Answers to Problem Set 6

1.- A. The overall length of the cell cycle is equivalent to the time it takes for the entire population of cells to double in number. To find the length of the cell cycle, select any two points on the graph in Figure 1 between which the number of cells has doubled. The time separating those two points is the length of the cell cycle. For example, the first two data points in Fig. 1 are 3 x 10⁵ cells (10 hours) and 6 x 10⁵ cells (30 hours). Since the population of mouse L cells doubled in 20 hours (30-10), the length of the cell cycle is 20 hours.

B. In outline, the length of G₂ can be derived from the data in Figure 2A, the length of S from the data in Figure 2B, and the length of G₁, from the overall length of the cell cycle (A) minus (M+S+G₂).

³H-thymidine is incorporated only during S phase. Thus no label will be present in cells undergoing mitosis until the cells that were at the very tail end of S phase when the label was added have traversed the G₂ portion of the cell cycle. The appearance of the first labeled mitotic cells at 3 hours after ³H-thymidine addition (Fig 2A) suggests that G₂ is 3 hours-long. The majority of mitotic cells, however, did not become labeled until 4 hours after addition of label (and a few did not become labeled until 5 hours after addition of label). This variation suggests that there is some variability in the length of G₂; if the length of G₂ were precisely defined, 100% of mitoses would be labeled when labeling was first observed. Thus, the length of G₂ in mouse L cells is 3 to 4 hours.

The length of S phase can be deduced from the number of silver grains over labeled mitotic cells (Fig 2B). Cells that were at the very end of S phase when label was added will have incorporated very little ³H-thymidine and thus will have very few silver grains, whereas cells that were at the beginning of S phase will have incorporated much more label and thus will have many more silver grains. The important realization is that cells at the beginning of S phase and cells that were in G₁ will have the same number of silver grains, since they incorporated label for the same length of time (that is, throughout S phase). Thus the beginning of S phase is the point in Fig 2B at which the number of silver grains per labeled cell reaches a plateau, which is about 10 hours before mitosis. Since G₂ is about 3 hours-long, S phase must be about 7 hours long.

Given that M phase is 1 hour, S phase is 7 hours, G₂ is 3 to 4 hours, and the overall length of the cell cycle is 20 hours, G₁ must be 8 to 9 hours-long.

2.- A. The state of phosphorylation of Wee1 and Cdc25 is the result of the balance between the protein kinase and the protein phosphatase activities that regulate them. By inhibiting the protein phosphatases, okadaic acid causes Wee1 and Cdc25 to accumulate in their phosphorylated forms as shown in the figure below. Since this change activates MPF, Wee1 and Cdc25 must have originally been present in the extract in their nonphosphorylated forms. Thus active Wee1 kinase is nonphosphorylated as is inactive Cdc25 phosphatase. Knowing which forms are phosphorylated allows you to label the arrows that correspond to the kinases and phosphatases that control Wee1 and Cdc25 phosphorylation (figure below).

B. The protein kinases and phosphatases that control phosphorylation of Wee1 and Cdc25 must be specific for serine/threonine residues because they are affected by okadaic acid, which is specific for serine/threonine phosphatases.

C. Okadaic acid has no effect on Cdc2 phosphorylation because it is phosphorylated on a tyrosine residue. Tyrosine phosphatases are unaffected by okadaic acid. The decrease in Cdc2 phosphorylation is a consequence of the change in activation of Wee1 kinase and Cdc25 phosphatase.

D. As soon as some active MPF appears it would begin to phosphorylate Wee1 and Cdc25, inactivating the kinase and activating the phosphatase. The resultant decrease in Wee1 kinase activity and increase in Cdc25 phosphatase activity would lead to dephosphorylation (and activation) of more MPF. This in turn would further decrease the activity of Wee1 kinase and further increase the activity of Cdc25 phosphatase, leading to still more MPF activity. Thus the initial appearance of a little MPF activity would rapidly lead to its complete activation.

This sort of activation is referred to as a positive feedback loop. It is a common means of regulation when it is advantageous for a system to flip rapidly from one state to another without lingering in the intermediate steps.

3.- A. Several of the experiments in Table 1 indicate that the stimulated binding due to the anti-b1 antibody occurs by the interaction of fibronectin with a5b1 integrin. The antibody the a5 chain blocks fibronectin binding in the presence or absence of the anti-b1 antibody. Anti-fibronectin antibody interferes with anti-b1 stimulated binding. And most conclusively, a peptide containing the RGD motif (through which integrins bind fibronectin) blocks the stimulated binding, whereas a similar peptide without the complete RGD motif has no effect.

B. Apparently, the anti-b1 antibody by binding to a specific site on the b1 chain induces a conformational change in the a5b1 integrin that flips it into its high-affinity state.