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    Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.

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    Authors
    Hagan, Iain M
    Grallert, Agnes
    Simanis, V
    Affiliation
    CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX,
    Issue Date
    2016
    
    Metadata
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    Abstract
    Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive β-tubulin mutation was optimized by Mitsuhiro Yanagida's laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of β-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of β-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase.
    Citation
    Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release. 2016, 2016 (8):pdb.prot091256 Cold Spring Harb Protoc
    Journal
    Cold Spring Harbor Protocols
    URI
    http://hdl.handle.net/10541/619920
    DOI
    10.1101/pdb.prot091256
    PubMed ID
    27480719
    Type
    Article
    Language
    en
    ISSN
    1559-6095
    ae974a485f413a2113503eed53cd6c53
    10.1101/pdb.prot091256
    Scopus Count
    Collections
    All Paterson Institute for Cancer Research

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