| Inference of cell cycle-dependent proteolysis by laser scanning cytometry. | |
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MedLine Citation:
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PMID: 19331831 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods. |
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Authors:
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Abhishek A Chakraborty; William P Tansey |
Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2009-01-27 |
Journal Detail:
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Title: Experimental cell research Volume: 315 ISSN: 1090-2422 ISO Abbreviation: Exp. Cell Res. Publication Date: 2009 Jun |
Date Detail:
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Created Date: 2009-05-12 Completed Date: 2009-05-26 Revised Date: 2011-09-26 |
Medline Journal Info:
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Nlm Unique ID: 0373226 Medline TA: Exp Cell Res Country: United States |
Other Details:
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Languages: eng Pagination: 1772-8 Citation Subset: IM |
Affiliation:
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Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Cell Cycle* Cell Cycle Proteins / metabolism Cell Line, Tumor Cyclin E / metabolism F-Box Proteins / metabolism Gene Expression Regulation, Neoplastic Humans Laser Scanning Cytometry* Phosphorylation Protein Processing, Post-Translational* Proto-Oncogene Proteins c-myc / genetics, metabolism RNA, Messenger / genetics, metabolism Ubiquitin-Protein Ligases / metabolism |
| Grant Support | |
ID/Acronym/Agency:
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CA-13106/CA/NCI NIH HHS; CA45508/CA/NCI NIH HHS; P01 CA013106-310027/CA/NCI NIH HHS; P01 CA013106-360026/CA/NCI NIH HHS; P01 CA013106-370026/CA/NCI NIH HHS; S10 RR021174-01/RR/NCRR NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Cell Cycle Proteins; 0/Cyclin E; 0/F-Box Proteins; 0/Proto-Oncogene Proteins c-myc; 0/RNA, Messenger; EC 6.3.2.19/FBXW7 protein, human; EC 6.3.2.19/Ubiquitin-Protein Ligases |
| Comments/Corrections | |
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