Incorporation of Drosophila CID/CENP-A and CENP-C into Centromeres during Early Embryonic Anaphase

Abstract

The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. Here we show by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, our findings uncover a strikingly dynamic behavior of centromere components in anaphase.

Centromere identity is specified by a single centromeric nucleosome in budding yeast

“…It will therefore be critical to determine the histone/nonhistone components associated with budding yeast CenH3 by examining their localization to the CEN at precise cell cycle intervals. Such an experiment is also particularly noteworthy in light of several groups reporting the deposition of CenH3 outside of S phase (42,(46)(47)(48), a detail that has not been observed in budding yeast (19,49). Because of the ability to achieve single-nucleosome resolution, utilization of our modified ChIP protocol should clarify the protein content of the centromeric nucleosome.…”

Chromatin and DNA Replication

“…During replication of centromeric chromatin, CenH3 nucleosomes become diluted to half the initial concentration on daughter chromatin (Shelby et al 2000;Jansen et al 2007). It is not until the next G 1 phase that new CenH3 gets incorporated again (Jansen et al 2007;Schuh et al 2007). This case illustrates a situation in which the disruption of chromatin during replication is clearly separated from the functional reassembly of the chromatin outside of S phase.…”

In vivo analysis of MT‐based vesicle transport by confocal reflection microscopy

“…Droplets that moved in the same direction but burst (could be followed for short period and the speed exceeded the average stream velocity 1 13SD, 458 1 156 nm/s) were clustered to the plus end tracking category. To test that lipid droplets participate in all the above mentioned motions considering the excellent but not 100% selectivity of CRM for lipid droplets, we visualized them by using Histone2Av-RFP [Schuh et al, 2007] that accumulates transiently in the lipid droplets [Cermelli et al, 2006]. The particles that were visualized by RFP fluorescence are involved in all of the motion categories (Supp.…”