Fig 1.
Simplified mechanism of cell cycle progression and START transition.
(A) Progression through the cell cycle in budding yeast. Cln3 and Bck2 are activators of START (turn on SBF, MBF needed for Cln1,2; Clb5,6). Cln1,2 phosphorylates and inhibits Sic1, a stoichiometric inhibitor of Clb5,6, thus allowing DNA replication to occur. S-phase cyclins, Clb5,6, inhibit Cdh1, an antagonist of the mitotic cyclins, Clb1,2, thus allowing progression through the mitotic events, and finally exit from mitosis leading back to G1. (B) The earlier hypothesis that Whi5 phosphorylation is crucial for SBF activation. This model for START includes (i) activation of SBF by Clns (Cln3, Cln1,2, Clb5,6) by inactivation of Whi5 (by phosphorylating free and SBF-bound Whi5) in late G1, (ii) activation of SBF by Bck2 to an alternate form independent of Whi5 and CDK, and (iii) inactivation of SBF by Clb1,2 in late S phase. (C) Earlier hypothesis on association, dissociation, and translocation events underlying SBF activation. Inactive SBF-Whi5 trimer gets phosphorylated by Cln/CDK on both Swi6 and Whi5, followed by the trimer dissociation to give active SBF (with Swi6 phosphorylated) and phosphorylated Whi5 that can get exported to the cytoplasm (by transport protein, Msn5).
Fig 2.
(A) The core model of SBF activation and inactivation by Clns and Clbs. Presented in the figure are the most important interactions considered in START-BYCC for SBF activation and inactivation. The core components are Swi4 (orange icon), Swi6 (turquoise icon), Whi5 (red icon), the promoter (purple bar), kinases (Cln3, Cln1,2, Clb5,6, Clb1,2) and export protein (Msn5) (white box). The nucleus is represented with a blue-gray background, while the white space corresponds to the cytoplasm. Promoter-bound complexes enclosed in boxes with borders in dark green, light green, and red represent complexes with maximal, residual, and no activity, respectively. White-filled circles represent activatory phosphorylations done by Cln3, Cln1,2, and Clb5,6; whereas the black-filled circles represent the inactivating phosphorylations by Clbs (Clb5,6 & Clb1,2 for Swi6 phosphorylation and Clb1,2 for Swi4 phosphorylation). The Cln (white) phosphorylation on Whi5 and Clb (black) phosphorylation on Swi6 (S160) are needed for export to the cytoplasm. To avoid overcrowding, remaining complexes corresponding to modifications on other free forms are not included in the figure. All the concerned equations are listed in S2 Text. Key facts about the abundance, regulation, and localization of all the components are described in S2 Table. (B) Ratios of the promoter to Swi4/Mbp1, Swi6, and Whi5 (Roughly based on Ghaemmaghami et al. [84]; e.g., when rescaled, we would have four promoters, 11 each of Swi4/Mbp1, 20 Whi5, and 60 Swi6 molecules).
Fig 3.
Other mechanisms involved in the START transition.
(A) Role of Bck2 in the activation of SBF. Bck2 acts on Swi4 and modifies it to a less active form (indicated by the enclosing light green box). The cartoon is a concise representation of regulation by Bck2 (intermediate steps not included). Black-filled circles with ‘B’ represent the Bck2-induced modification, and active forms are enclosed in a light green box (since these complexes are not as active as Cln-activated forms). Note that only promoter-bound forms are active. Clb2 causes complex inactivation (see equations in S2 Text). (B) MBF regulation. MBF alone is inactive in a repressed state (indicated by the enclosing dark red box). It is activated either by cyclins (Cln3, Cln1,2, Clb5,6) or by Bck2 (more active, as indicated by the dark green box). MBF is primarily inactivated by its transcriptional target, Nrm1, resulting in the negative feedback highlighted by the ‘–’ sign. Clb2 is a secondary and minor inhibitor of MBF.
Table 1.
Model Predictions and Validations.
(A more exhaustive list of experimentally determined phenotypes is shown in S5 Table). Single letter notations in column one denote: v: validated, p: predicted, and c: contradictory. Grey rows indicate parent groups of mutants, with variations that follow.
Fig 4.
Simulation of wildtype cells in glucose.
Each panel tracks the following proteins/components for ~3 cell cycles in glucose with mass doubling time 90 min and daughter cycle time ~107 min. Mass acts as a proxy for the growing and dividing cell; here we follow the daughter cells (~0.46 x total_size_at_division; see Equations in S2 Text). (A) Cyclins (Cln2, Clb5 (active), Clb2 (active)), (B) Cyclin antagonists (Cdh1, Sic1 (active), Cdc6 (active)), (C) Transcription factors (SBF, MBF, MCM1, Swi5), (D) Markers (CDK targets (BUD, ORI, SPN) used to indicate the occurrence of physiological events via concentration threshold (BUD = 1 (bud emergence), ORI = 1 (DNA synthesis initiation), SPN = 1 (spindle alignment in metaphase)), (E) Active SBF and MBF complexes. Each form shown (SBFa1-a5, MBFacln, MBFabck) is the activity of SBF or MBF contributed by a particular form. The complexes contributing to the different forms (all bound to promoter) are as follows: SBFa1 = unmodified SBF (SBFB in the model); SBFa2 = SBF phosphorylated on Swi6 (SBFB6P+SBFB6PQ); SBFa3 = SBF-Whi5 complex phosphorylated on Swi6 (WSB6P+WSB6PQ); SBFa4 = SBF-Whi5 complex phosphorylated on Whi5 (WSB5P); SBFa5 = Swi4dimers activated by Bck2 (Swi4B); MBFa = MBF activated by Clns or Bck2. Check and cross marks denote the presence or absence (due to mutation) of specific complexes. The absence of any sign denotes that the specified complex is absent in the simulation of that strain. The black curve in all panels denotes mass (corresponding to exponential cell growth and division). As shown in (E), for wildtype cells, the dominant form of SBF is SBFa2, the form of SBF phosphorylated on Swi6, whereas the dominant form of MBF is the Cln-activated MBF.
Fig 5.
Cell size as a function of growth rate in budding yeast.
The blue curve connecting red circles shows cell size (mass) at division at specific mass doubling times (MDT) from 90–300 min (30 min interval) and corresponding specific growth rates (ln2/MDT) from START-BYCC simulations. This curve shows the same trend as the experimental graph in Lord and Wheals (1980), where median cell volumes for populations are plotted at different growth rates.
Fig 6.
Simulation of the timing of localization (export) of different monomers.
The cytoplasmic fraction of monomers Swi4, Swi6 and Whi5 are plotted against time. The bar at the bottom of the graph shows timing w.r.t. phases of the cell cycle. In the model, the onset of S and M phases correspond to DNA synthesis (ORI = 1) and spindle assembly checkpoint (SPN = 1), respectively. In compliance with experiments, Whi5 enters the cytoplasm (exits the nucleus) in late G1, followed by Swi6 in S-phase. Both Swi6 and Whi5 are cytoplasmic until mitotic exit. Swi4 is mostly nuclear at all times. Details are discussed in the main text.
Fig 7.
Simulation of non-phosphorylable mutants.
The simulations shown here track the cell size and key active SBF/MBF fractions, in specific non-phosphorylable START mutants, and the descriptions are as in Fig 4. (A) WHI5-12A (SBF-Whi5 complex phosphorylated on Swi6 and MBF are the primary active forms–SBFa3, MBFa; cells are ~WT size), (B) SWI6-SA4 (SBF activated by Bck2, SBF-Whi5 complex phosphorylated on Whi5, and MBF are the primary active forms–SBFa4, MBFa; cells are ~WT size), (C) WHI5-12A SWI6-SA4 (only very little of Bck2 activated MBF forms are present–MBFa; cells are larger than WT).
Fig 8.
Simulation results of a few important START mutants.
Simulations of the following mutants and their steady state sizes (viability/inviability) and the active SBF/MBF complexes present are shown: (A) cln3Δ (only Bck2 activated forms are present; cells are very large), (B) bck2Δ (only Cln-activated forms are present; cells are slightly larger than WT), (C) swi6Δ (only Swi4dimers (SBFa5) present; cells are viable yet large), (D) swi4Δ swi6Δ (no SBF/MBF; cells are inviable), and (E) cln3Δ swi6Δ (only Swi4dimers (SBFa5) present; cells are viable yet large), (F) bck2Δ swi6Δ (no active SBF/MBF; cells are inviable), (G) swi4Δ (only MBF present; cells are very large), (H) mbp1Δ (only SBF present; cells are slightly larger than WT), (I) swi4Δ mbp1Δ (no SBF or MBF; cells are inviable), (J) GAL-WHI5-12A SWI6-SA4 (excess non-phosphorylable Whi5 inhibits MBF; cells are inviable), (K) whi5Δ (cells begin the cycle with less active SBF (SBFa1) instead of Whi5-bound inactive SBF, and get converted into more active forms by Clns (SBFa3). Active MBF is also present. Hence, cells are smaller than WT.), (L) bck2Δ cln3Δ (no active SBF/MBF; cells are inviable), (M) bck2Δ cln3Δ whi5Δ (deletion of Whi5 relieves SBF and the unmodified form of SBF (SBFa1) is consistently present and cells become viable), (N) CLN3-1 swi6Δ (only Swi4dimers (SBFa5) present; cells are viable yet large), (O) bck2Δ swi6Δ-SA4 (SBFa1, SBFa4, and MBFa present; cells are slightly larger than WT).