64 research outputs found
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 Communityâs 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 âSFB Chromosome Dynamicsâ, 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 MĂŒller fellowship.2014-04-0
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
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
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
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
The Cul3âKLHL21 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
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 Commissionâs 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
Tubulin polyglutamylation stimulates spastin-mediated microtubule severing
Microtubules with long polyglutamylated C-terminal tails are more prone to severing by spastin, establishing the importance of tubulin posttranslational modifications
Molecular excitation in the Interstellar Medium: recent advances in collisional, radiative and chemical processes
We review the different excitation processes in the interstellar mediumComment: Accepted in Chem. Re
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