Distinct states of nucleolar stress induced by anticancer drugs.

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress.

Superresolution Microscopy for Visualization of Physical Contacts Between Chromosomes at Nanoscale Resolution.

This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches-structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci.

Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes.

The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes.

Ribosomal DNA and the nucleolus in the context of genome organization.

The nucleolus constitutes a prominent nuclear compartment, a membraneless organelle that was first documented in the 1830s. The fact that specific chromosomal regions were present in the nucleolus was recognized by Barbara McClintock in the 1930s, and these regions were termed nucleolar organizing regions, or NORs. The primary function of ribosomal DNA (rDNA) is to produce RNA components of ribosomes.

Transcriptome analysis of tetraploid cells identifies cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53.

Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates changes in gene expression evoked in acute and adapted tetraploid cells and their effect on cell-cycle progression. Acute polyploidy was generated by knockdown of the essential regulator of cytokinesis anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology.

Karyotyping human and mouse cells using probes from single-sorted chromosomes and open source software.

Multispectral karyotyping analyzes all chromosomes in a single cell by labeling them with chromosome-specific probes conjugated to unique combinations of fluorophores. Currently available multispectral karyotyping systems require the purchase of specialized equipment and reagents. However, conventional laser scanning confocal microscopes that are capable of separating multiple overlapping emission spectra through spectral imaging and linear unmixing can be utilized for classifying chromosomes painted with multicolor probes.

Aneuploidy and chromosomal instability: a vicious cycle driving cellular evolution and cancer genome chaos.

Aneuploidy and chromosomal instability frequently co-exist, and aneuploidy is recognized as a direct outcome of chromosomal instability. However, chromosomal instability is widely viewed as a consequence of mutations in genes involved in DNA replication, chromosome segregation, and cell cycle checkpoints. Telomere attrition and presence of extra centrosomes have also been recognized as causative for errors in genomic transmission.

Cohesion fatigue induces chromatid separation in cells delayed at metaphase.

Background: Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together.

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited.

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms.

Expression of HPV16 E5 produces enlarged nuclei and polyploidy through endoreplication.

Anogenital cancers and head and neck cancers are causally associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently.

Ska3 is required for spindle checkpoint silencing and the maintenance of chromosome cohesion in mitosis.

The mitotic spindle checkpoint monitors proper bipolar attachment of chromosomes to the mitotic spindle. Previously, depletion of the novel kinetochore complex Ska1/Ska2 was found to induce a metaphase delay. By using bioinformatics, we identified C13orf3, predicted to associate with kinetochores.

Fine tuning the cell cycle: activation of the Cdk1 inhibitory phosphorylation pathway during mitotic exit.

Inactivation of cyclin-dependent kinase (Cdk) 1 promotes exit from mitosis and establishes G1. Proteolysis of cyclin B is the major known mechanism that turns off Cdk1 during mitotic exit. Here, we show that mitotic exit also activates pathways that catalyze inhibitory phosphorylation of Cdk1, a mechanism previously known to repress Cdk1 only during S and G2 phases of the cell cycle.

The reversibility of mitotic exit in vertebrate cells.

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation.