Katanin-like protein Katnal2 is required for ciliogenesis and brain development in Xenopus embryos.

Microtubule remodeling is critical for cellular and developmental processes underlying morphogenetic changes and for the formation of many subcellular structures. Katanins are conserved microtubule severing enzymes that are essential for spindle assembly, ciliogenesis, cell division, and cellular motility. We have recently shown that a related protein, Katanin-like 2 (KATNAL2), is similarly required for cytokinesis, cell cycle progression, and ciliogenesis in cultured mouse cells. However, its developmental expression pattern, localization, and in vivo role during organogenesis have yet to be characterized. Here, we used Xenopus embryos to reveal that Katnal2 (1) is expressed broadly in ciliated and neurogenic tissues throughout embryonic development; (2) is localized to basal bodies, ciliary axonemes, centrioles, and mitotic spindles; and (3) is required for ciliogenesis and brain development. Since human KATNAL2 is a risk gene for autism spectrum disorders, our functional data suggest that Xenopus may be a relevant system for understanding the relationship of mutations in this gene to autism and the underlying molecular mechanisms of pathogenesis.

Analyzing Mitotic Chromosome Structural Defects After Topoisomerase II Inhibition or Mutation.

For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction we have adapted classical cell cycle experiments, classical cytological techniques and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rational for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II takes place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization. We also put these observations into the context of the two topo II-dependent cell cycle checkpoints. In addition, we have devised a system to analyze phenotypes that result when topo II is mutated in human cells. This serves as an alternative strategy to the use of topo II inhibitors to perturb topo II function.

Monitoring the DNA Topoisomerase II Checkpoint in Saccharomyces cerevisiae.

Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control which responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpoint using spindle morphology, chromosome condensation using fluorescently labeled chromosomal loci and cell cycle progression by flow cytometry. Further characterization of this novel checkpoint is warranted so that we can further our understanding of the cell cycle, genomic stability, and the possibility of identifying novel drug targets.

Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening.

CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency.

A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation.

DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes. Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation.

By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.

Genome instability: does genetic diversity amplification drive tumorigenesis?

Recent data show that catastrophic events during one cell cycle can cause massive genome damage producing viable clones with unstable genomes. This is in contrast with the traditional view that tumorigenesis requires a long-term process in which mutations gradually accumulate over decades. These sudden events are likely to result in a large increase in genomic diversity within a relatively short time, providing the opportunity for selective advantages to be gained by a subset of cells within a population. This genetic diversity amplification, arising from a single aberrant cell cycle, may drive a population conversion from benign to malignant. However, there is likely a period of relative genome stability during the clonal expansion of tumors - this may provide an opportunity for therapeutic intervention, especially if mechanisms that limit tolerance of aneuploidy are exploited.