CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

Abstract

Current models of centromere/kinetochore architecture are not sufficient to explain the number of molecules of histone H3 variant CENP-A observed in quantitative microscopy.

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

“…Given ∼1,500 molecules of Mid1p (17) in ∼140 nodes around the equator, cytokinesis nodes have ∼10 Mid1p molecules and equal numbers of the Cdc15p, Rng2p, and myosin II dimers. These ratios agree with previously reported quantitative confocal microscopy measurements of the total number of Myo2p, Rlc1p, and Mid1p at the division site (17) and estimates of the numbers of Myo2p, Rlc1p, Cdc15p, and Mid1p per node (22,41,42).…”

“…This validates the assumption in the computer model of constriction that Myo2 is organized in clusters (21). This finding is somewhat surprising, given that anillin Mid1p is important for organizing Myo2 in nodes but leaves the contractile ring before it constricts (22,41,42). Thus, our data indicate that Myo2 and other node proteins remain clustered together without Mid1p after the ring assembles.…”

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

“…2). Our measurement showed that the increase in GFP intensity is much less significant (2-fold that of XL556) than that reported by Valerie et al in GFP intensity (4.4-fold that of XL556) (35). Quantification of GFP(S65T) copy number, using budding yeast Ndc80-GFP(S65T) as the reference (39) (7 Ndc80 molecules/kinetochore or a 112 molecule/kinetochore cluster in anaphase), showed that there are on average 154 Ϯ 40 Cnp1-GFP(S65T) molecules/kinetochore cluster in G 2 /M phase, equivalent to ϳ13 Cnp1 nucleosomes/centromere.…”

“…At least one reason for the low estimation of Cnp1 copy number in our earlier study may be due to unexpected complication in the GFP-tagging construction (34,35). The previous yeast strain carrying Cnp1-GFP (S65T) is at the cnp1 ϩ endogenous locus (strain XL556) (39).…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…This model appears true in the "point" centromeres of some budding yeasts, including Saccharomyces cerevisiae, which has a defined 125-bp centromeric DNA sequence consisting of three centromere-DNA elements (CDEs) that are necessary and sufficient for centromere specification and kinetochore assembly (Clarke and Carbon 1980;Fitzgerald-Hayes et al 1982;Cottarel et al 1989). A single CENP-A Cse4 nucleosome is positioned at CDEII in a centromeric DNA sequence-dependent manner (Spencer and Hieter 1992;Stoler et al 1995;Meluh et al 1998;Furuyama and Biggins 2007), although recent evidence suggests multiple accessory CENP-A Cse4 nucleosomes, proposed to not contribute to kinetochore formation, may exist at centromeres (Coffman et al 2011;Lawrimore et al 2011;Haase et al 2013). In contrast to budding yeast, fission yeast and higher eukaryotes, including humans, have "regional" centromeres that span kilo-to megabases of highly repetitive DNA.…”