Effects of Cdc5 kinase and of Cdc14 and PP2A phosphatase activities on Cdc14 re-sequestration.

<p>A stabilized version of Cdc5 (3×<i>CDC5ΔN70</i>) causes a delay in Cdc14 re-sequestration both in wild-type cells (panel A) and <i>bub2Δ</i> background (panel B). (<b>A</b>) Cdc20 block-and-release was pre-simulated for 180 min with no degradation of Cdc5 (<i>k</i>_d,polo′ = 0, <i>k</i>_s,polo = 0.011). (<b>B</b>) Simulation was done as in A with the rates of inactivation of Tem1 set to zero (<i>k</i>_i,tem′ = <i>k</i>_i,tem″ = 0). (<b>C</b>) Cdc14 stays released from the nucleolus in <i>cdc14-1</i> cells arrested in telophase. Cdc20 block-and-release was presimulated with no Cdc14 activity (<i>effc14</i> = 0). (<b>D</b>, <b>E</b> and <b>F</b>) Cdc20 block-and-release in wild-type cells; after 24 min (when cells start to enter G1 phase), either PP2A activity (panel D, <i>effpa</i> = 0) or Cdc14 activity (panel E, <i>effc14</i> = 0) was inhibited. In either case, Cdc14 is re-sequestered into the nucleoulus. In panel F, when both PP2A and Cdc14 phosphatase activities are inhibited after 24 min (<i>effpa</i> = <i>effc14</i> = 0), Cdc14 does not return to the nucleolus.</p

<i>clb2Δ</i> and <i>bub2Δ cdh1Δ</i> mutants.

<p>(<b>A</b>) When Clb2 is inhibited Cdc14 is released with a delay. Simulation was started at metaphase Cdc20 block for 80 min with rate of synthesis of Clb2 in the model decreased to 1/3 of baseline due to residual Clb1 activity (<i>k</i>_s,b2 = 0.1) and Cdc20 was added back at time zero. (<b>B</b>) In <i>bub2Δ cdh1Δ</i> cells, Cdc14 stays released after ME. Simulation was done as wild-type cells except that rates of inactivation of Tem1 and total concentration of Cdh1 were set to zero (<i>k</i>_i,tem′ = <i>k</i>_i,tem″ = <i>CDH1T</i> = 0).</p

Flux diagrams in wild-type cells.

<p>Initially cells are in the metaphase steady state by Cdc20 deprivation. Cdc20 activation at time zero (<i>k</i>_s,20 = 0.015) induces mitotic progression through anaphase, telophase and G1. Flux definitions are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030810#pone.0030810.s001" target="_blank">Table S1</a>.</p

Mitotic progression of cells containing an <i>ESP1</i> mutation.

<p>(<b>A</b>) In metaphase arrested cells at 23°C, overexpression of Esp1 induces Cdc14 release; however, cells do not exit from mitosis, and Cdh1 stays inactive. Cells are presimulated in metaphase arrest by Cdc20 deprivation, then at <i>t</i> = 0 the rate of synthesis of Esp1 is increased 60-fold (<i>k</i>_s,esp = 0.078), with the rate of Clb2 degradation at 23°C assumed to be half its basal value (<i>k</i>_d,b2″ = 1.5). (<b>B</b>) Cdc14 release is dependent upon Cdc5 in nocodazole-arrested cells; when <i>CDC5</i> is deleted, overexpressed Esp1 can no longer induce Cdc14 release. Cells are presimulated in metaphase arrest by nocodazole (<i>N</i> = 1) with no synthesis of Cdc5 (<i>k</i>_s,polo = 0) and no initial Cdc5 proteins. Then at <i>t</i> = 0 the rate of synthesis of Esp1 is increased 60-fold (<i>k</i>_s,esp = 0.078). (<b>C</b>) When <i>CLB5</i> is deleted, overexpressed Esp1 can induce ME. Reduction in Cdk activity by Clb5 deprivation allows for ME by increasing the phosphatase-to-kinase ratio, leading to activation of Cdh1. Simulation was done as in panel A, except that the synthesis rate of Clb2 was set to 80% of its basal value (<i>k</i>_s,b2 = 0.024). (<b>D</b>) When Esp1 is inactive, Cdc14 cannot be released and the cell cannot exit from mitosis. It is assumed that separase is absent in <i>esp1-2^td</i> mutant cells (<i>k_s,esp</i> = 0). During the 120 min pre-simulation of Cdc20 block in metaphase, the rate of degradation of Esp1 was increased 10-fold, and the activity of Esp1 was lowered 10-fold. (<i>effesp</i> = 0.1, <i>k_d,esp</i> = 0.028,). At <i>t</i> = 0, Cdc20 synthesis was induced, as usual.</p

Model predicts that Cdc14 is responsible for its own re-sequestration after ME.

<p>(<b>A–C</b>) All simulations were done similar to <i>cdc15-2</i> mutant simulations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030810#pone-0030810-g005" target="_blank">Figure 5A</a> except that after 20 min either Cdc14 (in A, <i>effc14</i> = 0) or PP2A (in B, <i>effppa</i> = 0) or both (in C, <i>effc14</i> = <i>effppa</i> = 0)) were inactivated by setting their corresponding activity factors to zero.</p

Flux diagrams and temporal changes of RENT, Net1 forms in <i>NET1-6cdk</i> mutants.

<p>Simulation was done similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030810#pone-0030810-g005" target="_blank">Figure 5C</a>.</p

Simulation of mitotic progression of cells containing overexpressed <i>CDC5</i> and inactive <i>cdc5</i> mutations.

<p>(<b>A</b>) Cdc5 is necessary for ME. Cdc20 block-and-release was simulated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030810#pone-0030810-g002" target="_blank">Figure 2</a> with inactive Cdc5 (<i>cdc5-as1</i>; <i>effpol</i> = 0). Cdc14 is not released, nor is Cdh1 activated. (<b>B</b>) The MEN requirement for ME can be bypassed by overexpressed Cdc5. Cdc20 block-and-release was simulated as usual, with inactive Cdc15 (<i>cdc15-2</i>; <i>effc15</i> = 0) and with Cdc5 overexpressed 30-fold (<i>GAL-CDC5</i>; <i>k</i>_s,polo = 0.3). (<b>C</b>) Overexpressed Cdc5 is sufficient for Cdc14 release when FEAR and MEN are inactive. Simulation was started in an arrested steady state with initial conditions of Clb2 and Polo were set less than metaphase values to represent an earlier stage of the arrest by hydroxyurea (Clb2 = 0.8, Polo = 0.6, Poloi = 0.2, <i>k</i>_s,b2 = 0.024, <i>k</i>_s,polo = 0.006) and with inactive Cdc15 (<i>effc15</i> = 0) for 15 min. Then Cdc5 and Pds1 overexpressions were induced at time zero (<i>k</i>_s,polo = 0.3, <i>k</i>_s,pds = 0.45, <i>k</i>_d,pds′ = 0). (<b>D</b>) The Cdc5 requirement for Cdc14 release and ME can be bypassed by overexpression of a truncated version of Cdc15. Cdc20 block-and-release was pre-simulated for 60 min with no synthesis of either Cdc20 or Cdc5 (<i>k</i>_s,polo = <i>k</i>_s,20 = 0; setting also the initial conditions for Cdc5 active and inactive forms to zero) while the total concentration of Cdc15 was increased 20-fold and inhibition of Cdc15 by Cdk was reduced 1000-fold (<i>k</i>_i,c15′ = 0.00009, <i>CDC15T</i> = 20). At <i>t</i> = 0, Cdc20 synthesis is induced as usual (<i>k</i>_s,20 = 0.015). (<b>E</b>) Cdc14 is not released in <i>cdc5-1</i> and <i>cdc5-1 cdc14-1</i> cells in E and F. Therefore, Cdc14 release in the <i>cdc14-1</i> mutant may be attributable solely to Net1 phosphorylation by Cdc5. Simulation in E was done similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030810#pone-0030810-g004" target="_blank">Figure 4A</a> except that <i>effpol</i> was set to 0.1 for the small residual activity of Cdc5. (<b>F</b>) Simulation in F was done similar to A except that activity of Cdc14 was set to zero (<i>effc14</i> = 0).</p

Temporal changes of RENT, Net1 forms and fluxes in <i>cdc15-2</i> cells blocked at telophase in mitosis.

<p>Simulation was done similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030810#pone-0030810-g005" target="_blank">Figure 5A</a>.</p

Inactive Clb2 is not required, whereas Polo inactivation is sufficient for Cdc14 re-sequestration to the nucleolus.

<p>(<b>A</b>) Cells of the triple-deletion strain <i>cdc20Δ pds1Δ cdh1Δ</i> arrest in telophase with Cdc14 released from the nucleolus. Simulation was done by setting to zero the rate of synthesis of Pds1, the total concentration of Cdh1, and the initial conditions of Cdh1, Pds1 and PE complex (<i>k</i>_s,pds = <i>CDH1T</i> = 0). (<b>B</b>) After 6 hours of telophase arrest, <i>cdc20Δ pds1Δ cdh1Δ</i> cells are subjected to Sic1 overexpression, and Cdc14 does not completely return to the nucleolus. Simulation was done as in panel A; after 6 hours <i>INH</i> was set to 5 to implement Cdk inhibition by Sic1. (<b>C</b>) In <i>cdc20Δ pds1Δ cdh1Δ</i> cells arrested in telophase, deprivation of Cdc5 causes return of Cdc14 to the nucleolus. Simulation was done as in panel A; after 40 min the rate of synthesis of Cdc5 was set to zero and the basal degradation rate of Cdc5 was increased 10-fold (<i>k</i>_s,polo = 0, <i>k</i>_d,polo = 0.1).</p

Mitotic progression of cells containing <i>CDC55</i>, <i>CLB2</i> and <i>BUB2</i> mutations.

<p>(<b>A</b>) In the <i>CDC55</i> deletion strain, Cdc14 is re-sequestered with a delay. Cdc20 block-and-release was simulated as usual, with [PP2A]_total = 0. (<b>B</b>) Cdc14 is released prematurely in <i>bub2Δ</i> cells. Cdc20 block-and-release was pre-simulated with the rates of Tem1 inactivation by Cdc14 and PP2A set to zero (<i>k</i>_i,tem′ = <i>k</i>_i,tem″ = 0). (<b>C</b>) In a nocodazole-arrested cell, overexpression of Clb2 induces Cdc14 release, and the cell arrests in telophase. Pre-simulation was done by setting <i>N</i> = 1 for 15 min. At <i>t</i> = 0, the rate of synthesis of Clb2 was increased 20-fold and the rates of degradation of Clb2 were set to zero (<i>N</i> = 1, <i>k</i>_d,b2′ = <i>k</i>_d,b2″ = 0, <i>k</i>_s,b2 = 0.6). (<b>D</b>) Cdc14 is not released when Clb2 is overexpressed in G1 cells with Cdc5 inactive. This simulation was started from G1 initial conditions (low levels of Clb2, Cdc5 and Cdc14). At <i>t</i> = 0, the initial condition of Polo is set to 0.01, the synthesis rate of Clb2 is set to a large value and its degradation rate is set to zero (<i>k</i>_d,b2′ = <i>k</i>_d,b2″ = <i>k</i>_s,polo = 0, <i>k</i>_s,b2 = 0.6).</p