Held Michael

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Held
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Michael
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  • Preprint
    CellCognition : time-resolved phenotype annotation in high-throughput live cell imaging
    ( 2010-07) Held, Michael ; Schmitz, Michael H. A. ; Fischer, Bernd ; Walter, Thomas ; Neumann, Beate ; Olma, Michael H. ; Peter, Matthias ; Ellenberg, Jan ; Gerlich, Daniel W.
    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.
  • Preprint
    Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells
    ( 2010-07) Schmitz, Michael H. A. ; Held, Michael ; Janssens, Veerle ; Hutchins, James R. A. ; Hudecz, Otto ; Ivanova, Elitsa ; Goris, Jozef ; Trinkle-Mulcahy, Laura ; Lamond, Angus I. ; Poser, Ina ; Hyman, Anthony A. ; Mechtler, Karl ; Peters, Jan-Michael ; Gerlich, Daniel W.
    When vertebrate cells exit mitosis, they reorganize various cellular structures to build functional interphase cells1. This depends on Cdk1 inactivation and subsequent dephosphorylation of its substrates2-4. Members of PP1 and PP2A phosphatase families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5, 6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we used a live imaging assay to screen by RNAi a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identified a trimeric PP2A-B55α complex as a key factor for postmitotic reassembly of the nuclear envelope, the Golgi apparatus, and decondensed chromatin, as well as for mitotic spindle breakdown. Using a chemically-induced mitotic exit assay, we found that PP2A-B55α functions downstream of Cdk1 inactivation. PP2A-B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate histone H1 and it was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor Importin β1, and RNAi depletion of Importin β1 delayed mitotic exit synergistically with PP2A-B55α. This demonstrates that PP2A-B55α and Importin β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.