Mobility of kinetochore proteins measured by FRAP analysis in living cells

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.

De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays)

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.