Molecular Analysis of Mitotic Chromosome Condensation Using a Quantitative Time-Resolved Fluorescence Microscopy Assay

Chromosomes condense during mitotic entry to facilitate their segregation. Condensation is typically assayed in fixed preparations, limiting analysis of contributing factors. Here, we describe a quantitative method to monitor condensation kinetics in living cells expressing GFP fused to a core histo...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2006-10, Vol.103 (41), p.15097-15102
Main Authors: Maddox, Paul S., Portier, Nathan, Desai, Arshad, Oegema, Karen
Format: Article
Language:English
Subjects:
Analysis
Animals
Autoantigens - genetics
Biological Sciences
Caenorhabditis elegans
Caenorhabditis elegans - chemistry
Caenorhabditis elegans - embryology
Caenorhabditis elegans - genetics
Cells
Centromere Protein A
Chromatids
Chromatin
Chromosomal Proteins, Non-Histone - deficiency
Chromosomal Proteins, Non-Histone - genetics
Chromosomes
Chromosomes - chemistry
Chromosomes - metabolism
Condensation
Embryos
Female
Fluorescence
Kinetics
Kinetochores
Male
Microscopy
Microscopy, Fluorescence
Mitosis - genetics
Pixels
Prophase
Time Factors
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Summary:Chromosomes condense during mitotic entry to facilitate their segregation. Condensation is typically assayed in fixed preparations, limiting analysis of contributing factors. Here, we describe a quantitative method to monitor condensation kinetics in living cells expressing GFP fused to a core histone. We demonstrate the utility of this method by using it to analyze the molecular requirements for the condensation of holocentric chromosomes during the first division of the Caenorhabditis elegans embryo. In control embryos, the fluorescence intensity distribution for nuclear GFP:histone changes during two distinct time intervals separated by a plateau phase. During the first interval, primary condensation converts diffuse chromatin into discrete linear chromosomes. After the plateau, secondary condensation compacts the curvilinear chromosomes to form shorter bar-shaped structures. We quantitatively compared the consequences on this characteristic profile of depleting the condensin complex, the mitosis-specific histone H3 kinase Aurora B, the centromeric histone CENP-A, and CENP-C, a conserved protein required for kinetochore assembly. Both condensin and CENP-A play critical but distinct roles in primary condensation. In contrast, depletion of CENP-C slows but does not prevent primary condensation. Finally, Aurora B inhibition has no effect on primary condensation, but slightly delays secondary condensation. These results provide insights into the process of condensation, help resolve apparent contradictions from prior studies, and indicate that CENP-A chromatin has an intrinsic role in the condensation of holocentric chromosomes that is independent of its requirement for kinetochore assembly.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0606993103