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Question: in fission yeast entry into mitosis is regulated by the...

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In fission yeast entry into mitosis is regulated by the phosphorylation status of Tyrsin-15 (=Y15) in Cdc2. This residue can be phosphorylated by the two kinases Wee1 and Mik1, and de-phosphorylated by the phosphatase Cdc25. Mitosis is triggered when the phosphatase activity of Cdc25 overcomes the kinase activity of Wee1/Mik1.
To determine if the checkpoint pathway inhibits mitosis by targeting Cdc2 Y15 phosphorylation, cells with a Cdc2-Y15F mutation were studied. These cell cells are viable but “wee” (small), because Cdc2 cannot be phosphorylated on F15. Y15F cells and wild type cells were synchronized in G2 by elutriation (a technique that allows isolation of the small newborn cells) and irradiated, and the kinetics of completion of mitosis was monitored (Figure 9-1). Unirradiated cells of both strains were included as controls.

YISF irradiated continuously 1-1 : 一ーwild type irradiated continuously 80 t9-wildtype unirradiaied 3 50 10 180 Minutes after elutriation 120 240 300

In order to investigate if Cdc25 is part of the checkpoint response, cells deleted for mik1 and carrying a temperature-sensitive mutation in wee1 (wee1-50 ∆mik1) were studied (Figure 9-2).  

wee1-60k 100 T 90 70 60 50 40 30Shilft!o 20 35 C 10 0 60 120 180 240 Minutes after elutriation Figure 2 G2 cells (grown at 25C) were isolated by elutriation and released at 35C (the restrictive temperature for wee1-50; to prevent new Y15 phosphorylation on Cdc2). The culture was divide in two, and one half was irradiated with gamma (+y), while the other was left untreated (-V), and the kinetics of completion of mitosis was monitored. As a negative control a weel-50 Δmik1 Achk1 strain was included. This strain has no functional checkpoint due to a deletion of the Chk1 kinase. QUESTION 9-5 What can you conclude from the observed mitotic delay in irradiated wee1-50 Amik1 cells?

In subsequent experiments it was shown that Chk1 can directly phosphorylate Cdc25, but this does not appear to affect the Cdc25 protein level (data not shown). Therefore, the subcellular localization of Cdc25 was investigated in the absence (-IR) or presences (+IR) of irradiation (Figure 9-3). Upper panel shows MYCtagged Cdc25 visualized by indirect immunofluorescence, while the lower panel shows DNA by DAPIstaining.
chk1+ Achk -IR +IR -IR +IR Myc12-Cdc25 DAP Figure 9-3 QUESTION 9-6 Describe the localization of Cdc25 in unirradiated and irradiated cells. What effect does checkpoint activation have on Cdc25 localization? When Cdc2 is activating mitosis, it is mainly located to the nucleus Explain how the observed dynamics of Cdc25 localization potentially could contribute to cell cycle arrest following checkpoint activation. QUESTION 9-7

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