Show simple item record

dc.contributor.authorHagan, Iain M
dc.contributor.authorGrallert, Agnes
dc.contributor.authorSimanis, V
dc.date.accessioned2016-10-04T15:10:43Z
dc.date.available2016-10-04T15:10:43Z
dc.date.issued2016
dc.identifier.citationSynchronizing 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 Protocen
dc.identifier.issn1559-6095
dc.identifier.pmid27480719
dc.identifier.doi10.1101/pdb.prot091256
dc.identifier.urihttp://hdl.handle.net/10541/619920
dc.description.abstractHere, 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.
dc.language.isoenen
dc.rightsArchived with thanks to Cold Spring Harbor protocolsen
dc.titleSynchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.en
dc.typeArticleen
dc.contributor.departmentCRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX,en
dc.identifier.journalCold Spring Harbor Protocolsen
html.description.abstractHere, 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.


This item appears in the following Collection(s)

Show simple item record