Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies.
Most proteins of the Mitotic Exit Network (MEN) and their upstream regulators localize at spindle pole bodies (SPBs) at least in some stages of the cell cycle. Studying the SPB localization of MEN factors has been extremely useful to elucidate their biological roles, organize them in a hierarchical pathway, and define their dynamics under different conditions.Recruitment to SPBs of the small GTPase Tem1 and the downstream kinases Cdc15 and Mob1/Dbf2 is thought to be essential for Cdc14 activation and mitotic exit, while that of the upstream Tem1 regulators (the Kin4 kinase and the GTPase activating protein Bub2-Bfa1) is important for MEN inhibition upon spindle mispositioning. Here, we describe the detailed fluorescence microscopy procedures that we use in our lab to analyze the localization at SPBs of Mitotic Exit Network (MEN) components tagged with GFP or HA epitopes.
Centre de Recherche en Biologie Cellulaire de Montpellier (CRBM), CNRS, 1919 Route de Mende, Montpellier, 34293, France.|LPCV, iRTSV, CEA Grenoble, 17 Rue des martyrs, Grenoble, 38054, France.
Ilaria
I
Scarfone
Centre de Recherche en Biologie Cellulaire de Montpellier (CRBM), CNRS, 1919 Route de Mende, Montpellier, 34293, France. simonetta.piatti@crbm.cnrs.fr.
Simonetta
S
Piatti
D018797:Cell Cycle Proteins / Q000032:analysis*; D005404:Fixatives; D005455:Fluorescent Antibody Technique / Q000379:methods; D005656:Fungal Proteins / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis; D059566:M Phase Cell Cycle Checkpoints; D008856:Microscopy, Fluorescence / Q000379:methods*; D008938:Mitosis; D064048:Spindle Pole Bodies / Q000648:ultrastructure*; D015003:Yeasts / Q000166:cytology* / Q000648:ultrastructure
C082279:CDC15 protein; D018797:Cell Cycle Proteins; D005404:Fixatives; D005656:Fungal Proteins; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins
Fluorescence microscopy; Indirect immunofluorescence; Mitotic exit network; Protein tagging; Spindle pole body