Detrimental incorporation of excess Cenp-A/Cid and Cenp-C intoDrosophilacentromeres is prevented by limiting amounts of the bridging factor Cal1

Abstract

SummaryPropagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere-specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp-A/Cid is required for the recruitment of Cenp-C, another conserved centromere protein. With yeast three-hybrid experiments, we demonstrate that the essential Drosophila centromere protein Cal1 can link Cenp-A/Cid and Cenp-C. Cenp-A/Cid and Cenp-C interact with the N-and C-terminal domains of Cal1, respectively. These Cal1 domains are sufficient for centromere localization and function, but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores, we demonstrate that centromeric Cal1 levels are far lower than those of Cenp-A/Cid, Cenp-C and other conserved kinetochore components, which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore, Cal1 limits centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis. We demonstrate that the low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp-A/Cid and Cenp-C are present in excess.

Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells

“…1). The fluorescent centromere protein variants were previously shown to be functional (Schuh et al, 2007;Schittenhelm et al, 2010;Schittenhelm et al, 2009). Expression of these variants was controlled by the cis-regulatory regions of the corresponding centromere protein genes (Fig.…”

“…Localization of the few centromere components Cid, Cenp-C and Cal1 has been shown to be interdependent (Goshima et al, 2007;Erhardt et al, 2008;Schittenhelm et al, 2010). Cid serves as an epigenetic centromere mark (Mendiburo et al, 2011;Raychaudhuri et al, 2012), Cenp-C as a structural platform for kinetochore assembly in mitosis (Przewloka et al, 2011;Gascoigne et al, 2011) and Cal1 is proposed to function as a Cid loading factor rather than serving a structural role (Phansalkar et al, 2012;Schittenhelm et al, 2010).…”

“…Experimentally enforced alterations in the level of centromeric Cid in sperm are maintained during development of the next generation (Raychaudhuri et al, 2012). Acute overloading of Cid at centromeres during embryonic cell division cycles causes severe mitotic defects (Schittenhelm et al, 2010), emphasizing the relevance of quantitative control.…”

A Novel Approach for Studying Histone H1 Function in Vivo

“…Consistent with this possibility, we have been unable to express mutant forms of the histone H1 protein lacking the winged-helix domain required for nucleosome binding (data not shown). Histone H1 stability could also be dependent on a limiting interaction partner, as has been observed for the centromere-associated histone H3 variant CENP-A, which is unstable unless it is bound to the CENP-A assembly factor Cal1 (Schittenhelm et al 2010;Chen et al 2014).…”

Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function

“…After maximum projection, kinetochore signals were quantified as described previously (Schittenhelm et al 2010).…”