Figure 1.
A minimal model for SIN asymmetry establishment.
(A) Direct antagonistic interactions between molecule X and Y at the two SPBs. Both molecules induce the removal of the other from the SPB they are both bound. Solid lines are transitions, dashed arrows show catalytic effects. (B) A less than 0.1% difference in the SPB binding rates or in initial conditions (not shown) can induce quick asymmetry establishment. Solid lines for molecules at old SPB, dashed lines for molecules at new SPB, time is in arbitrary units. (C) The proposed antagonistic double-negative ( = positive) feedback between SIN components and Cdc16-Byr4. (D) Merging ideas from panels A and C to create a minimal molecular model of asymmetry establishment.
Figure 2.
Behavior of the minimal molecular model of SIN asymmetry establishment.
(A) A small bias in Byr4 binding to SPB is enough to establish asymmetry from an initial condition corresponding to metaphase-anaphase transition. Solid lines for molecules at old SPB, dashed lines for molecules at new SPB, time in arbitrary units. (B–E) Timing of transition (reaching the inflection point in the SINNew curve) greatly depends on total level of each of the investigated proteins (plotted on a log2 scale). In each plot the basal (wild type) parameter is normalized to 1 (dashed lines) and the final phenotype of the effect of increase and decrease are noted with the multinucleate and multiseptate S. pombe cartoons. SIN level cannot be varied in either direction (A), Byr4 cannot be increased, while major reduction has also a deleterious effect. (B) Cdc11 and SIP can be changed also in small regimes (C,D). The observed multiseptate phenotype at reduced Cdc11 levels might come from the fact that we start simulations with an initial mitotic high SIN state, which might not be even reached in this mutant, while the multinucleate phenotype of Cdc11 overexpression contradicts literature data [10], [34].
Figure 3.
Model expansion on Cdc11 regulation.
The minimal model was extended by multiple phosphorylation forms of Cdc11 (A). It can be phosphorylated by SIN (green), Cdk (light blue) and both. The Cdk sites are assumed to be dephosphorylated by the unknown phosphatase “ppC”, while the SIN sites are dephosphorylated by an unknown phosphatase, “ppS”. (B) Simulation time course of SIN and Byr4 activities at the two SPBs (solid for Old, dashed for New). (C) Changes in the various phosphorylated forms of Cdc11. Notations and color code on forms on panel A.
Figure 4.
Sensitivity of asymmetry establishment timing on Cdc11 modification efficiencies.
Efficiencies of SIN and Cdk phosphorylation (A) and ppC and ppS dephosphorylation (B) on the time it takes to reach asymmetry in SIN activity (inflection point in Byr4Old curve). Small decrease in SIN efficiency on Cdc11 phosphorylation advances asymmetry (this is what was observed for the cdc11-S5A mutant, noted with a green arrow), while major decrease in this efficiency delays the transitions and eventually leads to high Byr4 (∼multinucleate) phenotype. Increase in this efficiency leads to SIN hyperactivation (∼multisetpate) phenotype. Decrease in Cdk efficiency has no major effect on asymmetry, but increase in this delays the transition and can lead to SIN hyperactivation. Increase in ppC seems to have no effect on asymmetry timing, while increase in ppS can lead to Byr4 hyperactivation. All wild type parameter values are normalized to 1, thus horizontal dotted lines show the wild type timing of asymmetry establishment.
Figure 5.
Predictions and experimental tests on collective effects of multiple mutations on SIN asymmetry establishment timing.
(A–C) Simulations of interactions of cdc16ts (A) and cdc16ts sip− (C) mutations with mutations in cdc11 phosphorylation sites. Reduced level of Cdc16 activity was simulated by the indicated reduction in Byr4 efficiency on SIN inactivation. Mutations in SIP was captured by 25% reduction in both ppC and ppS efficiency. As shown on Fig. 4 we assume that 50% SIN efficiency corresponds to the cdc11-S5A mutation. Time courses of Byr4 level changes at the old SPB are plotted as a representative proxy of SIN asymmetry establishment (other variables follow this as on Fig. 3). (B–D) Spot assays: The indicated cultures were serially diluted and spotted on YES agar medium, and grown at the specified temperatures. (B) At 32°C cdc11-S8A can partially compensate effects of the temperature sensitive cdc16-116 mutation, while cdc11-S5A makes it even more severe. (D) cdc11-S5A decreases while cdc11-S8A minimally increases viability of cdc16ts sip− mutants. (E) Phenotypes observed in the colonies of panel D at 25°C. n>300 cells for each strain.
Figure 6.
Simulations of the most peculiar observations in SIN asymmetry establishment.
(A) We simulated the laser ablation of the new SPB after anaphase (top), what leads to SIN activation at the old SPB [42]. At 200 time steps (horizontal dotted line) we stopped transport towards the new SPB and let all its content diffuse into the cytoplasm. (B) Simulation of the termination of SIN activity. At 200 time steps we induced the production (or reduced degradation) of new Byr4 molecules (as a proxy for the unknown signal that turns off SIN). At the same time we cut the communication between the two SPBs as it happens at the end of cytokinesis (“separated”, lighter color curves) or let the two SPBs communicate through the cytoplasm as it happens in some dikarions [43] (Non-separated, darker color lines and dots on top panel). If the cells are separated the newly formed Byr4 goes to the only existing new SPB, while if the cells did not separate it will be constantly recruited to the old SPB, thus SIN at the new SPB will turn off much later. (C) Seesaw metaphors of the two cases of panel B (seesaws are common examples of antagonistic interactions with two opposing steady states). The right arm of the seesaws represent SIN activity at the two SPBs, and they are connected to each other (water can flow between them in the metaphor - molecules can diffuse between SPBs in cells). The situation where the active and inactive SPBs are separated is captured on the left, where both SPBs are active, water is poured in (signals induce SIN inactivation) they both can turn together. On the right (non-separated active and inactive SPB) one SPB has high SIN, the other has low SIN. When water is poured in, first it flows to the lower (already inactive SIN) bucket and the upper seesaw will turn only if the lower bucket and the pipe are full.