Assays for mitotic chromosome condensation in live yeast and mammalian cells

The dynamic reorganization of chromatin into rigid and compact mitotic chromosomes is of fundamental importance for faithful chromosome segregation. Owing to the difficulty of investigating this process under physiological conditions, the exact morphological transitions and the molecular machinery driving chromosome condensation remain poorly defined. Here, we review how imaging-based methods can be used to quantitate chromosome condensation in vivo, focusing on yeast and animal tissue culture cells as widely used model systems. We discuss approaches how to address structural dynamics of condensing chromosomes and chromosome segments, as well as to probe for mechanical properties of mitotic chromosomes. Application of such methods to systematic perturbation studies will provide a means to reveal the molecular networks underlying the regulation of mitotic chromosome condensatio

Kinetic framework of spindle assembly checkpoint signaling

Author Posting. © The Author(s), 2013. 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 Cell Biology 15 (2013): 1370-1377, doi:10.1038/ncb2842.The mitotic spindle assembly checkpoint (SAC) delays anaphase onset until all chromosomes have attached to both spindle poles1, 2. Here, we investigated SAC signaling kinetics in response to acute detachment of individual chromosomes using laser microsurgery. Most detached chromosomes delayed anaphase until they had realigned to the metaphase plate. A substantial fraction of cells, however, entered anaphase in the presence of unaligned chromosomes. We identify two mechanisms by which cells can bypass the SAC: First, single unattached chromosomes inhibit the anaphase promoting complex/cyclosome (APC/C) less efficiently than a full complement of unattached chromosomes. Second, because of the relatively slow kinetics of reimposing APC/C inhibition during metaphase, cells were unresponsive to chromosome detachment up to several minutes before anaphase onset. Our study defines when cells irreversibly commit to enter anaphase and shows that the SAC signal strength correlates with the number of unattached chromosomes. Detailed knowledge about SAC signaling kinetics is important for understanding the emergence of aneuploidy and the response of cancer cells to chemotherapeutics targeting the mitotic spindle.Research in the Gerlich laboratory has received funding from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreements n° 241548 (MitoSys) and n° 258068 (Systems Microscopy), from an ERC Starting Grant (agreement n° 281198), from the EMBO Young Investigator Programme, from the Swiss National Science Foundation, from the Austrian Science Fund (FWF)-funded project “SFB Chromosome Dynamics”, and from a Summer Research Award of the Marine Biology Laboratory Woods Hole (Laura and Arthur Colwin Endowed Summer Research Fellowship Fund). A.E.D. is a fellow of the Zurich Ph.D. Program in Molecular Life Sciences and has received funding from a PhD fellowship by the Boehringer Ingelheim Fonds and from a Peter Müller fellowship.2014-04-0

Nuclear Envelope Breakdown Proceeds by Microtubule-Induced Tearing of the Lamina

AbstractThe mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor

CellH5: a format for data exchange in high-content screening

Summary: High-throughput microscopy data require a diversity of analytical approaches. However, the construction of workflows that use algorithms from different software packages is difficult owing to a lack of interoperability. To overcome this limitation, we present CellH5, an HDF5 data format for cell-based assays in high-throughput microscopy, which stores high-dimensional image data along with inter-object relations in graphs. CellH5Browser, an interactive gallery image browser, demonstrates the versatility and performance of the file format on live imaging data of dividing human cells. CellH5 provides new opportunities for integrated data analysis by multiple software platforms. Availability: Source code is freely available at www.github.com/cellh5 under the GPL license and at www.bioconductor.org/packages/release/bioc/html/rhdf5.html under the Artistic-2.0 license. Demo datasets and the CellH5Browser are available at www.cellh5.org. A Fiji importer for cellh5 will be released soon. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15–20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis

Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) [2]. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit

Profiling DNA damage response following mitotic perturbations

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes.This work was supported by the Novo Nordisk Foundation (NNF14CC0001 to J.L. and NNF12OC0002088 to C.L.), Danish Cancer Society (R72-A4436 to J.L.), the European Community 6th Framework Programme MitoCheck (LSHG-CT-2004-503464 to J.E.) and European Community 7th Framework Program MitoSys (241548 to J.E.).Peer Reviewe

The N2D+/N2H+ ratio as an evolutionary tracer of Class 0 protostars

Deuterated ions are abundant in cold (T=10 K), dense (n=10^5 cm^-3) regions, in which CO is frozen out onto dust grains. In such environments, the deuterium fractionation of such ions can exceed the elemental abundance ratio of D/H by a factor of 10^4. In this paper we use the deuterium fractionation to investigate the evolutionary state of Class 0 protostars. In a sample of 20 protostellar objects, we found a clear correlation between the N2D+/N2H+ ratio and evolutionary tracers. As expected, the coolest, i.e. the youngest, objects show the largest deuterium fractionation. Furthermore, we find that sources with a high N2D+/N2H+ ratio show clear indication for infall.Comment: 19 pages, 12 figures, accepted by A&

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