Cdc14 Localization as a Marker for Mitotic Exit: In Vivo Quantitative Analysis of Cdc14 Release.
To complete cell division and to exit from mitosis into the next G1 phase, eukaryotic cells need to inactivate the cyclin-dependent kinase (Cdk) and reverse Cdk-phosphorylation events. In budding yeast mitotic exit depends on the phosphatase Cdc14. During the majority of the cell cycle Cdc14 is sequestered and kept inactive in the nucleolus. Activation of Cdc14 at anaphase onset coincides with its release from the nucleolus into the nucleus and subsequently into the cytoplasm. Here we describe a microscopy method, originally developed in the laboratory of Frederick Cross (Lu and Cross, Cell 141:268-279, 2010), that allows quantifying Cdc14 release in live cells using the open source software FIJI. We adapted this method and show that it is able to distinguish between Cdc14 activation defects caused by mutations in the "cdcFourteen Early Anaphase Release"-(FEAR) and the mitotic exit network (MEN) using slk19∆ and cdc15-1 mutant strains.
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona, 08003, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, 08003, Spain.|Massachusetts Institute of Technology (MIT), 500 Main Street, Cambridge, MA, 02139, USA.
Gabriel
G
Neurohr
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona, 08003, Spain. manuel.mendoza@crg.es.|Universitat Pompeu Fabra (UPF), Barcelona, 08003, Spain. manuel.mendoza@crg.es.
Manuel
M
Mendoza
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*; D008938:Mitosis; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D012984:Software
C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D017027:Protein Tyrosine Phosphatases
Anaphase; Budding yeast; Cdc14; FEAR; Live cell microscopy; MEN