title: Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.
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.
authors.affiliation: CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom;
authors.forename: Iain M
authors.initials: IM
authors.lastname: Hagan
authors.affiliation: CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom;
authors.forename: Agnes
authors.initials: A
authors.lastname: Grallert
authors.affiliation: EPFL SV ISREC UPSIM, SV2.1830, CH-1015 Lausanne, Switzerland.
authors.forename: Viesturs
authors.initials: V
authors.lastname: Simanis
mesh_terms: D003080:Cold Temperature; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011418:Prophase; D016196:S Phase; D012568:Schizosaccharomyces / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins; D014404:Tubulin