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1
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Earl CP, Cobbaut M, Barros-Carvalho A, Ivanova ME, Briggs DC, Morais-de-Sá E, Parker PJ, McDonald NQ. Capture, mutual inhibition and release mechanism for aPKC-Par6 and its multisite polarity substrate Lgl. Nat Struct Mol Biol 2025; 32:729-739. [PMID: 39762628 PMCID: PMC11996676 DOI: 10.1038/s41594-024-01425-0] [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: 01/30/2024] [Accepted: 10/15/2024] [Indexed: 02/23/2025]
Abstract
The mutually antagonistic relationship of atypical protein kinase C (aPKC) and partitioning-defective protein 6 (Par6) with the substrate lethal (2) giant larvae (Lgl) is essential for regulating polarity across many cell types. Although aPKC-Par6 phosphorylates Lgl at three serine sites to exclude it from the apical domain, aPKC-Par6 and Lgl paradoxically form a stable kinase-substrate complex, with conflicting roles proposed for Par6. We report the structure of human aPKCι-Par6α bound to full-length Llgl1, captured through an aPKCι docking site and a Par6PDZ contact. This complex traps a phospho-S663 Llgl1 intermediate bridging between aPKC and Par6, impeding phosphorylation progression. Thus, aPKCι is effectively inhibited by Llgl1pS663 while Llgl1 is captured by aPKCι-Par6. Mutational disruption of the Lgl-aPKC interaction impedes complex assembly and Lgl phosphorylation, whereas disrupting the Lgl-Par6PDZ contact promotes complex dissociation and Lgl phosphorylation. We demonstrate a Par6PDZ-regulated substrate capture-and-release model requiring binding by active Cdc42 and the apical partner Crumbs to drive complex disassembly. Our results suggest a mechanism for mutual regulation and spatial control of aPKC-Par6 and Lgl activities.
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Affiliation(s)
- Christopher P Earl
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK
| | - Mathias Cobbaut
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK.
- Protein Phosphorylation Laboratory, Francis Crick Institute, London, UK.
| | - André Barros-Carvalho
- Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal
| | - Marina E Ivanova
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK
- Imperial College, London, UK
| | - David C Briggs
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK
| | - Eurico Morais-de-Sá
- Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal
| | - Peter J Parker
- Protein Phosphorylation Laboratory, Francis Crick Institute, London, UK
- School of Cancer and Pharmaceutical Sciences, King's College London, Guy's Campus, London, UK
| | - Neil Q McDonald
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK.
- Institute of Structural and Molecular Biology, School of Natural Sciences, Birkbeck College, London, UK.
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2
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Chinthalapudi K, Heissler SM. Structure, regulation, and mechanisms of nonmuscle myosin-2. Cell Mol Life Sci 2024; 81:263. [PMID: 38878079 PMCID: PMC11335295 DOI: 10.1007/s00018-024-05264-6] [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: 03/11/2024] [Revised: 04/24/2024] [Accepted: 04/30/2024] [Indexed: 06/23/2024]
Abstract
Members of the myosin superfamily of molecular motors are large mechanochemical ATPases that are implicated in an ever-expanding array of cellular functions. This review focuses on mammalian nonmuscle myosin-2 (NM2) paralogs, ubiquitous members of the myosin-2 family of filament-forming motors. Through the conversion of chemical energy into mechanical work, NM2 paralogs remodel and shape cells and tissues. This process is tightly controlled in time and space by numerous synergetic regulation mechanisms to meet cellular demands. We review how recent advances in structural biology together with elegant biophysical and cell biological approaches have contributed to our understanding of the shared and unique mechanisms of NM2 paralogs as they relate to their kinetics, regulation, assembly, and cellular function.
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Affiliation(s)
- Krishna Chinthalapudi
- Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, Columbus, OH, 43210, USA
| | - Sarah M Heissler
- Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, Columbus, OH, 43210, USA.
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3
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Bii VM, Rudoy D, Klezovitch O, Vasioukhin V. Lethal giant larvae gene family ( Llgl1 and Llgl2 ) functions as a tumor suppressor in mouse skin epidermis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.06.531408. [PMID: 36945368 PMCID: PMC10028895 DOI: 10.1101/2023.03.06.531408] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/11/2023]
Abstract
Loss of cell polarity and tissue disorganization occurs in majority of epithelial cancers. Studies in simple model organisms identified molecular mechanisms responsible for the establishment and maintenance of cellular polarity, which play a pivotal role in establishing proper tissue architecture. The exact role of these cell polarity pathways in mammalian cancer is not completely understood. Here we analyzed the mammalian orthologs of drosophila apical-basal polarity gene lethal giant larvae ( lgl ), which regulates asymmetric stem cell division and functions as a tumor suppressor in flies. There are two mammalian orthologs of lgl ( Llgl1 and Llgl2 ). To determine the role of the entire lgl signaling pathway in mammals we generated mice with ablation of both Llgl1 and Llgl2 in skin epidermis using K14-Cre ( Llgl1/2 -/- cKO mice). Surprisingly, we found that ablation of Llgl1/2 genes does not impact epidermal polarity in adult mice. However, old Llgl1/2 cKO mice present with focal skin lesions which are missing epidermal layer and ripe with inflammation. To determine the role of lgl signaling pathway in cancer we generated Trp53 -/- /Llgl1/2 -/- cKO and Trp53 -/+ /Llgl1/2 -/- cKO mice. Loss of Llgl1/2 promoted squamous cell carcinoma (SCC) development in Trp53 -/- cKO and caused SCC in Trp53 -/+ cKO mice, while no cancer was observed in Trp53 -/+ cKO controls. Mechanistically, we show that ablation of Llgl1/2 causes activation of aPKC and upregulation of NF-kB signaling pathway, which may be necessary for SCC in Trp53 -/+ /Llgl1/2 -/- cKO mice. We conclude that Lgl signaling pathway functions as a tumor suppressor in mammalian skin epidermis.
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4
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HBXIP blocks myosin-IIA assembly by phosphorylating and interacting with NMHC-IIA in breast cancer metastasis. Acta Pharm Sin B 2022; 13:1053-1070. [PMID: 36970214 PMCID: PMC10031283 DOI: 10.1016/j.apsb.2022.11.025] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 10/10/2022] [Accepted: 10/19/2022] [Indexed: 11/27/2022] Open
Abstract
Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.
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5
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Endocytosis at the Crossroad of Polarity and Signaling Regulation: Learning from Drosophila melanogaster and Beyond. Int J Mol Sci 2022; 23:ijms23094684. [PMID: 35563080 PMCID: PMC9101507 DOI: 10.3390/ijms23094684] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 04/19/2022] [Accepted: 04/21/2022] [Indexed: 02/06/2023] Open
Abstract
Cellular trafficking through the endosomal–lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal–lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.
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6
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Ma R, Gong D, You H, Xu C, Lu Y, Bergers G, Werb Z, Klein OD, Petritsch CK, Lu P. LGL1 binds to Integrin β1 and inhibits downstream signaling to promote epithelial branching in the mammary gland. Cell Rep 2022; 38:110375. [PMID: 35172155 PMCID: PMC9113222 DOI: 10.1016/j.celrep.2022.110375] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Revised: 10/08/2021] [Accepted: 01/20/2022] [Indexed: 11/29/2022] Open
Abstract
Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct. Ma et al. show that Lgl1 is essential for mammary gland branching morphogenesis but not epithelial polarity. Lgl1 is required for directional migration by regulating Integrin β1 signaling levels and focal adhesion strengths. Finally, branching mechanisms are distinct between mammary gland and Drosophila systems where directional migration is indispensable.
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Affiliation(s)
- Rongze Ma
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Difei Gong
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Huanyang You
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Chongshen Xu
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Yunzhe Lu
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Gabriele Bergers
- VIB-KU Leuven Center for Cancer Biology, Department of Oncology, KU Leuven, 3000 Leuven, Belgium
| | - Zena Werb
- Department of Anatomy and Program in Developmental and Stem Cell Biology, University of California, San Francisco, San Francisco, CA 94143-0452, USA
| | - Ophir D Klein
- Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, UCSF Box 0422, 513 Parnassus Avenue, HSE1508, San Francisco, CA 94143-0422, USA
| | - Claudia K Petritsch
- Department of Neurological Surgery, Stanford University, Palo Alto, CA 94305, USA
| | - Pengfei Lu
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
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7
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Wang Y, Zhang Y, Sang B, Zhu X, Yu R, Zhou X. Human giant larvae-1 promotes migration and invasion of malignant glioma cells by regulating N-cadherin. Oncol Lett 2021; 21:167. [PMID: 33552285 PMCID: PMC7798033 DOI: 10.3892/ol.2021.12428] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 12/01/2020] [Indexed: 12/13/2022] Open
Abstract
Human giant larvae-1 (Hugl-1) is a human homologue of Drosophila tumor suppressor lethal (2)-giant larvae and has been reported to be involved in the development of human malignancies. Previous studies performed by our group demonstrated that Hugl-1 inhibits glioma cell proliferation in an intracranial model of nude mice. However, the exact molecular mechanisms underlying the participation of Hugl-1 in glioma invasion and migration, and in the depolarizing process remain largely unknown. Utilizing the U251-MG cells with stable expression of Hugl-1, the present study used wound healing, Transwell invasion and western blot assays to explore the role and specific mechanism of Hugl-1 in glioma invasion and migration. The results of the present study demonstrated that overexpression of Hugl-1 decreased cell-cell adhesion and increased cell-cell extracellular matrix adhesion. In addition, overexpression of Hugl-1 promoted pseudopodia formation, glioma cell migration and invasion. The molecular mechanism of action involved the negative regulation of N-cadherin protein levels by Hugl-1. Overexpression or knockdown of N-cadherin partially suppressed or enhanced the effects of Hugl-1 on glioma cell migration and invasion, respectively. Furthermore, Hugl-1 inhibited cell proliferation, while promoting cell migration, which suggests that it may serve a two-sided biological role in cellular processes. Taken together, these results suggest that Hugl-1 promotes the migration and invasion of malignant glioma cells by decreasing N-cadherin expression. Thus, Hugl-1 may be applied in the development of targeted and personalized treatment.
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Affiliation(s)
- Yan Wang
- The Graduate School, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.,Institute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.,Department of Neurosurgery, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China
| | - Yu Zhang
- The Graduate School, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.,Institute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China
| | - Ben Sang
- The Graduate School, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China
| | - Xianlong Zhu
- The Graduate School, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China
| | - Rutong Yu
- Institute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.,Department of Neurosurgery, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China
| | - Xiuping Zhou
- Institute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.,Department of Neurosurgery, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China
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8
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Shen H, Meng Y, Hu T, Li S, Du M, Xin J, Gu D, Wang M, Fu Z. Genetic variants in Hippo signalling pathway-related genes affect the risk of colorectal cancer. Arch Toxicol 2020; 95:271-281. [PMID: 33011827 DOI: 10.1007/s00204-020-02910-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2019] [Accepted: 09/10/2020] [Indexed: 01/12/2023]
Abstract
The Hippo signalling pathway plays a crucial role in carcinogenesis. Therefore, we hypothesized that genetic variants in genes related to this pathway are associated with the colorectal cancer risk. A case-control study including 1150 patients and 1342 controls was performed to assess the association of genetic variants of genes involved in the Hippo signalling pathway with the risk of colorectal cancer. The results were corrected for multiple comparisons using the false discovery rate (FDR). We used a regression model to determine the effects of single-nucleotide polymorphisms (SNPs) on the survival of patients with colorectal cancer in The Cancer Genome Atlas (TCGA) datasets. An expression quantitative trait loci (eQTL) analysis was performed using TCGA datasets and the Genotype-Tissue Expression (GTEx) project. Gene Expression Omnibus (GEO) datasets were used to provide additional data on the expression of genes in colorectal cancer. The SCRIB rs13251492 G allele was associated with a significantly decreased risk of colorectal cancer (odds ratio (OR) = 0.79, 95% confidence interval (CI) = 0.70-0.89, P = 7.76 × 10-5, P(FDR) = 6.98 × 10-4). Patients with the rs13251492 AG/GG allele experienced a longer recurrence-free survival (RFS) time (hazard ratio (HR) = 0.64, 95% CI = 0.42-0.99, P = 0.049) than patients with the rs13251492 A allele. The eQTL analysis revealed a significant association between rs13251492 and the expression of the SCRIB mRNA in colorectal tumors. Dual-luciferase reporter assays in DLD-1 and HCT116 cells revealed a lower enhancer activity of the rs13251492 G allele than the A allele. In addition, the SCRIB mRNA was expressed at markedly higher levels in colorectal cancer tissues than in normal tissues. Therefore, we identified the SCRIB rs13251492 variant as a novel colorectal cancer susceptibility locus and provided evidence of its functional relevance.
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Affiliation(s)
- Hengyang Shen
- The First School of Clinical Medicine, Nanjing Medical University, Nanjing, China.,Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu, 210029, People's Republic of China
| | - Yixuan Meng
- Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Jiangsu Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China.,Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, Center for Global Health, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu, 211166, People's Republic of China
| | - Tao Hu
- The First School of Clinical Medicine, Nanjing Medical University, Nanjing, China.,Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu, 210029, People's Republic of China
| | - Shuwei Li
- Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Jiangsu Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China.,Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, Center for Global Health, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu, 211166, People's Republic of China
| | - Mulong Du
- Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Jiangsu Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China.,Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, Center for Global Health, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu, 211166, People's Republic of China
| | - Junyi Xin
- Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Jiangsu Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China.,Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, Center for Global Health, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu, 211166, People's Republic of China
| | - Dongying Gu
- Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
| | - Meilin Wang
- Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Jiangsu Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China. .,Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, Center for Global Health, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu, 211166, People's Republic of China.
| | - Zan Fu
- The First School of Clinical Medicine, Nanjing Medical University, Nanjing, China. .,Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu, 210029, People's Republic of China.
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9
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Zhu YX, Li CH, Li G, Feng H, Xia T, Wong CH, Fung FKC, Tong JHM, To KF, Chen R, Chen Y. LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma. Cell Mol Gastroenterol Hepatol 2020; 10:811-828. [PMID: 32615164 PMCID: PMC7505810 DOI: 10.1016/j.jcmgh.2020.06.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 06/22/2020] [Accepted: 06/22/2020] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells. METHODS Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1. RESULTS Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966-0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription. CONCLUSIONS LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR-extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681).
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Affiliation(s)
- Yin-Xin Zhu
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Chi Han Li
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Guolin Li
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Guangzhou, China
| | - Huiyi Feng
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Tian Xia
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Chi Hin Wong
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Frederic Khe Cheong Fung
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Joanna Hung-Man Tong
- Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Ka-Fai To
- Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Rufu Chen
- Guangdong Provincial People's Hospital, Guangzhou, Guangdong Province, China.
| | - Yangchao Chen
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
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10
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Liu N, Cui W, Chen M, Zhang X, Song X, Pan C. A 21-bp indel within the LLGL1 gene is significantly associated with litter size in goat. Anim Biotechnol 2019; 32:213-218. [PMID: 31646948 DOI: 10.1080/10495398.2019.1677682] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The scribble cell polarity complex component (LLGL1) is part of the cytoskeletal network and is involved in maintaining cell polarity and epithelial integrity. Based on the whole-genome sequencing analysis in goat, LLGL1 gene is suggested as a putative important candidate gene affecting litter size in Shaanbei White Cashmere Goats (SBWC). Therefore, the objective of this study was to uncover the possible novel insertion/deletion (Indel) variant in goat LLGL1 gene and to evaluate its association with litter size of SBWC (n = 827). Using the PCR detection and DNA sequencing, the 21-bp indel in the upstream of LLGL1 was firstly founded and two genotypes were identified: II (insertion/insertion) and ID (insertion/deletion), respectively. Association analyses revealed that the 21-bp indel was significantly correlated with litter size (p = 0.017). Notably, the individuals with II genotype were significantly greater than that of the genotype ID, and the 'I' allele was dominant. Additionally, the remarkable influence of the indel on traits might be related to the change of DEAF-1-related (NUDR) binding site through bioinformatics analysis. Briefly, the 21-bp indel within the goat LLGL1 gene could be an effective DNA molecular marker and provide valuable theoretical basis for marker-assisted selection (MAS) in goat industry.
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Affiliation(s)
- Nuan Liu
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Wenbo Cui
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Mingyue Chen
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Xuelian Zhang
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Xiaoyue Song
- Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin, Shaanxi, China.,Life Science Research Center, Yulin University, Yulin, Shaanxi, China
| | - Chuanying Pan
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
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11
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Choi J, Troyanovsky RB, Indra I, Mitchell BJ, Troyanovsky SM. Scribble, Erbin, and Lano redundantly regulate epithelial polarity and apical adhesion complex. J Cell Biol 2019; 218:2277-2293. [PMID: 31147384 PMCID: PMC6605793 DOI: 10.1083/jcb.201804201] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Revised: 11/05/2018] [Accepted: 05/10/2019] [Indexed: 12/22/2022] Open
Abstract
The basolateral protein Scribble (Scrib), a member of the LAP protein family, is essential for epithelial apicobasal polarity (ABP) in Drosophila However, a conserved function for this protein in mammals is unclear. Here we show that the crucial role for Scrib in ABP has remained obscure due to the compensatory function of two other LAP proteins, Erbin and Lano. A combined Scrib/Erbin/Lano knockout disorganizes the cell-cell junctions and the cytoskeleton. It also results in mislocalization of several apical (Par6, aPKC, and Pals1) and basolateral (Llgl1 and Llgl2) identity proteins. These defects can be rescued by the conserved "LU" region of these LAP proteins. Structure-function analysis of this region determined that the so-called LAPSDb domain is essential for basolateral targeting of these proteins, while the LAPSDa domain is essential for supporting the membrane basolateral identity and binding to Llgl. In contrast to the key role in Drosophila, mislocalization of Llgl proteins does not appear to be critical in the scrib ABP phenotype.
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Affiliation(s)
- Jongho Choi
- Department of Dermatology, Northwestern University, The Feinberg School of Medicine, Chicago, IL
| | - Regina B Troyanovsky
- Department of Dermatology, Northwestern University, The Feinberg School of Medicine, Chicago, IL
| | - Indrajyoti Indra
- Department of Dermatology, Northwestern University, The Feinberg School of Medicine, Chicago, IL
| | - Brian J Mitchell
- Department of Cell and Molecular Biology, The Feinberg School of Medicine, Chicago, IL
| | - Sergey M Troyanovsky
- Department of Dermatology, Northwestern University, The Feinberg School of Medicine, Chicago, IL
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12
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Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2. Proc Natl Acad Sci U S A 2019; 116:10804-10812. [PMID: 31088962 DOI: 10.1073/pnas.1821514116] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Metazoan cell polarity is controlled by a set of highly conserved proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity through phosphorylation-dependent interactions with several other proteins as well as the plasma membrane. Phosphorylation of Lgl by atypical protein kinase C (aPKC), a component of the partitioning-defective (Par) complex in epithelial cells, excludes Lgl from the apical membrane, a crucial step in the establishment of epithelial cell polarity. We present the crystal structures of human Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. Lgl2 adopts a double β-propeller structure that is unchanged by aPKC phosphorylation of an unstructured loop in its second β-propeller, ruling out models of phosphorylation-dependent conformational change. We demonstrate that phosphorylation controls the direct binding of purified Lgl2 to negative phospholipids in vitro. We also show that a coil-helix transition of this region that is promoted by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylation-dependent, implying a highly effective phosphorylative switch for membrane association.
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13
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Daynac M, Chouchane M, Collins HY, Murphy NE, Andor N, Niu J, Fancy SPJ, Stallcup WB, Petritsch CK. Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis. Nat Commun 2018; 9:2862. [PMID: 30131568 PMCID: PMC6104045 DOI: 10.1038/s41467-018-05099-3] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2017] [Accepted: 06/13/2018] [Indexed: 12/29/2022] Open
Abstract
Oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to generate one OPC and one differentiating oligodendrocyte (OL) progeny. Loss of pro-mitotic proteoglycan and OPC marker NG2 in the OL progeny is the earliest immunophenotypic change of unknown mechanism that indicates differentiation commitment. Here, we report that expression of the mouse homolog of Drosophila tumor suppressor Lethal giant larvae 1 (Lgl1) is induced during OL differentiation. Lgl1 conditional knockout OPC progeny retain NG2 and show reduced OL differentiation, while undergoing more symmetric self-renewing divisions at the expense of asymmetric divisions. Moreover, Lgl1 and hemizygous Ink4a/Arf knockouts in OPC synergistically induce gliomagenesis. Time lapse and total internal reflection microscopy reveals a critical role for Lgl1 in NG2 endocytic routing and links aberrant NG2 recycling to failed differentiation. These data establish Lgl1 as a suppressor of gliomagenesis and positive regulator of asymmetric division and differentiation in the healthy and demyelinated murine brain.
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Affiliation(s)
- Mathieu Daynac
- Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Malek Chouchane
- Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Hannah Y Collins
- Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA
- Department of Neurobiology, Stanford University, Stanford, CA, 94305, USA
| | - Nicole E Murphy
- Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Noemi Andor
- Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA
- Department of Medicine, Stanford University, Stanford, CA, 94305, USA
| | - Jianqin Niu
- Department of Pediatrics, University of California San Francisco, San Francisco, CA, 94158, USA
| | - Stephen P J Fancy
- Department of Pediatrics, University of California San Francisco, San Francisco, CA, 94158, USA
- Department of Neurology, University of California San Francisco, San Francisco, CA, 94158, USA
| | - William B Stallcup
- Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA 92037, USA
| | - Claudia K Petritsch
- Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA.
- Brain Tumor Center, University of California San Francisco, San Francisco, CA, 94158, USA.
- Helen Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, 94158, USA.
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, 94143, USA.
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14
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Stephens R, Lim K, Portela M, Kvansakul M, Humbert PO, Richardson HE. The Scribble Cell Polarity Module in the Regulation of Cell Signaling in Tissue Development and Tumorigenesis. J Mol Biol 2018; 430:3585-3612. [PMID: 29409995 DOI: 10.1016/j.jmb.2018.01.011] [Citation(s) in RCA: 97] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2017] [Revised: 01/19/2018] [Accepted: 01/19/2018] [Indexed: 01/22/2023]
Abstract
The Scribble cell polarity module, comprising Scribbled (Scrib), Discs-large (Dlg) and Lethal-2-giant larvae (Lgl), has a tumor suppressive role in mammalian epithelial cancers. The Scribble module proteins play key functions in the establishment and maintenance of different modes of cell polarity, as well as in the control of tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. Whilst molecular details are known regarding the roles of Scribble module proteins in cell polarity regulation, their precise mode of action in the regulation of other key cellular processes remains enigmatic. An accumulating body of evidence indicates that Scribble module proteins play scaffolding roles in the control of various signaling pathways, which are linked to the control of tissue growth, differentiation and cell migration. Multiple Scrib, Dlg and Lgl interacting proteins have been discovered, which are involved in diverse processes, however many function in the regulation of cellular signaling. Herein, we review the components of the Scrib, Dlg and Lgl protein interactomes, and focus on the mechanism by which they regulate cellular signaling pathways in metazoans, and how their disruption leads to cancer.
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Affiliation(s)
- Rebecca Stephens
- Department of Biochemistry and Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, Australia
| | - Krystle Lim
- Department of Biochemistry and Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, Australia
| | - Marta Portela
- Department of Molecular, Cellular and Developmental Neurobiology, Cajal Institute (CSIC), Avenida Doctor Arce, 37, Madrid 28002, Spain
| | - Marc Kvansakul
- Department of Biochemistry and Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, Australia
| | - Patrick O Humbert
- Department of Biochemistry and Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, Australia; Department of Biochemistry & Molecular Biology, University of Melbourne, Melbourne, Victoria 3010, Australia; Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia
| | - Helena E Richardson
- Department of Biochemistry and Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, Australia; Department of Biochemistry & Molecular Biology, University of Melbourne, Melbourne, Victoria 3010, Australia; Department of Anatomy & Neurobiology, University of Melbourne, Melbourne, Victoria 3010, Australia.
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15
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Beattie R, Hippenmeyer S. Mechanisms of radial glia progenitor cell lineage progression. FEBS Lett 2017; 591:3993-4008. [PMID: 29121403 PMCID: PMC5765500 DOI: 10.1002/1873-3468.12906] [Citation(s) in RCA: 86] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2017] [Revised: 10/31/2017] [Accepted: 11/06/2017] [Indexed: 12/11/2022]
Abstract
The mammalian cerebral cortex is responsible for higher cognitive functions such as perception, consciousness, and acquiring and processing information. The neocortex is organized into six distinct laminae, each composed of a rich diversity of cell types which assemble into highly complex cortical circuits. Radial glia progenitors (RGPs) are responsible for producing all neocortical neurons and certain glia lineages. Here, we discuss recent discoveries emerging from clonal lineage analysis at the single RGP cell level that provide us with an inaugural quantitative framework of RGP lineage progression. We further discuss the importance of the relative contribution of intrinsic gene functions and non‐cell‐autonomous or community effects in regulating RGP proliferation behavior and lineage progression.
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Affiliation(s)
- Robert Beattie
- Institute of Science and Technology Austria, Klosterneuburg, Austria
| | - Simon Hippenmeyer
- Institute of Science and Technology Austria, Klosterneuburg, Austria
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16
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Jossin Y, Lee M, Klezovitch O, Kon E, Cossard A, Lien WH, Fernandez TE, Cooper JA, Vasioukhin V. Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells. Dev Cell 2017; 41:481-495.e5. [PMID: 28552558 DOI: 10.1016/j.devcel.2017.05.002] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2016] [Revised: 04/11/2017] [Accepted: 05/01/2017] [Indexed: 10/19/2022]
Abstract
Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1fl/fl), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells.
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Affiliation(s)
- Yves Jossin
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Mammalian Development & Cell Biology Unit, Institute of Neuroscience, Université Catholique de Louvain, 1200 Brussels, Belgium.
| | - Minhui Lee
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA
| | - Olga Klezovitch
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Elif Kon
- Mammalian Development & Cell Biology Unit, Institute of Neuroscience, Université Catholique de Louvain, 1200 Brussels, Belgium
| | - Alexia Cossard
- Mammalian Development & Cell Biology Unit, Institute of Neuroscience, Université Catholique de Louvain, 1200 Brussels, Belgium
| | - Wen-Hui Lien
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA
| | - Tania E Fernandez
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Jonathan A Cooper
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA
| | - Valera Vasioukhin
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA; Department of Pathology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.
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17
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Heng S, Evans J, Salamonsen LA, Jobling TW, Nie G. The significance of post-translational removal of α-DG-N in early stage endometrial cancer development. Oncotarget 2017; 8:81942-81952. [PMID: 29137235 PMCID: PMC5669861 DOI: 10.18632/oncotarget.17286] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Accepted: 04/11/2017] [Indexed: 01/11/2023] Open
Abstract
Endometrial cancer is one of the most common gynecological malignancies affecting post-menopausal women, yet the underlying mechanisms are not well understood. Dystroglycan (DG) is a large glycoprotein, consisting of α- and β-subunits that are non-covalently associated with each other. Modifications to α-DG have been linked to a variety of cancers, where the N-terminus of α-DG (α-DG-N) is post-translationally removed by a furin-like enzyme. However, the functional significance of α-DG-N removal is unknown. Our previous studies have established that the α-DG cleavage enzyme furin is significantly up-regulated in endometrial cancer. This study aimed to investigate the importance of α-DG-N removal in post-menopausal endometrial cancer. We demonstrated that α-DG-N removal predominantly occurred in early stage endometrial cancer tissues, and that the cleaved α-DG-N was significantly elevated in the uterine lavage of early grade endometrial cancer patients. Furthermore, α-DG-N removal significantly decreased the tight junction integrity and polarity of the endometrial epithelial cells, promoting the loss of polarity markers scribble and atypical protein kinase C (aPKC) and reducing the trans-epithelial electrical resistance. The removal of α-DG-N also sensitized the cells for estrogen-dependent proliferation. These results strongly suggest that α-DG-N removal plays an important role in early stage development of endometrial cancer, and that the elevated levels of α-DG-N in uterine fluid may provide a biomarker for early detection of endometrial cancer.
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Affiliation(s)
- Sophea Heng
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia.,Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia.,Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
| | - Jemma Evans
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia.,Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia.,Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
| | - Lois A Salamonsen
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia.,Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia.,Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
| | - Tom W Jobling
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia.,Epworth Research Institute, Epworth Health Care, Richmond, Victoria, Australia
| | - Guiying Nie
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia.,Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia.,Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
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18
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Visco I, Hoege C, Hyman AA, Schwille P. In vitro Reconstitution of a Membrane Switch Mechanism for the Polarity Protein LGL. J Mol Biol 2016; 428:4828-4842. [PMID: 27720986 DOI: 10.1016/j.jmb.2016.10.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2016] [Revised: 09/28/2016] [Accepted: 10/01/2016] [Indexed: 12/24/2022]
Abstract
Cell polarity arises from a combination of interactions between biological molecules, such as activation, inhibition, and positive or negative feedback between specific polarity units. Activation and inhibition often take place in the form of a membrane binding switch. Lethal giant larvae (LGL), a conserved regulator of cell polarity in animals, was suggested to function as such a switch. LGL localizes to both the cytoplasm and, asymmetrically, the membrane. However, the spatial regulation mechanism of LGL membrane localization has remained unclear. For systematic elucidation, we set out to reconstitute a minimal polarity unit using a model membrane, Caenorhabditis elegans LGL (LGL-1), and atypical protein kinase C (aPKC) supposed to activate the membrane switch. We identified a membrane binding sequence (MBS) in LGL-1 by a screen in vivo, reconstituted LGL-1 membrane binding in vitro, and successfully implemented the membrane switch by aPKC phosphorylation activity, detaching LGL from membranes. Upon membrane binding, LGL-1 MBS folds into an alpha-helix in which three regions can be identified: a positively charged patch, a switch area containing the three aPKC phosphorylation sites, and a hydrophobic area probably buried in the membrane. Phosphorylation by aPKC dramatically reduces the binding affinity of the LGL-1 MBS to negatively charged model membranes, inducing its detachment. Specific residues in the MBS are critical for LGL-1 function in C. elegans.
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Affiliation(s)
- Ilaria Visco
- Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
| | - Carsten Hoege
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Anthony A Hyman
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Petra Schwille
- Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
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19
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Johnson SA, Zitserman D, Roegiers F. Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells. Mol Biol Cell 2016; 27:2857-66. [PMID: 27466320 PMCID: PMC5025272 DOI: 10.1091/mbc.e15-11-0751] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2015] [Accepted: 07/21/2016] [Indexed: 11/11/2022] Open
Abstract
Steady-state and pulse-labeling techniques are used to follow Notch receptors in sensory organ precursor cells in Drosophila. Numb and L(2)gl antagonize a pool of Notch receptors, and Numb promotes Notch targeting to late endosomes in Drosophila neural progenitors to regulate Notch signaling and cell fate. The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.
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Affiliation(s)
- Seth A Johnson
- Fox Chase Cancer Center, Philadelphia, PA 19111 Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
| | - Diana Zitserman
- Fox Chase Cancer Center, Philadelphia, PA 19111 University of Bridgeport, Bridgeport, CT 06604
| | - Fabrice Roegiers
- Fox Chase Cancer Center, Philadelphia, PA 19111 Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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20
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Mruk DD, Cheng CY. The Mammalian Blood-Testis Barrier: Its Biology and Regulation. Endocr Rev 2015; 36:564-91. [PMID: 26357922 PMCID: PMC4591527 DOI: 10.1210/er.2014-1101] [Citation(s) in RCA: 448] [Impact Index Per Article: 44.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2014] [Accepted: 09/03/2015] [Indexed: 12/31/2022]
Abstract
Spermatogenesis is the cellular process by which spermatogonia develop into mature spermatids within seminiferous tubules, the functional unit of the mammalian testis, under the structural and nutritional support of Sertoli cells and the precise regulation of endocrine factors. As germ cells develop, they traverse the seminiferous epithelium, a process that involves restructuring of Sertoli-germ cell junctions, as well as Sertoli-Sertoli cell junctions at the blood-testis barrier. The blood-testis barrier, one of the tightest tissue barriers in the mammalian body, divides the seminiferous epithelium into 2 compartments, basal and adluminal. The blood-testis barrier is different from most other tissue barriers in that it is not only comprised of tight junctions. Instead, tight junctions coexist and cofunction with ectoplasmic specializations, desmosomes, and gap junctions to create a unique microenvironment for the completion of meiosis and the subsequent development of spermatids into spermatozoa via spermiogenesis. Studies from the past decade or so have identified the key structural, scaffolding, and signaling proteins of the blood-testis barrier. More recent studies have defined the regulatory mechanisms that underlie blood-testis barrier function. We review here the biology and regulation of the mammalian blood-testis barrier and highlight research areas that should be expanded in future studies.
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Affiliation(s)
- Dolores D Mruk
- Center for Biomedical Research, Population Council, New York, New York 10065
| | - C Yan Cheng
- Center for Biomedical Research, Population Council, New York, New York 10065
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21
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Liu X, Lu D, Ma P, Liu H, Cao Y, Sang B, Zhu X, Shi Q, Hu J, Yu R, Zhou X. Hugl-1 inhibits glioma cell growth in intracranial model. J Neurooncol 2015; 125:113-21. [PMID: 26341367 DOI: 10.1007/s11060-015-1901-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2015] [Accepted: 08/29/2015] [Indexed: 12/28/2022]
Abstract
Drosophila lethal (2) giant larvae (lgl) has been reported as a tumor suppressor and could regulate the Drosophila hippo signaling. Human giant larvae-1(Hugl-1), one human homologue of Drosophila lgl, also has been reported to be involved in the development of some human cancers. However, whether Hugl-1 is associated with the pathogenesis of malignant gliomas remains poorly understood. In the present work, we examined the effect of Hugl-1 on glioma cell growth both in vitro and in vivo. Firstly, we found that Hugl-1 protein levels decreased in the human glioma tissues, suggesting that Hugl-1 is involved in glioma progression. Unfortunately, either stably or transiently over-expressing Hugl-1 did not affect glioma cell proliferation in vitro. In addition, Hugl-1 over-expression did not regulate hippo signaling pathway. Interestingly, over-expression of Hugl-1 not only inhibited gliomagenesis but also markedly inhibited cell proliferation and promoted the apoptosis of U251 cells in an orthotopic model of nude mice. Taken together, this study provides the evidence that Hugl-1 inhibits glioma cell growth in intracranial model of nude mice, suggesting that Hugl-1 might be a potential tumor target for glioma therapy.
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Affiliation(s)
- Xuejiao Liu
- Institute of Nervous System Diseases, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
- Brain Hospital, Affiliated Hospital of Xuzhou Medical College, 99 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
| | - Dong Lu
- Institute of Nervous System Diseases, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
- Brain Hospital, Affiliated Hospital of Xuzhou Medical College, 99 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
| | - Peng Ma
- The Graduate School, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China
| | - Huaqiang Liu
- The Graduate School, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China
| | - Yuewen Cao
- The Graduate School, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China
| | - Ben Sang
- The Graduate School, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China
| | - Xianlong Zhu
- The Graduate School, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China
| | - Qiong Shi
- Institute of Nervous System Diseases, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
- Brain Hospital, Affiliated Hospital of Xuzhou Medical College, 99 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
| | - Jinxia Hu
- Institute of Nervous System Diseases, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
- Brain Hospital, Affiliated Hospital of Xuzhou Medical College, 99 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China
| | - Rutong Yu
- Institute of Nervous System Diseases, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China.
- Brain Hospital, Affiliated Hospital of Xuzhou Medical College, 99 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China.
| | - Xiuping Zhou
- Institute of Nervous System Diseases, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China.
- Brain Hospital, Affiliated Hospital of Xuzhou Medical College, 99 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China.
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22
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Watson K, Rossi G, Temple B, Brennwald P. Structural basis for recognition of the Sec4 Rab GTPase by its effector, the Lgl/tomosyn homologue, Sro7. Mol Biol Cell 2015. [PMID: 26202462 PMCID: PMC4569318 DOI: 10.1091/mbc.e15-04-0228] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Members of the tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to-cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in tomosyn vertebrate homologues.
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Affiliation(s)
- Kelly Watson
- Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 Graduate Program in Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599
| | - Guendalina Rossi
- Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599
| | - Brenda Temple
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 R. L. Juliano Structural Bioinformatics Core, University of North Carolina School of Medicine, Chapel Hill, NC 27599
| | - Patrick Brennwald
- Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 Graduate Program in Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599
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Chen S, Zhang D. Friend or foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer. FEBS Open Bio 2015; 5:91-8. [PMID: 25709888 PMCID: PMC4329646 DOI: 10.1016/j.fob.2015.01.004] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2014] [Revised: 01/21/2015] [Accepted: 01/21/2015] [Indexed: 12/12/2022] Open
Abstract
ERp29 regulates epithelial cell plasticity and the mesenchymal–epithelial transition. ERp29 shows a tumor suppressive function in primary tumor development. ERp29 is potentially associated with distant metastasis in cancer. ERp29 modulates cell survival against genotoxic stress. Thus, ERp29 displays dual functions as a “friend or foe” in epithelial cancer. The endoplasmic reticulum (ER) protein 29 (ERp29) is a molecular chaperone that plays a critical role in protein secretion from the ER in eukaryotic cells. Recent studies have also shown that ERp29 plays a role in cancer. It has been demonstrated that ERp29 is inversely associated with primary tumor development and functions as a tumor suppressor by inducing cell growth arrest in breast cancer. However, ERp29 has also been reported to promote epithelial cell morphogenesis, cell survival against genotoxic stress and distant metastasis. In this review, we summarize the current understanding on the biological and pathological functions of ERp29 in cancer and discuss the pivotal aspects of ERp29 as “friend or foe” in epithelial cancer.
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Affiliation(s)
- Shaohua Chen
- Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China
| | - Daohai Zhang
- Cancer Research Group, The Canberra Hospital, ANU Medical School, Australia National University, ACT 2605, Australia
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Dulyaninova NG, Bresnick AR. The heavy chain has its day: regulation of myosin-II assembly. BIOARCHITECTURE 2015; 3:77-85. [PMID: 24002531 DOI: 10.4161/bioa.26133] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain.
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Betapudi V. Life without double-headed non-muscle myosin II motor proteins. Front Chem 2014; 2:45. [PMID: 25072053 PMCID: PMC4083560 DOI: 10.3389/fchem.2014.00045] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2014] [Accepted: 06/19/2014] [Indexed: 11/20/2022] Open
Abstract
Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients' life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.
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Affiliation(s)
- Venkaiah Betapudi
- Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Cleveland, OH, USA ; Department of Physiology and Biophysics, Case Western Reserve University Cleveland, OH, USA
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The lethal giant larvae gene in Tribolium castaneum: molecular properties and roles in larval and pupal development as revealed by RNA interference. Int J Mol Sci 2014; 15:6880-96. [PMID: 24758930 PMCID: PMC4013667 DOI: 10.3390/ijms15046880] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2014] [Revised: 03/21/2014] [Accepted: 04/11/2014] [Indexed: 11/16/2022] Open
Abstract
We identified and characterized the TcLgl gene putatively encoding lethal giant larvae (Lgl) protein from the red flour beetle (Tribolium castaneum). Analyses of developmental stage and tissue-specific expression patterns revealed that TcLgl was constitutively expressed. To examine the role of TcLgl in insect development, RNA interference was performed in early (1-day) larvae, late (20-day) larvae, and early (1-day) pupae. The early larvae injected with double-stranded RNA of TcLgl (dsTcLgl) at 100, 200, and 400 ng/larva failed to pupate, and 100% mortality was achieved within 20 days after the injection or before the pupation. The late larvae injected with dsTcLgl at these doses reduced the pupation rates to only 50.3%, 36.0%, and 18.2%, respectively. The un-pupated larvae gradually died after one week, and visually unaffected pupae failed to emerge into adults and died during the pupal stage. Similarly, when early pupae were injected with dsTcLgl at these doses, the normal eclosion rates were reduced to only 22.5%, 18.0%, and 11.2%, respectively, on day 7 after the injection, and all the adults with abnormal eclosion died in two days after the eclosion. These results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.
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27
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Dahan I, Petrov D, Cohen-Kfir E, Ravid S. The tumor suppressor Lgl1 forms discrete complexes with NMII-A and Par6α-aPKCζ that are affected by Lgl1 phosphorylation. J Cell Sci 2013; 127:295-304. [PMID: 24213535 DOI: 10.1242/jcs.127357] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Non-muscle myosin IIA (NMII-A) and the tumor suppressor lethal giant larvae 1 (Lgl1) play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. Here we show that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition of NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. We further show that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ, and that the formation of these complexes is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, we show that aPKCζ and NMII-A compete to bind directly to Lgl1 at the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α and aPKCζ in polarized migrating cells.
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Affiliation(s)
- Inbal Dahan
- Department of Biochemistry and Molecular Biology, The Institute of Medical Research Israel-Canada, The Hebrew University - Hadassah Medical School, Jerusalem 91120, Israel
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28
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Song J, Peng XL, Ji MY, Ai MH, Zhang JX, Dong WG. Hugl-1 induces apoptosis in esophageal carcinoma cells both in vitro and in vivo. World J Gastroenterol 2013; 19:4127-4136. [PMID: 23864775 PMCID: PMC3710414 DOI: 10.3748/wjg.v19.i26.4127] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2013] [Revised: 04/15/2013] [Accepted: 05/10/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To determine whether the human giant larvae homolog 1 gene (Hugl-1/Llg1/Lgl1) exerts tumor suppressor effects in esophageal cancer.
METHODS: We constructed a Hugl-1 expression plasmid, pEZ-M29-Hugl1, for gene transfection. We transfected the pEZ-M29-Hugl1 plasmid into Eca109 esophageal cancer cell lines with Lipofectamine 2000 to overexpress Hugl-1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the effects of the plasmid on Hugl-1 expression. In vitro cell proliferation and apoptosis were examined separately by cell counting Kit-8 (CCK-8) assay, flow cytometry, and Western blotting before and after the transfection of the plasmid into Eca109 cells. Cell cycle distribution was assessed with flow cytometry. The effect of Hugl-1 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. Expression of Hugl-1 in xenograft tumor was analyzed by immunohistochemistry. The transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to detect and quantitate apoptotic cell.
RESULTS: The transfection efficiency was confirmed with real-time RT-PCR and Western blotting. Our results show that compared with control groups the mRNA levels and protein levels of Hugl-1 in pEZ-M29-Hugl1-treated group were remarkably increased (P < 0.05). The CCK-8 assay demonstrated that the growth of cells overexpressing Hugl-1 was significantly lower than control cells. Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing Hugl-1 (64.09% ± 3.14% vs 50.32% ± 4.60%, 64.09% ± 3.14% vs 49.13% ± 2.24%). Annexin V-fluorescein isothiocyanate revealed that apoptosis was significantly increased in cells overexpressing Hugl-1 compared with control group (17.33% ± 4.76% vs 6.90% ± 1.61%, 17.33% ± 4.76% vs 6.27% ± 0.38%). Moreover, we found that Hugl-1 changes the level of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax and the activation of both caspase-3 and caspase-9. With a TUNEL assay, we found that Hugl-1 markedly increased the apoptosis rate of Eca109 cells in vivo (60.50% ± 9.11% vs 25.00% ± 12.25%). It was shown that Hugl-1 represents a significantly more effective tumor suppressor gene alone in a xenograft tumor mouse model. This data suggest that Hugl-1 inhibited tumor growth and induced cell apoptosis in vivo.
CONCLUSION: These results suggest that Hugl-1 induces growth suppression and apoptosis in a human esophageal squamous cell carcinoma cell line both in vitro and in vivo.
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Quéré N, Desmarais E, Tsigenopoulos CS, Belkhir K, Bonhomme F, Guinand B. Gene flow at major transitional areas in sea bass (Dicentrarchus labrax) and the possible emergence of a hybrid swarm. Ecol Evol 2012; 2:3061-78. [PMID: 23301173 PMCID: PMC3539001 DOI: 10.1002/ece3.406] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2012] [Revised: 09/18/2012] [Accepted: 09/25/2012] [Indexed: 11/08/2022] Open
Abstract
The population genetic structure of sea bass (Dicentrarchus labrax) along a transect from the Atlantic Ocean (AO) to the Eastern Mediterranean (EM) Sea differs from that of most other marine taxa in this area. Three populations (AO, Western Mediterranean [WM], EM) are recognized today, which were originally two allopatric populations. How two ancestral genetic units have evolved into three distinct units has not been addressed yet. Therefore, to investigate mechanisms that lead to the emergence of the central WM population, its current status, and its connectivity with the two parental populations, we applied 20 nuclear loci that were either gene associated or gene independent. Results confirmed the existence of three distinct gene pools, with higher differentiation at two transitional areas, the Almeria-Oran Front (AOF) and of the Siculo-Tunisian Strait (STS), than within any population. Significant linkage disequilibrium and heterozygote excess indicated that the STS is probably another tension zone, as already described for the AOF. Neutrality tests fail to reveal marker loci that could be driven by selection within or among metapopulations, except for locus DLA0068. Collectively, results support that the central WM population arose by trapping two tensions zones at distinct geographic locations of limited connectivity. Population assignment further revealed that WM individuals were more introgressed than individuals from the other two metapopulations. This suggests that this population might result from hybrid swarming, and was or is still seeded by genes received through the filter of each tension zone.
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Affiliation(s)
- Nolwenn Quéré
- Institut des Sciences de l'Évolution de Montpellier, CNRS-UMR 5554, Université Montpellier 2cc63, 34095, Montpellier Cedex 5, France
- Station Méditerranéenne de l'Environnement Littoral2 Avenue des chantiers, 34200, Sète, France
| | - Erick Desmarais
- Institut des Sciences de l'Évolution de Montpellier, CNRS-UMR 5554, Université Montpellier 2cc63, 34095, Montpellier Cedex 5, France
- LabEx CeMEB, Université Montpellier IIplace E. Bataillon, cc63, 34095, Montpellier Cedex 5, France
| | - Costas S Tsigenopoulos
- Hellenic Center for Marine Research, Institute of Marine Biology and GeneticsPO Box 2214, Gournes Pediados, 71500, Heraklion, Crete, Greece
| | - Khalid Belkhir
- Institut des Sciences de l'Évolution de Montpellier, CNRS-UMR 5554, Université Montpellier 2cc63, 34095, Montpellier Cedex 5, France
| | - François Bonhomme
- Institut des Sciences de l'Évolution de Montpellier, CNRS-UMR 5554, Université Montpellier 2cc63, 34095, Montpellier Cedex 5, France
- Station Méditerranéenne de l'Environnement Littoral2 Avenue des chantiers, 34200, Sète, France
| | - Bruno Guinand
- Institut des Sciences de l'Évolution de Montpellier, CNRS-UMR 5554, Université Montpellier 2cc63, 34095, Montpellier Cedex 5, France
- Station Méditerranéenne de l'Environnement Littoral2 Avenue des chantiers, 34200, Sète, France
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Abstract
Technological advances in biology have begun to dramatically change the way we think about evolution, development, health and disease. The ability to sequence the genomes of many individuals within a population, and across multiple species, has opened the door to the possibility of answering some long-standing and perplexing questions about our own genetic heritage. One such question revolves around the nature of cellular hyperproliferation. This cellular behavior is used to effect wound healing in most animals, as well as, in some animals, the regeneration of lost body parts. Yet at the same time, cellular hyperproliferation is the fundamental pathological condition responsible for cancers in humans. Here, I will discuss why microevolution, macroevolution and developmental biology all have to be taken into consideration when interpreting studies of both normal and malignant hyperproliferation. I will also illustrate how a synthesis of evolutionary sciences and developmental biology through the study of diverse model organisms can inform our understanding of both health and disease.
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31
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Clark BS, Cui S, Miesfeld JB, Klezovitch O, Vasioukhin V, Link BA. Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis. Development 2012; 139:1599-610. [PMID: 22492354 DOI: 10.1242/dev.078097] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.
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Affiliation(s)
- Brian S Clark
- Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA
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32
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Wan Q, Liu J, Zheng Z, Zhu H, Chu X, Dong Z, Huang S, Du Q. Regulation of myosin activation during cell-cell contact formation by Par3-Lgl antagonism: entosis without matrix detachment. Mol Biol Cell 2012; 23:2076-91. [PMID: 22496418 PMCID: PMC3364173 DOI: 10.1091/mbc.e11-11-0940] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila lethal(2)giant larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell–cell contact formation in Madin-Darby canine kidney cells. Altering the counteraction between Par3 and Lgl1/2 leads to entosis without matrix detachment. Cell–cell contact formation following cadherin engagement requires actomyosin contraction along the periphery of cell–cell contact. The molecular mechanisms that regulate myosin activation during this process are not clear. In this paper, we show that two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila Lethal (2) Giant Larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell–cell contact formation in Madin-Darby canine kidney cells. While overexpression of Lgl1/2 or depletion of endogenous Par3 leads to enhanced myosin II activation, knockdown of Lgl1/2 does the opposite. Intriguingly, altering the counteraction between Par3 and Lgl1/2 induces cell–cell internalization during early cell–cell contact formation, which involves active invasion of the lateral cell–cell contact underneath the apical-junctional complexes and requires activation of the Rho–Rho-associated, coiled-coil containing protein kinase (ROCK)–myosin pathway. This is followed by predominantly nonapoptotic cell-in-cell death of the internalized cells and frequent aneuploidy of the host cells. Such effects are reminiscent of entosis, a recently described process observed when mammary gland epithelial cells were cultured in suspension. We propose that entosis could occur without matrix detachment and that overactivation of myosin or unbalanced myosin activation between contacting cells may be the driving force for entosis in epithelial cells.
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Affiliation(s)
- Qingwen Wan
- Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
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33
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Dahan I, Yearim A, Touboul Y, Ravid S. The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration. Mol Biol Cell 2012; 23:591-601. [PMID: 22219375 PMCID: PMC3279388 DOI: 10.1091/mbc.e11-01-0015] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical-basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. In this study we show direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). We demonstrate that Lgl1 and NMII-A form a complex in vivo and provide data that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, we show that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly.
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Affiliation(s)
- Inbal Dahan
- Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
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Ruthala K, Gadi J, Lee JY, Yoon H, Chung HJ, Kim MH. Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion. Mol Cells 2011; 32:273-9. [PMID: 21773674 PMCID: PMC3887630 DOI: 10.1007/s10059-011-0069-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2011] [Revised: 06/15/2011] [Accepted: 06/20/2011] [Indexed: 12/30/2022] Open
Abstract
Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ~2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5' upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.
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Affiliation(s)
| | - Jogeswar Gadi
- Institute of Endocrinology, Yonsei University College of Medicine, Seoul 120-752, Korea
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35
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Forsmark A, Rossi G, Wadskog I, Brennwald P, Warringer J, Adler L. Quantitative proteomics of yeast post-Golgi vesicles reveals a discriminating role for Sro7p in protein secretion. Traffic 2011; 12:740-53. [PMID: 21477180 DOI: 10.1111/j.1600-0854.2011.01186.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.
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Affiliation(s)
- Annabelle Forsmark
- Department of Cell and Molecular Biology, Microbiology, University of Gothenburg, Box 462, SE-405 30 Gothenburg, Sweden
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36
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Chalasani K, Brewster RM. N-cadherin-mediated cell adhesion restricts cell proliferation in the dorsal neural tube. Mol Biol Cell 2011; 22:1505-15. [PMID: 21389116 PMCID: PMC3084673 DOI: 10.1091/mbc.e10-08-0675] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Neural progenitors are organized as a pseudostratified epithelium held together by adherens junctions (AJs), multiprotein complexes composed of cadherins and α- and β-catenin. Catenins are known to control neural progenitor division; however, it is not known whether they function in this capacity as cadherin binding partners, as there is little evidence that cadherins themselves regulate neural proliferation. We show here that zebrafish N-cadherin (N-cad) restricts cell proliferation in the dorsal region of the neural tube by regulating cell-cycle length. We further reveal that N-cad couples cell-cycle exit and differentiation, as a fraction of neurons are mitotic in N-cad mutants. Enhanced proliferation in N-cad mutants is mediated by ligand-independent activation of Hedgehog (Hh) signaling, possibly caused by defective ciliogenesis. Furthermore, depletion of Hh signaling results in the loss of junctional markers. We therefore propose that N-cad restricts the response of dorsal neural progenitors to Hh and that Hh signaling limits the range of its own activity by promoting AJ assembly. Taken together, these observations emphasize a key role for N-cad-mediated adhesion in controlling neural progenitor proliferation. In addition, these findings are the first to demonstrate a requirement for cadherins in synchronizing cell-cycle exit and differentiation and a reciprocal interaction between AJs and Hh signaling.
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Affiliation(s)
- Kavita Chalasani
- Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA
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37
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Beatty A, Morton D, Kemphues K. The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo. Development 2010; 137:3995-4004. [PMID: 21041363 DOI: 10.1242/dev.056028] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.
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Affiliation(s)
- Alexander Beatty
- Department of Molecular Biology and Genetics, Cornell University, 433 Biotechnology Building, Ithaca, NY 14850, USA
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38
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LGL Can Partition the Cortex of One-Cell Caenorhabditis elegans Embryos into Two Domains. Curr Biol 2010; 20:1296-303. [DOI: 10.1016/j.cub.2010.05.061] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2010] [Revised: 05/12/2010] [Accepted: 05/18/2010] [Indexed: 12/29/2022]
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Jang TJ. Lethal Giant Larvae2 Expression Is Reduced or Localized at Cytoplasm in Colon Adenomas and Adenocarcinomas. KOREAN JOURNAL OF PATHOLOGY 2010. [DOI: 10.4132/koreanjpathol.2010.44.5.488] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Affiliation(s)
- Tae Jung Jang
- Department of Pathology, Dongguk University College of Medicine, Gyeongju, Korea
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40
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Choi SC, Sokol SY. The involvement of lethal giant larvae and Wnt signaling in bottle cell formation in Xenopus embryos. Dev Biol 2009; 336:68-75. [PMID: 19782678 DOI: 10.1016/j.ydbio.2009.09.033] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2009] [Revised: 09/18/2009] [Accepted: 09/21/2009] [Indexed: 12/29/2022]
Abstract
Lethal giant larvae (Lgl) plays a critical role in establishment of cell polarity in epithelial cells. While Frizzled/Dsh signaling has been implicated in the regulation of the localization and activity of Lgl, it remains unclear whether specific Wnt ligands are involved. Here we show that Wnt5a triggers the release of Lgl from the cell cortex into the cytoplasm with the concomitant decrease in Lgl stability. The observed changes in Lgl localization were independent of atypical PKC (aPKC), which is known to influence Lgl distribution. In ectodermal cells, both Wnt5a and Lgl triggered morphological and molecular changes characteristic of apical constriction, whereas depletion of their functions prevented endogenous and ectopic bottle cell formation. Furthermore, Lgl RNA partially rescued bottle cell formation in embryos injected with a dominant negative Wnt5a construct. These results suggest a molecular link between Wnt5a and Lgl that is essential for apical constriction during vertebrate gastrulation.
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Affiliation(s)
- Sun-Cheol Choi
- Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029, USA
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41
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Sabherwal N, Tsutsui A, Hodge S, Wei J, Chalmers AD, Papalopulu N. The apicobasal polarity kinase aPKC functions as a nuclear determinant and regulates cell proliferation and fate during Xenopus primary neurogenesis. Development 2009; 136:2767-77. [PMID: 19633170 PMCID: PMC2730405 DOI: 10.1242/dev.034454] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/17/2009] [Indexed: 12/25/2022]
Abstract
During neurogenesis in Xenopus, apicobasally polarised superficial and non-polar deep cells take up different fates: deep cells become primary neurons while superficial cells stay as progenitors. It is not known whether the proteins that affect cell polarity also affect cell fate and how membrane polarity information may be transmitted to the nucleus. Here, we examine the role of the polarity components, apically enriched aPKC and basolateral Lgl2, in primary neurogenesis. We report that a membrane-tethered form of aPKC (aPKC-CAAX) suppresses primary neurogenesis and promotes cell proliferation. Unexpectedly, both endogenous aPKC and aPKC-CAAX show some nuclear localisation. A constitutively active aPKC fused to a nuclear localisation signal has the same phenotypic effect as aPKC-CAAX in that it suppresses neurogenesis and enhances proliferation. Conversely, inhibiting endogenous aPKC with a dominant-negative form that is restricted to the nucleus enhances primary neurogenesis. These observations suggest that aPKC has a function in the nucleus that is important for cell fate specification during primary neurogenesis. In a complementary experiment, overexpressing basolateral Lgl2 causes depolarisation and internalisation of superficial cells, which form ectopic neurons when supplemented with a proneural factor. These findings suggest that both aPKC and Lgl2 affect cell fate, but that aPKC is a nuclear determinant itself that might shuttle from the membrane to the nucleus to control cell proliferation and fate; loss of epithelial cell polarity by Lgl2 overexpression changes the position of the cells and is permissive for a change in cell fate.
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Affiliation(s)
- Nitin Sabherwal
- Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester, UK
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42
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Bultje RS, Castaneda-Castellanos DR, Jan LY, Jan YN, Kriegstein AR, Shi SH. Mammalian Par3 regulates progenitor cell asymmetric division via notch signaling in the developing neocortex. Neuron 2009; 63:189-202. [PMID: 19640478 PMCID: PMC2736606 DOI: 10.1016/j.neuron.2009.07.004] [Citation(s) in RCA: 271] [Impact Index Per Article: 16.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2008] [Revised: 07/01/2009] [Accepted: 07/01/2009] [Indexed: 11/26/2022]
Abstract
Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. Here, we found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex.
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Affiliation(s)
- Ronald S. Bultje
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
- Department of Pharmacology, Weill Medical College of Cornell University, 445 East 69th Street, New York, NY 10065, USA
| | - David R. Castaneda-Castellanos
- Department of Physiology and Cellular Biophysics, Columbia University, 630 West 168th Street, New York, NY 10032, USA
- Department of Neurology, School of Medicine, University of California at San Francisco, 513 Parnassus Avenue, HSW 1201-0525, San Francisco, CA 94143, USA
| | - Lily Yeh Jan
- Howard Hughes Medical Institute, Departments of Physiology, Biochemistry, and Biophysics, University of California at San Francisco, 1550 4th Street, San Francisco, CA 94143, USA
| | - Yuh-Nung Jan
- Howard Hughes Medical Institute, Departments of Physiology, Biochemistry, and Biophysics, University of California at San Francisco, 1550 4th Street, San Francisco, CA 94143, USA
| | - Arnold R. Kriegstein
- Department of Neurology, School of Medicine, University of California at San Francisco, 513 Parnassus Avenue, HSW 1201-0525, San Francisco, CA 94143, USA
| | - Song-Hai Shi
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
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Dodd ME, Hatzold J, Mathias JR, Walters KB, Bennin DA, Rhodes J, Kanki JP, Look AT, Hammerschmidt M, Huttenlocher A. The ENTH domain protein Clint1 is required for epidermal homeostasis in zebrafish. Development 2009; 136:2591-600. [PMID: 19570844 DOI: 10.1242/dev.038448] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Epidermal hyperproliferation and inflammation are hallmarks of the human condition psoriasis. Here, we report that a zebrafish line with a mutation in the cargo adaptor protein Clint1 exhibits psoriasis-like phenotypes including epithelial hyperproliferation and leukocyte infiltration. Clint1 is an ENTH domain-containing protein that binds SNARE proteins and functions in vesicle trafficking; however, its in vivo function in animal models has not been reported to date. The clint1 mutants exhibit chronic inflammation characterized by increased Interleukin 1beta expression, leukocyte infiltration, bidirectional trafficking and phagocytosis of cellular debris. The defects in clint1 mutants can be rescued by expression of zebrafish clint1 and can be phenocopied with clint1-specific morpholinos, supporting an essential role for Clint1 in epidermal development. Interaction studies suggest that Clint1 and Lethal giant larvae 2 function synergistically to regulate epidermal homeostasis. Accordingly, clint1 mutants show impaired hemidesmosome formation, loss of cell-cell contacts and increased motility suggestive of epithelial to mesenchymal transition. Taken together, our findings describe a novel function for the ENTH domain protein Clint1 in epidermal development and inflammation and suggest that its deficiency in zebrafish generates a phenotype that resembles the human condition psoriasis.
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Affiliation(s)
- M Ernest Dodd
- Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA
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Lu X, Feng X, Man X, Yang G, Tang L, Du D, Zhang F, Yuan H, Huang Q, Zhang Z, Liu Y, Strand D, Chen Z. Aberrant splicing of Hugl-1 is associated with hepatocellular carcinoma progression. Clin Cancer Res 2009; 15:3287-96. [PMID: 19447873 DOI: 10.1158/1078-0432.ccr-08-2078] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay. RESULTS In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short "direct repeat" sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice. CONCLUSIONS We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.
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Affiliation(s)
- Xuefeng Lu
- State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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Kienle N, Kloepper TH, Fasshauer D. Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi. BMC Evol Biol 2009; 9:19. [PMID: 19166604 PMCID: PMC2639358 DOI: 10.1186/1471-2148-9-19] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2008] [Accepted: 01/23/2009] [Indexed: 12/29/2022] Open
Abstract
Background In eukaryotic cells, directional transport between different compartments of the endomembrane system is mediated by vesicles that bud from a donor organelle and then fuse with an acceptor organelle. A family of integral membrane proteins, termed soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, constitute the key machineries of these different membrane fusion events. Over the past 30 years, the yeast Saccharomyces cerevisiae has served as a powerful model organism for studying the organization of the secretory and endocytic pathways, and a few years ago, its entire set of SNAREs was compiled. Results Here, we make use of the increasing amount of genomic data to investigate the history of the SNARE family during fungi evolution. Moreover, since different SNARE family members are thought to demarcate different organelles and vesicles, this approach allowed us to compare the organization of the endomembrane systems of yeast and animal cells. Our data corroborate the notion that fungi generally encompass a relatively simple set of SNARE proteins, mostly comprising the SNAREs of the proto-eukaryotic cell. However, all fungi contain a novel soluble SNARE protein, Vam7, which carries an N-terminal PX-domain that acts as a phosphoinositide binding module. In addition, the points in fungal evolution, at which lineage-specific duplications and diversifications occurred, could be determined. For instance, the endosomal syntaxins Pep12 and Vam3 arose from a gene duplication that occurred within the Saccharomycotina clade. Conclusion Although the SNARE repertoire of baker's yeast is highly conserved, our analysis reveals that it is more deviated than the ones of basal fungi. This highlights that the trafficking pathways of baker's yeast are not only different to those in animal cells but also are somewhat different to those of many other fungi.
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Affiliation(s)
- Nickias Kienle
- Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
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Abstract
The epithelial-to-mesenchymal transition (EMT) is a crucial process in tumour progression providing tumour cells with the ability to escape from the primary tumour, to migrate to distant regions and to invade tissues. EMT requires a loss of cell-cell adhesion and apical-basal polarity, as well as the acquisition of a fibroblastoid motile phenotype. Several transcription factors have emerged in recent years that induce EMT, with important implications for tumour progression. However, their effects on cell polarity remain unclear. Here, we have re-examined the data available related to the effect of EMT related transcription factors on epithelial cell plasticity, focusing on their impact on cell polarity. Transcriptional and post-transcriptional regulatory mechanisms mediated by several inducers of EMT, in particular the ZEB and Snail factors, downregulate the expression and/or functional organization of core polarity complexes. We also summarize data on the expression of cell polarity genes in human tumours and analyse genetic interactions that highlight the existence of complex regulatory networks converging on the regulation of cell polarity by EMT inducers in human breast carcinomas. These recent observations provide new insights into the relationship between alterations in cell polarity components and EMT in cancer, opening new avenues for their potential use as therapeutic targets to prevent tumour progression.
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Webb AE, Driever W, Kimelman D. psoriasis regulates epidermal development in zebrafish. Dev Dyn 2008; 237:1153-64. [PMID: 18351656 DOI: 10.1002/dvdy.21509] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The zebrafish epidermis completely envelopes the embryo by 14 hours postfertilization, providing an essential barrier between the internal organs and the environment. As the embryo increases in size, keratinocytes in the epidermis must proliferate and differentiate to form the three epidermal layers present in the adult. The mechanisms controlling growth, differentiation, and maintenance of the fish epidermis are mostly unknown. Here, we describe psoriasis, an epidermal mutant that exhibits widespread overproliferation of the epidermis at 3 days postfertilization and a defect in keratinocyte differentiation. Based on mosaic analysis, we show that psoriasis acts non-cell-autonomously, suggesting that psoriasis encodes a secreted factor. Our analysis of the psoriasis mutant indicates that keratinocyte differentiation and proliferation are tightly regulated to maintain a cohesive epidermal sheet around the embryo and that disruptions in these processes result in the formation of epidermal aggregates.
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Affiliation(s)
- Ashley E Webb
- University of Washington, Department of Biochemistry, Seattle, Washington 98195-7350, USA
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Kloepper TH, Kienle CN, Fasshauer D. SNAREing the basis of multicellularity: consequences of protein family expansion during evolution. Mol Biol Evol 2008; 25:2055-68. [PMID: 18621745 DOI: 10.1093/molbev/msn151] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Vesicle trafficking between intracellular compartments of eukaryotic cells is mediated by conserved protein machineries. In each trafficking step, fusion of the vesicle with the acceptor membrane is driven by a set of distinctive soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that assemble into tight 4-helix bundle complexes between the fusing membranes. During evolution, about 20 primordial SNARE types were modified independently in different eukaryotic lineages by episodes of duplication and diversification. Here we show that 2 major changes in the SNARE repertoire occurred in the evolution of animals, each reflecting a main overhaul of the endomembrane system. In addition, we found several lineage-specific losses of distinct SNAREs, particularly in nematodes and platyhelminthes. The first major transformation took place during the transition to multicellularity. The primary event that occurred during this transformation was an increase in the numbers of endosomal SNAREs, but the SNARE-related factor lethal giant larvae also emerged. Apparently, enhanced endosomal sorting capabilities were an advantage for early multicellular animals. The second major transformation during the rise of vertebrates resulted in a robust expansion of the secretory set of SNAREs, which may have helped develop a more versatile secretory apparatus.
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Affiliation(s)
- Tobias H Kloepper
- Research Group Structural Biochemistry, Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
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Abstract
Specification of the anteroposterior (AP) axis in Drosophila oocytes requires proper organization of the microtubule and actin cytoskeleton. The establishment and regulation of cytoskeletal polarity remain poorly understood, however. Here, we show important roles for the tumor suppressor Lethal (2) giant larvae (Lgl) and atypical protein kinase C (aPKC) in regulating microtubule polarity and setting up the AP axis of the oocyte. Lgl in the germline cells regulates the localization of axis-specifying morphogens. aPKC phosphorylation of Lgl restricts Lgl activity to the oocyte posterior, thereby dividing the cortex into different domains along the AP axis. Active Lgl promotes the formation of actin-rich projections at the oocyte cortex and the posterior enrichment of the serine/threonine kinase Par-1, a key step for oocyte polarization. Our studies suggest that Lgl and its phosphorylation by aPKC may form a conserved regulatory circuitry in polarization of various cell types.
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Affiliation(s)
- Ai-Guo Tian
- Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA
| | - Wu-Min Deng
- Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA
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Grzeschik NA, Amin N, Secombe J, Brumby AM, Richardson HE. Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye. Dev Biol 2007; 311:106-23. [PMID: 17870065 PMCID: PMC2974846 DOI: 10.1016/j.ydbio.2007.08.025] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2007] [Revised: 08/06/2007] [Accepted: 08/07/2007] [Indexed: 01/16/2023]
Abstract
In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl- clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl- tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl- mutants are separable. Furthermore, lgl- mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl- and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl- tissue shows less apoptosis.
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Affiliation(s)
- Nicola A. Grzeschik
- Peter MacCallum Cancer Center, Melbourne, Victoria, Australia
- Anatomy and Cell Biology Department, University of Melbourne, Melbourne, Victoria, Australia
| | - Nancy Amin
- Peter MacCallum Cancer Center, Melbourne, Victoria, Australia
- Anatomy and Cell Biology Department, University of Melbourne, Melbourne, Victoria, Australia
| | - Julie Secombe
- Genetics Department, University of Adelaide, Adelaide, South Australia, Australia
| | - Anthony M. Brumby
- Peter MacCallum Cancer Center, Melbourne, Victoria, Australia
- Anatomy and Cell Biology Department, University of Melbourne, Melbourne, Victoria, Australia
- Genetics Department, University of Adelaide, Adelaide, South Australia, Australia
| | - Helena E. Richardson
- Peter MacCallum Cancer Center, Melbourne, Victoria, Australia
- Anatomy and Cell Biology Department, University of Melbourne, Melbourne, Victoria, Australia
- Genetics Department, University of Adelaide, Adelaide, South Australia, Australia
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