3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin
Daniel G. Booth5, Alison J. Beckett, Oscar Molina, Itaru Samejima, Hiroshi Masumoto, Natalay Kouprina, Vladimir Larionov, Ian A. Prior, William C. Earnshaw
Published Online: November 10, 2016
Open access funded by Wellcome Trust
Published: November 10, 2016 Accepted: October 5, 2016
Received in revised form: August 24, 2016 Received: April 4, 2016
Creative Commons Attribution (CC BY 4.0)
• 3D-CLEM combines light and serial block-face scanning electron microscopy
• The complete architecture of all 46 human chromosomes has been defined
• A large portion of mitotic chromosomes is not composed of chromatin
• Chromosome volumes determined by light and electron microscopy differ dramatically
Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%–47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.
Keywords: 3D-CLEM, chromosome periphery, Ki-67, electron microscopy, CLEM
Received: April 4, 2016; Received in revised form: August 24, 2016; Accepted: October 5, 2016; Published: November 10, 2016
© 2016 The Author(s). Published by Elsevier Inc.
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