Inference of cell cycle-dependent proteolysis by laser scanning cytometry

Chakraborty, A. A. , Tansey, W. P. (June 2009) Inference of cell cycle-dependent proteolysis by laser scanning cytometry. Experimental Cell Research, 315 (10). pp. 1772-1778.

URL: http://www.ncbi.nlm.nih.gov/pubmed/19331831
DOI: 10.1016/j.yexcr.2009.01.011

Abstract

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.

Item Type: Paper
Uncontrolled Keywords: Laser scanning cytometry Cell cycle Proteolysis Myc Cyclin E
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > proteolysis
CSHL Authors:
Communities: CSHL labs > Tansey lab
Depositing User: Matt Covey
Date: 10 June 2009
Date Deposited: 20 Feb 2013 21:27
Last Modified: 20 Feb 2013 21:27
PMCID: PMC2680938
Related URLs:
URI: http://repository.cshl.edu/id/eprint/27397

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