Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.
Hagan, Iain M Grallert, Agnes Simanis, V
Hagan, Iain M
Grallert, Agnes
Simanis, V
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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.
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2016
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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