression by downregulating SCARA5 through modifying β-catenin degradation. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2018; 37:229. [PMID: 30249289 PMCID: PMC6154423 DOI: 10.1186/s13046-018-0891-3] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/22/2018] [Accepted: 08/13/2018] [Indexed: 02/06/2023]
Abstract
BACKGROUND The sperm-associated antigen 5 (SPAG5) plays a key role in controlling various cellular phenomena, including cell cycle progression and proliferation. However, the role of SPAG5 in hepatocellular carcinoma (HCC) remains unknown. METHODS This study investigated the function and clinical significance of SPAG5 protein expression in hepatocellular carcinoma. We analyzed SPAG5 expression in surgical specimens from 136 HCC patients. The correlation between the clinical characteristics and prognosis was also determined. Furthermore, the SPAG5 was overexpressed in HCC cell and silenced with shRNA in HCC cells. Moreover, cell proliferation and apoptosis were measured using Edu assay and flow cytometry and a molecular mechanism of SPAG5 promotes HCC progression was explored. RESULTS Herein, our study showed that upregulation of SPAG5 was detected frequently in primary HCC tissues, and was associated with significantly worse survival among the HCC patients. Multivariate analyses revealed that high SPAG5 expression was an independent predictive marker for the poor prognosis of HCC. SPAG5 silence effectively abolished the proliferation abilities of SPAG5 in vivo and in vitro, while induced apoptosis in HCC cells. Furthermore, our results indicate that SPAG5 promoted cell progression by decreasing SCARA5 expression, which has been reported to control the progression of HCC, and our data demonstrated that SCARA5 is crucial for SPAG5-mediated HCC cell progression in vitro and in vivo. Moreover, we found that the expression of SPAG5 and SCARA5 are inversely correlated in HCC tissues. In addition, we demonstrated that SPAG5 promoted progression in HCC via downregulating SCARA5 depended on the β-catenin/TCF4 signaling pathway. Interestingly, the underlying mechanism is which SPAG5 regulates SCARA5 expression by modulating β-catenin degradation. CONCLUSIONS Taken together, our data provide a novel evidence for the biological and clinical significance of SPAG5 as a potential biomarker, and we demonstrate that SPAG5-β-catenin-SCARA5 might be a novel pathway involved in HCC progression.
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Affiliation(s)
- Hongliang Liu
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China
| | - Junwen Hu
- Department of Hepatobiliary Surgery, Tumor Hospital of Guanxi Medical University, Nanning, China.,Department of General Surgery, Third Affiliated Hospital of Nanchang University, Nanchang, China
| | - Ran Wei
- The First Clinical Medical College of Nanchang University, Nanchang, China
| | - Longfei Zhou
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China
| | - Hua Pan
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China
| | - Hongchao Zhu
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China
| | - Mingwen Huang
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China
| | - Jun Luo
- Department of Rehabilitation Medicine, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China.
| | - Wei Xu
- Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Min De Road, Nanchang, 330006, Jiangxi Province, China.
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Platani M, Samejima I, Samejima K, Kanemaki MT, Earnshaw WC. Seh1 targets GATOR2 and Nup153 to mitotic chromosomes. J Cell Sci 2018; 131:jcs.213140. [PMID: 29618633 PMCID: PMC5992584 DOI: 10.1242/jcs.213140] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 03/23/2018] [Indexed: 12/27/2022] Open
Abstract
In metazoa, the Nup107 complex (also known as the nucleoporin Y-complex) plays a major role in formation of the nuclear pore complex in interphase and is localised to kinetochores in mitosis. The Nup107 complex shares a single highly conserved subunit, Seh1 (also known as SEH1L in mammals) with the GATOR2 complex, an essential activator of mTORC1 kinase. mTORC1/GATOR2 has a central role in the coordination of cell growth and proliferation. Here, we use chemical genetics and quantitative chromosome proteomics to study the role of the Seh1 protein in mitosis. Surprisingly, Seh1 is not required for the association of the Nup107 complex with mitotic chromosomes, but it is essential for the association of both the GATOR2 complex and nucleoporin Nup153 with mitotic chromosomes. Our analysis also reveals a role for Seh1 at human centromeres, where it is required for efficient localisation of the chromosomal passenger complex (CPC). Furthermore, this analysis detects a functional interaction between the Nup107 complex and the small kinetochore protein SKAP (also known as KNSTRN). Highlighted Article: The nucleoporin Seh1 is essential for the association of both the GATOR2 complex and the nucleoporin Nup153, but not the Nup107 complex, with mitotic chromosomes.
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Affiliation(s)
- Melpomeni Platani
- Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Itaru Samejima
- Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Kumiko Samejima
- Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK
| | - Masato T Kanemaki
- Division of Molecular Cell Engineering, National Institute of Genetics, ROIS, and Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan
| | - William C Earnshaw
- Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK
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SPAG5 promotes proliferation and suppresses apoptosis in bladder urothelial carcinoma by upregulating Wnt3 via activating the AKT/mTOR pathway and predicts poorer survival. Oncogene 2018; 37:3937-3952. [PMID: 29662193 DOI: 10.1038/s41388-018-0223-2] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Revised: 01/27/2018] [Accepted: 02/27/2018] [Indexed: 01/07/2023]
Abstract
Sperm-associated antigen 5 (SPAG5) is involved in various biological processes. However, the roles of SPAG5 in bladder urothelial carcinoma (BUC) are unknown. This study showed that upregulation of SPAG5 was detected frequently in primary BUC tissues, and was associated with significantly worse survival among the 112 patients that underwent radical cystectomy (RC). Up and downregulating the expression of SPAG5 enhanced or inhibited, respectively, the proliferation of BUC cells in vitro and in vivo, and suppressed or enhanced, respectively, apoptosis in vitro and in vivo. Moreover, SPAG5 increased the resistance of BUC cells to chemotherapy-induced apoptosis. Mechanistic investigations showed that SPAG5 promotes proliferation and suppresses apoptosis in BUC at least partially via upregulating Wnt3 through activating the AKT/mTOR signaling pathway. The importance of the SPAG5/AKT-mTOR/Wnt3 axis identified in BUC cell models was confirmed via immunohistochemical analysis of a cohort of human BUC specimens that underwent RC. Collectively, our data suggested that in patients with BUC who underwent RC, high SPAG5 expression is associated with poor survival. In addition, targeting SPAG5 might represent a novel therapeutic strategy to improve the survival of patients with BUC.
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38
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Kern DM, Monda JK, Su KC, Wilson-Kubalek EM, Cheeseman IM. Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80. eLife 2017; 6:26866. [PMID: 28841134 PMCID: PMC5602300 DOI: 10.7554/elife.26866] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2017] [Accepted: 08/24/2017] [Indexed: 12/15/2022] Open
Abstract
Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.
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Affiliation(s)
- David M Kern
- Whitehead Institute for Biomedical Research, Cambridge, United States.,Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
| | - Julie K Monda
- Whitehead Institute for Biomedical Research, Cambridge, United States.,Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
| | - Kuan-Chung Su
- Whitehead Institute for Biomedical Research, Cambridge, United States
| | | | - Iain M Cheeseman
- Whitehead Institute for Biomedical Research, Cambridge, United States.,Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
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39
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Shrestha RL, Conti D, Tamura N, Braun D, Ramalingam RA, Cieslinski K, Ries J, Draviam VM. Aurora-B kinase pathway controls the lateral to end-on conversion of kinetochore-microtubule attachments in human cells. Nat Commun 2017; 8:150. [PMID: 28751710 PMCID: PMC5532248 DOI: 10.1038/s41467-017-00209-z] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2016] [Accepted: 06/12/2017] [Indexed: 12/29/2022] Open
Abstract
Human chromosomes are captured along microtubule walls (lateral attachment) and then tethered to microtubule-ends (end-on attachment) through a multi-step end-on conversion process. Upstream regulators that orchestrate this remarkable change in the plane of kinetochore-microtubule attachment in human cells are not known. By tracking kinetochore movements and using kinetochore markers specific to attachment status, we reveal a spatially defined role for Aurora-B kinase in retarding the end-on conversion process. To understand how Aurora-B activity is counteracted, we compare the roles of two outer-kinetochore bound phosphatases and find that BubR1-associated PP2A, unlike KNL1-associated PP1, plays a significant role in end-on conversion. Finally, we uncover a novel role for Aurora-B regulated Astrin-SKAP complex in ensuring the correct plane of kinetochore-microtubule attachment. Thus, we identify Aurora-B as a key upstream regulator of end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.Human chromosomes are captured along microtubule walls and then tethered to microtubule-ends through a multi-step end-on conversion process. Here the authors show that Aurora-B regulates end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.
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Affiliation(s)
- Roshan L Shrestha
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK
- Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Duccio Conti
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK
- School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK
| | - Naoka Tamura
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK
- Barts Cancer Institute, Queen Mary University of London, London, EC1M 6BQ, UK
| | - Dominique Braun
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK
| | - Revathy A Ramalingam
- School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK
| | - Konstanty Cieslinski
- European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Meyerhofstrasse 1, Heidelberg, Germany
| | - Jonas Ries
- European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Meyerhofstrasse 1, Heidelberg, Germany
| | - Viji M Draviam
- Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK.
- School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK.
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40
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Rattani A, Ballesteros Mejia R, Roberts K, Roig MB, Godwin J, Hopkins M, Eguren M, Sanchez-Pulido L, Okaz E, Ogushi S, Wolna M, Metson J, Pendás AM, Malumbres M, Novák B, Herbert M, Nasmyth K. APC/C Cdh1 Enables Removal of Shugoshin-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity. Curr Biol 2017; 27:1462-1476.e5. [PMID: 28502659 PMCID: PMC5457479 DOI: 10.1016/j.cub.2017.04.023] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2016] [Revised: 02/23/2017] [Accepted: 04/12/2017] [Indexed: 01/06/2023]
Abstract
In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata.
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Affiliation(s)
- Ahmed Rattani
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Randy Ballesteros Mejia
- Newcastle Fertility Centre, Centre for Life, Times Square, Newcastle upon Tyne NE1 4EP, UK; Wellcome Trust Centre for Mitochondrial Research, Institute for Genetic Medicine, Newcastle University, Newcastle upon Tyne NE4 5PL, UK
| | - Katherine Roberts
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Maurici B Roig
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Jonathan Godwin
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Michael Hopkins
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Manuel Eguren
- Cell Division and Cancer Group, Spanish National Cancer Research Center (CNIO), 28029 Madrid, Spain
| | - Luis Sanchez-Pulido
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Elwy Okaz
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Sugako Ogushi
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Magda Wolna
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Jean Metson
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Alberto M Pendás
- Instituto de Biología Molecular y Celular del Cáncer de Salamanca, CSIC-Universidad de Salamanca, 37007 Salamanca, Spain
| | - Marcos Malumbres
- Cell Division and Cancer Group, Spanish National Cancer Research Center (CNIO), 28029 Madrid, Spain
| | - Béla Novák
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Mary Herbert
- Newcastle Fertility Centre, Centre for Life, Times Square, Newcastle upon Tyne NE1 4EP, UK; Wellcome Trust Centre for Mitochondrial Research, Institute for Genetic Medicine, Newcastle University, Newcastle upon Tyne NE4 5PL, UK
| | - Kim Nasmyth
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
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41
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Critical roles of Astrin in the mitosis of immature rat Sertoli cells. Biochem Biophys Res Commun 2017; 486:958-964. [PMID: 28351621 DOI: 10.1016/j.bbrc.2017.03.137] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Accepted: 03/25/2017] [Indexed: 11/21/2022]
Abstract
Male hypogonadism (hgn/hgn) rats show testicular hypoplasia accompanied by dysplastic development of seminiferous tubules due to loss-of-function mutation of the gene encoding Astrin, which is required for mitotic progression in the division cycle of HeLa cells. In the present study, we examined the cytological base leading to the decrease of Sertoli cells in hgn/hgn testes. In hgn/hgn testes on postnatal day 3, anti-phospho-histone H3 (Ser10) (pH3)-positive mitotic phase and TUNEL-positive apoptosis increased in GATA4-positive Sertoli cells. Isolated immature Sertoli cells from hgn/hgn testes showed increased pH3-assessed mitotic index accompanied by decreased 5-bromo-2'-deoxyuridine-incorporation and increased TUNEL-positive apoptosis, suggesting mitotic delay and cell death. In the visualization of mitotic progression by nocodazole (NOC)-mediated cell cycle arrest and subsequent release, hgn/hgn rat-derived Sertoli cells failed to make the transition from prometaphase to metaphase, and the cells with micronuclei and TUNEL-positive cells gradually increased in a time-dependent manner. Western blot analysis detected ≈142 kDa protein expected as Astrin in extracts of +/+ and +/hgn testes and cultured normal Sertoli cells but not in extracts of hgn/hgn testes. CLASP1 was detected in extracts of both normal and hgn/hgn testes, whereas it was localized in kinetochore of normal mitotic Sertoli cells but diffused in cytoplasm of hgn/hgn Sertoli cells. These results indicate that Astrin is required for normal mitotic progression in immature Sertoli cells and that the most severe type of testicullar dysplasia in hgn/hgn rats is caused by mitotic cell death of immature Sertoli cells due to lack of Astrin.
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42
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Chung HJ, Park JE, Lee NS, Kim H, Jang CY. Phosphorylation of Astrin Regulates Its Kinetochore Function. J Biol Chem 2016; 291:17579-92. [PMID: 27325694 PMCID: PMC5016155 DOI: 10.1074/jbc.m115.712745] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2015] [Revised: 06/16/2016] [Indexed: 01/09/2023] Open
Abstract
The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.
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Affiliation(s)
- Hee Jin Chung
- Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea and
| | - Ji Eun Park
- Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul 04310, Republic of Korea
| | - Nam Soo Lee
- Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea and
| | - Hongtae Kim
- Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea and From the Center for Neuroscience Imaging Research, Institute for Basic Science and
| | - Chang-Young Jang
- Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul 04310, Republic of Korea
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43
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Chu X, Chen X, Wan Q, Zheng Z, Du Q. Nuclear Mitotic Apparatus (NuMA) Interacts with and Regulates Astrin at the Mitotic Spindle. J Biol Chem 2016; 291:20055-67. [PMID: 27462074 DOI: 10.1074/jbc.m116.724831] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2016] [Indexed: 11/06/2022] Open
Abstract
The large nuclear mitotic apparatus (NuMA) protein is an essential player in mitotic spindle assembly and maintenance. We report here the identification of Astrin, a spindle- and kinetochore-associated protein, as a novel interactor of NuMA. We show that the C-terminal tail of NuMA can directly bind to the C terminus of Astrin and that this interaction helps to recruit Astrin to microtubules. Knockdown of NuMA by RNA interference dramatically impaired Astrin recruitment to the mitotic spindle. Overexpression of the N terminus of mammalian homologue of Drosophila Pins (LGN), which blocks the microtubule binding of NuMA and competes with Astrin for NuMA binding, also led to similar results. Furthermore, we found that cytoplasmic dynein is required for the spindle pole accumulation of Astrin, and dynein-mediated transport is important for balanced distribution of Astrin between spindle poles and kinetochores. On the other hand, if Astrin levels are reduced, then NuMA could not efficiently concentrate at the spindle poles. Our findings reveal a direct physical link between two important regulators of mitotic progression and demonstrate the critical role of the NuMA-Astrin interaction for accurate cell division.
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Affiliation(s)
- Xiaogang Chu
- From the Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia 30912
| | - Xuanyu Chen
- From the Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia 30912
| | - Qingwen Wan
- From the Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia 30912
| | - Zhen Zheng
- From the Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia 30912
| | - Quansheng Du
- From the Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia 30912
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44
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Kern DM, Nicholls PK, Page DC, Cheeseman IM. A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends. J Cell Biol 2016; 213:315-28. [PMID: 27138257 PMCID: PMC4862331 DOI: 10.1083/jcb.201510117] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2015] [Accepted: 03/30/2016] [Indexed: 12/14/2022] Open
Abstract
The Astrin/SKAP complex regulates mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, Kern et al. demonstrate that a previously unappreciated short SKAP isoform mediates mitotic spindle positioning at astral microtubule plus ends. The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning.
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Affiliation(s)
- David M Kern
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
| | - Peter K Nicholls
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142
| | - David C Page
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Howard Hughes Medical Institute, Chevy Chase, MD 20815
| | - Iain M Cheeseman
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
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Abstract
Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.
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Affiliation(s)
- Sabine Petry
- Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014;
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46
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Friese A, Faesen AC, Huis in 't Veld PJ, Fischböck J, Prumbaum D, Petrovic A, Raunser S, Herzog F, Musacchio A. Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin. Nat Commun 2016; 7:11407. [PMID: 27095104 PMCID: PMC4843017 DOI: 10.1038/ncomms11407] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Accepted: 03/22/2016] [Indexed: 12/18/2022] Open
Abstract
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule–kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore–microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins. SKAP and Astrin form a heterodimer that localizes to spindle microtubules and to mature microtubule-kinetochore attachments during mitosis. Here, the authors identify molecular requirements for the inter-subunit interaction of SKAP and Astrin and kinetochore recruitment.
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Affiliation(s)
- Alexandra Friese
- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
| | - Alex C Faesen
- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
| | - Pim J Huis in 't Veld
- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
| | - Josef Fischböck
- Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany
| | - Daniel Prumbaum
- Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
| | - Arsen Petrovic
- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
| | - Stefan Raunser
- Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
| | - Franz Herzog
- Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany
| | - Andrea Musacchio
- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.,Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, 45141 Essen, Germany
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47
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Gholkar AA, Senese S, Lo YC, Vides E, Contreras E, Hodara E, Capri J, Whitelegge JP, Torres JZ. The X-Linked-Intellectual-Disability-Associated Ubiquitin Ligase Mid2 Interacts with Astrin and Regulates Astrin Levels to Promote Cell Division. Cell Rep 2015; 14:180-8. [PMID: 26748699 DOI: 10.1016/j.celrep.2015.12.035] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Revised: 10/13/2015] [Accepted: 12/04/2015] [Indexed: 12/13/2022] Open
Abstract
Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409, and is degraded during cytokinesis. Mid2 depletion led to astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant astrin in astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of astrin on K409, which is critical for its degradation and proper cytokinesis. These results could help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.
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Affiliation(s)
- Ankur A Gholkar
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Silvia Senese
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Yu-Chen Lo
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; Program in Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Edmundo Vides
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Ely Contreras
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Emmanuelle Hodara
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Joseph Capri
- Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Julian P Whitelegge
- Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Jorge Z Torres
- Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
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48
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Morrison JL, Zhang S, Tellam RL, Brooks DA, McMillen IC, Porrello ER, Botting KJ. Regulation of microRNA during cardiomyocyte maturation in sheep. BMC Genomics 2015. [PMID: 26198574 PMCID: PMC4509559 DOI: 10.1186/s12864-015-1693-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022] Open
Abstract
Background There is a limited capacity to repair damage in the mammalian heart after birth, which is primarily due to the inability of cardiomyocytes to proliferate after birth. This is in contrast to zebrafish and salamander, in which cardiomyocytes retain the ability to proliferate throughout life and can regenerate their heart after significant damage. Recent studies in zebrafish and rodents implicate microRNA (miRNA) in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, miR-133a, the miR-15 family, miR-199a and miR-590. However, the significance of these miRNA and miRNA in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. To determine the involvement of miRNA in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated miRNA and target gene expression in sheep hearts before and after birth. The experimental approach included targeted transcriptional profiling of miRNA and target mRNA previously identified in rodent studies as well as genome-wide miRNA profiling using microarrays. Results The cardiac expression of miR-133a increased and its target gene IGF1R decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. The expression of the miR-15 family members was variable with age, however, four of their target genes decreased with age. These latter profiles are inconsistent with the direct involvement of this family of miRNA in cardiomyocyte quiescence in late gestation sheep. The expression patterns of ‘pro-proliferative’ miR-199a and miR-590 were also inconsistent with their involvement in cardiomyocyte quiescence. Consequently, miRNA microarray analysis was undertaken, which identified six discrete clusters of miRNA with characteristic developmental profiles. The functions of predicted target genes for the miRNA in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these miRNA in regulating cardiomyocyte proliferation. Conclusion The results of this study show that the expression of miR-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. The expression patterns of the miR-15 family, miR-199a and miR-590 were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. miRNA microarray analysis suggests a coordinated and potentially more complex role of multiple miRNA in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1693-z) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Janna L Morrison
- Early Origins of Adult Health Research Group, University of South Australia, Adelaide, SA, Australia.
| | - Song Zhang
- Early Origins of Adult Health Research Group, University of South Australia, Adelaide, SA, Australia.
| | - Ross L Tellam
- CSIRO Agriculture, CSIRO, Queensland Biosciences Precinct, St Lucia, QLD, Australia.
| | - Doug A Brooks
- Mechanisms in Cell Biology and Diseases Research Group, University of South Australia, Adelaide, SA, Australia.
| | - I Caroline McMillen
- Early Origins of Adult Health Research Group, University of South Australia, Adelaide, SA, Australia.
| | - Enzo R Porrello
- Laboratory for Cardiac Regeneration, School of Biomedical Sciences, University of Queensland, St Lucia, QLD, Australia.
| | - Kimberley J Botting
- Early Origins of Adult Health Research Group, University of South Australia, Adelaide, SA, Australia.
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O'Regan L, Sampson J, Richards MW, Knebel A, Roth D, Hood FE, Straube A, Royle SJ, Bayliss R, Fry AM. Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression. J Cell Biol 2015; 209:349-58. [PMID: 25940345 PMCID: PMC4427782 DOI: 10.1083/jcb.201409151] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Accepted: 03/31/2015] [Indexed: 12/18/2022] Open
Abstract
Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber-stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT-kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG-TACC3 complex.
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Affiliation(s)
- Laura O'Regan
- Department of Biochemistry, University of Leicester, Leicester LE1 9HN, England, UK
| | - Josephina Sampson
- Department of Biochemistry, University of Leicester, Leicester LE1 9HN, England, UK
| | - Mark W Richards
- Department of Biochemistry, University of Leicester, Leicester LE1 9HN, England, UK
| | - Axel Knebel
- Kinasource Ltd, The Sir James Black Center, Dundee DD1 5EH, Scotland, UK
| | - Daniel Roth
- Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, England, UK
| | - Fiona E Hood
- Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool L69 3BX, England, UK
| | - Anne Straube
- Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, England, UK
| | - Stephen J Royle
- Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, England, UK Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool L69 3BX, England, UK
| | - Richard Bayliss
- Department of Biochemistry, University of Leicester, Leicester LE1 9HN, England, UK
| | - Andrew M Fry
- Department of Biochemistry, University of Leicester, Leicester LE1 9HN, England, UK
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50
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Ferreira JG, Pereira AL, Maiato H. Microtubule plus-end tracking proteins and their roles in cell division. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2014; 309:59-140. [PMID: 24529722 DOI: 10.1016/b978-0-12-800255-1.00002-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Microtubules are cellular components that are required for a variety of essential processes such as cell motility, mitosis, and intracellular transport. This is possible because of the inherent dynamic properties of microtubules. Many of these properties are tightly regulated by a number of microtubule plus-end-binding proteins or +TIPs. These proteins recognize the distal end of microtubules and are thus in the right context to control microtubule dynamics. In this review, we address how microtubule dynamics are regulated by different +TIP families, focusing on how functionally diverse +TIPs spatially and temporally regulate microtubule dynamics during animal cell division.
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Affiliation(s)
- Jorge G Ferreira
- Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, University of Porto, Porto, Portugal; Cell Division Unit, Department of Experimental Biology, University of Porto, Porto, Portugal
| | - Ana L Pereira
- Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, University of Porto, Porto, Portugal
| | - Helder Maiato
- Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, University of Porto, Porto, Portugal; Cell Division Unit, Department of Experimental Biology, University of Porto, Porto, Portugal.
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