The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit.
In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.
Molk
Jeffrey N
JN
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Schuyler
Scott C
SC
Liu
Jenny Y
JY
Evans
James G
JG
Salmon
E D
ED
Pellman
David
D
Bloom
Kerry
K
D000705:Anaphase; D018797:Cell Cycle Proteins / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D020559:Monomeric GTP-Binding Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors
C070351:BUB2 protein, S cerevisiae; C082279:CDC15 protein; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; C089357:LTE1 protein, S cerevisiae; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C089965:TEM1 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins