The Cul3–KLHL21 E3 ubiquitin ligase targets Aurora B to midzone microtubules in anaphase and is required for cytokinesis

Selective ubiquitination of Aurora B by different Cul3 adaptors targets it at the correct time to the correct place during mitosis

Centriole movements in mammalian epithelial cells during cytokinesis

<p>Abstract</p> <p>Background</p> <p>In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules.</p> <p>Results</p> <p>Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent.</p> <p>Conclusions</p> <p>These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.</p

Chromosome Tips Damaged in Anaphase Inhibit Cytokinesis

Genome maintenance is ensured by a variety of biochemical sensors and pathways that repair accumulated damage. During mitosis, the mechanisms that sense and resolve DNA damage remain elusive. Studies have demonstrated that damage accumulated on lagging chromosomes can activate the spindle assembly checkpoint. However, there is little known regarding damage to DNA after anaphase onset. In this study, we demonstrate that laser-induced damage to chromosome tips (presumptive telomeres) in anaphase of Potorous tridactylis cells (PtK2) inhibits cytokinesis. In contrast, equivalent irradiation of non-telomeric chromosome regions or control irradiations in either the adjacent cytoplasm or adjacent to chromosome tips near the spindle midzone during anaphase caused no change in the eventual completion of cytokinesis. Damage to only one chromosome tip caused either complete absence of furrow formation, a prolonged delay in furrow formation, or furrow regression. When multiple chromosome tips were irradiated in the same cell, the cytokinesis defects increased, suggesting a potential dose-dependent mechanism. These results suggest a mechanism in which dysfunctional telomeres inhibit mitotic exit

CellCognition : time-resolved phenotype annotation in high-throughput live cell imaging

Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Methods 7 (2010): 747-754, doi:10.1038/nmeth.1486.Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here, we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. The incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions, and confusion between different functional states with similar morphology. We demonstrate generic applicability in a set of different assays and perturbation conditions, including a candidate-based RNAi screen for mitotic exit regulators in human cells. CellCognition is published as open source software, enabling live imaging-based screening with assays that directly score cellular dynamics.Work in the Gerlich laboratory is supported by Swiss National Science Foundation (SNF) research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation, an EMBO YIP fellowship, and a MBL Summer Research Fellowship to D.W.G., an ETH TH grant, a grant by the UBS foundation, a Roche Ph.D. fellowship to M.H.A.S, and a Mueller fellowship of the Molecular Life Sciences Ph.D. program Zurich to M.H. M.H. and M.H.A.S are fellows of the Zurich Ph.D. Program in Molecular Life Sciences. B.F. was supported by European Commission’s seventh framework program project Cancer Pathways. Work in the Ellenberg laboratory is supported by a European Commission grant within the Mitocheck consortium (LSHG-CT-2004-503464). Work in the Peter laboratory is supported by the ETHZ, Oncosuisse, SystemsX.ch (LiverX) and the SNF

Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour

The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1–2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories

Progressive Telomere Dysfunction Causes Cytokinesis Failure and Leads to the Accumulation of Polyploid Cells

Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy

Automated microscopy for high-content RNAi screening

Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs

A Conserved Role for Syndecan Family Members in the Regulation of Whole-Body Energy Metabolism

Syndecans are a family of type-I transmembrane proteins that are involved in cell-matrix adhesion, migration, neuronal development, and inflammation. Previous quantitative genetic studies pinpointed Drosophila Syndecan (dSdc) as a positional candidate gene affecting variation in fat storage between two Drosophila melanogaster strains. Here, we first used quantitative complementation tests with dSdc mutants to confirm that natural variation in this gene affects variability in Drosophila fat storage. Next, we examined the effects of a viable dSdc mutant on Drosophila whole-body energy metabolism and associated traits. We observed that young flies homozygous for the dSdc mutation had reduced fat storage and slept longer than homozygous wild-type flies. They also displayed significantly reduced metabolic rate, lower expression of spargel (the Drosophila homologue of PGC-1), and reduced mitochondrial respiration. Compared to control flies, dSdc mutants had lower expression of brain insulin-like peptides, were less fecund, more sensitive to starvation, and had reduced life span. Finally, we tested for association between single nucleotide polymorphisms (SNPs) in the human SDC4 gene and variation in body composition, metabolism, glucose homeostasis, and sleep traits in a cohort of healthy early pubertal children. We found that SNP rs4599 was significantly associated with resting energy expenditure (P = 0.001 after Bonferroni correction) and nominally associated with fasting glucose levels (P = 0.01) and sleep duration (P = 0.044). On average, children homozygous for the minor allele had lower levels of glucose, higher resting energy expenditure, and slept shorter than children homozygous for the common allele. We also observed that SNP rs1981429 was nominally associated with lean tissue mass (P = 0.035) and intra-abdominal fat (P = 0.049), and SNP rs2267871 with insulin sensitivity (P = 0.037). Collectively, our results in Drosophila and humans argue that syndecan family members play a key role in the regulation of body metabolism

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases