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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Hagan, Iain M. Grallert, Agnes Simanis, Viesturs |
| Description | Author Affiliation: Hagan IM ( CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom); Grallert A ( CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom); Simanis V ( EPFL SV ISREC UPSIM, SV2.1830, CH-1015 Lausanne, Switzerland.) |
| Abstract | Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive ß-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 ß-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of ß-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. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 8 |
| Journal | Cold Spring Harbor Protocols |
| Volume Number | 2016 |
| e-ISSN | 15596095 |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2016-08-01 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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