Yeast cells are well-known to undergo a developmental switch from nutrient-driven invasive-growth to pheromone-driven growth-arrest and mating. Previous investigations have highlighted the fact that these pathways share many, but not all, component proteins. However it remains unclear which pathway-specific components are required for signal fidelity as opposed to pathway-specific behaviors such as altered morphology or invasion. As an alternative approach, we investigated a distinct differentiation switch triggered by a common stimulus (). At high doses of pheromone, cells arrest in G1 and exhibit the characteristic shmoo morphology. At intermediate doses they grow slowly and appear elongated (). Given that these events require the same receptor and G protein, as well as many of the same effector kinases, this system provides an excellent opportunity to determine the function of pathway-specific components. Here we investigate the role of Ste5.
Initially we investigated dose-dependent responses to pheromone, using a variety of functional assays. Pheromone-induced transcription has been shown to exhibit a graded dose-response profile (Poritz et al., 2001
), whereas downstream processes such as G1 arrest and mating are switch-like. Switch-like behavior is an extreme example of ultrasensitivity, defined as any response with a Hill coefficient greater than one (Ferrell and Machleder, 1998
; Goldbeter and Koshland, 1981
). To determine if other aspects of the pheromone response exhibit ultrasensitivity, we monitored differentiation of cells exposed to a range of pheromone concentrations. Cells were classified into one of three categories: vegetative-growth, elongated-growth, or shmoo. Vegetative-growth occurs in the absence of pheromone. In this case cells divide rapidly and bud axially (new buds emerge from the daughter cell in the direction of the mother cell). At intermediate doses of pheromone the cells appear elongated, proliferate slowly, and divide in a bipolar fashion, opposite the mother cell (Dorer et al., 1995
; Erdman and Snyder, 2001
; Madden and Snyder, 1992
). At high doses of pheromone the cells arrest and form a shmoo. Each of these morphologies is illustrated in . As shown in , >85% of wild-type cells grow vegetatively at pheromone concentrations of 0.1 µM or less. At concentrations of 0.3 – 3 µM the population transitions from vegetative-growth to elongated-growth, even in the absence of a pheromone gradient. At 10 µM nearly all cells undergo growth-arrest and shmoo formation. The first transition occurs over a broader range of pheromone concentrations, as compared with the second transition. Thus the differentiation switch from elongated-growth to growth-arrest may exhibit ultrasensitivity.
Experimental measurements of yeast cell differentiation
We then investigated how the MAP kinases contribute to the two developmental transitions. It is well-established that mutants lacking Fus3 are unable to shmoo, but maintain the ability to transition from vegetative-growth to elongated-growth (, second row). Conversely, cells lacking Kss1 still undergo both transitions (, third row), but the dose-response profile for the transition from vegetative-growth to elongated-growth is significantly steeper than that of cells containing both MAP kinases or Kss1 alone. Signaling exclusively through Fus3 increases the pheromone concentration required for elongated-growth, thereby reducing the range of pheromone levels for which this behavior is observed. Kss1 broadens this range allowing cells to become elongated at lower doses of pheromone, as compared with cells that express both MAP kinases. Thus Kss1 and Fus3 confer distinct dose-response characteristics on cell growth: Kss1-mediated responses are graded while Fus3 responses appear ultrasensitive.
We next examined whether elongated-growth corresponds to chemotropic-growth, defined here as cell expansion and division in the direction of a stimulus gradient. It has been suggested that chemotropic-growth could allow yeast, which are otherwise non-motile, to orient new bud formation in the direction of a weak pheromone stimulus and thus towards a distant mating partner (Dorer et al., 1995
; Erdman and Snyder, 2001
; Madden and Snyder, 1992
). To this end we constructed a microfluidic chamber capable of exposing cells to a precisely-controlled linear concentration gradient. The gradient is achieved by passive diffusion between two parallel microchannels containing either no pheromone or a dose of pheromone sufficient to induce cell division arrest ( and Supplementary Materials
). There is no active flow within the growth chamber, thereby allowing the non-adherent cells to remain stationary during the course of the experiment. In this method the cells are at low density and the pheromone is constantly replenished, resulting in a lower dose-activity profile (see below).
As shown in , , as well as in Supplementary Fig. S1
and the accompanying movies (Supplementary Information
), cells within the growth chamber exhibit all three morphologies, depending on the local concentration of pheromone. Cells exposed to minimal doses of pheromone grow vegetatively and divide axially (Dorer et al., 1995
; Madden and Snyder, 1992
). Cells exposed to high doses of pheromone undergo cell division arrest and form a shmoo. At intermediate doses the cells appear elongated but continue to divide slowly and in the direction of increasing pheromone concentrations (). This chemotropic-growth response generally entails just one round of elongated-growth, after which the daughter cell detaches from the mother cell and forms a shmoo. This is in contrast to invasive-growth, where cells undergo several rounds of polarized budding to form a long filament. In wild-type cells the vast majority of new buds and cell projections emerge within +/− 60° of the gradient, and very few are outside +/− 90° (). Mutants lacking Fus3 or Kss1 retain the ability to undergo the elongated-growth response. Likewise, mutants lacking Kss1 are able to divide in the direction of the gradient, although Kss1 may contribute at very low doses of pheromone or in mutants that are supersensitive to pheromone () (Dorer et al., 1995
; Erdman and Snyder, 2001
; Paliwal et al., 2007
; Segall, 1993
). In cells lacking Fus3 however, newly formed buds emerge randomly with respect to the gradient and the cells no longer undergo growth-arrest and shmoo formation (). Thus Fus3 is specifically required for chemotropic-growth but is dispensable for elongated-growth.
Cellular responses mediated by Fus3 appear to be ultrasensitive, while responses mediated by Kss1 appear graded (). Measurements of cell morphology are somewhat qualitative, however, so we also measured changes that occur at a molecular level. Specifically, we asked if there is ultrasensitivity at the level of protein kinase activity. To this end we monitored Fus3 and Kss1 activation in cells treated with a range of pheromone concentrations, using antibodies that recognize the dually-phosphorylated (fully activated) form of each protein. As shown in and Fig. S2
the temporal-and dose-response profiles for Fus3 and Kss1 phosphorylation are distinct. At 1 µM pheromone (a dose sufficient to trigger chemotropic-growth but not shmoo formation) Fus3 is phosphorylated to 22% of maximum while Kss1 is phosphorylated to 48% of maximum. At 10 µM pheromone (a dose sufficient to trigger cell division arrest and shmoo formation) both kinases are maximally activated. Remarkably, we found that maximum kinase activity occurs at different times depending on the pheromone concentration; for example Fus3 activity peaks at 60 min in response to 10 µM pheromone whereas it peaks at 15 min in 1 µM pheromone (). Thus two different doses may produce identical kinase activity at a single (early) time point, but nevertheless exhibit dramatic differences in the duration and final maximum level of kinase activation. Accordingly, we quantified peak kinase activity () and area under the curve () for each dose of pheromone. The latter incorporates the elements of duration and activity into a single parameter. Both of these methods accurately account for the observed differences in activity, and yielded similar results. As shown in , the effective Hill coefficient for activation of Fus3 is substantially higher than for activation of Kss1 (nH
= 2.2 and 1.3, respectively). This difference mirrors the dose-response profiles for Fus3-and Kss1-mediated cell differentiation responses, as reported in . Note that we measured the MAP kinase activation of a population of cells. The response might be even more switch-like in individual cells.
Experimental and computational analysis of MAP kinase activation with or without Ste5
Fus3 and Kss1 are activated by the same upstream protein kinases, most immediately by the MAP kinase kinase Ste7. Nevertheless we have shown above that Fus3 and Kss1 exhibit very different temporal-and dose-dependent behaviors. Whereas Kss1 activity peaks quickly, Fus3 activity increases slowly at a constant rate (slope) that is independent of the pheromone level. In both cases, increasing the pheromone level delays the time at which MAP kinase activity begins to decline. One way to account for this unusual behavior is to assume that the predominant effect of increases in pheromone concentration is to prolong the duration
of Ste7 activity (). To address this possibility we constructed simple mathematical models of Fus3 and Kss1 activation and investigated each model’s response to varying durations of Ste7 activation. Both models assume that the phosphorylation and dephosphorylation reactions follow Michaelis-Menten kinetics. Each model further assumes that the νmax
for phosphorylation of the MAP kinase is proportional to the active Ste7 concentration. The difference between the models is that the rate constants describing phosphorylation and dephosphorylation of Kss1 are larger than those of Fus3 (Supplementary Materials
). shows the Ste7 activation profile used as the input signal for both MAP kinases (top panel), as well as the activity for Fus3 (middle panel) and Kss1 (bottom panel) predicted by the corresponding models. There is a very good qualitative agreement between these simulations () and the experimental data (). The models also demonstrate how a purely kinetic mechanism (slow activation) can be used to convert a graded response to one that is more switch-like (see Discussion
We then considered why Fus3 is activated more slowly than Kss1. This difference could be an inherent property of each kinase. Alternatively, slow activation of Fus3 could be due to binding of Ste5. Indeed, Ste5 was reported previously to diminish Fus3-mediated transcription responses (Bhattacharyya et al., 2006
). To investigate the contribution of Ste5 to Fus3 dynamics, we monitored kinase activation in the absence of Ste5-Fus3 interaction. Deletion of STE5
blocks signaling altogether, so as an alternative we used a mutant form of Ste5 in which Fus3 docking is disrupted (“non-docking” allele, Ste5ND
). This mutant binds poorly to Fus3 (Maeder et al., 2007
), yet produces an enhanced
transcription-induction response (Bhattacharyya et al., 2006
). Thus we investigated how Ste5ND
affects dose-and time-dependent signaling events including cell morphogenesis, gradient-sensing and kinase-activation. To eliminate any possibility that Kss1 can substitute for Fus3 or otherwise modulate Fus3 activity in the absence of Ste5, these experiments were conducted in a Kss1-deficient strain. As shown in , cells that express Ste5ND
are able to undergo the transitions from vegetative-growth to elongated-growth and then to growth-arrest. However, the transitions occur at lower pheromone concentrations and the dose-response profile is more graded than that seen in wild-type cells. Indeed we observed considerable overlap in the distribution of all three categories of cells within the gradient chamber. Moreover, cells that express Ste5ND
exhibit diminished elongation and orientation towards the source of pheromone (). Likewise, Fus3 becomes fully activated in the Ste5ND
strain, but activation occurs with faster kinetics (). Additionally, the Hill coefficient for activation of Fus3 is reduced from nH
=2.2 in wild-type cells to nH
=1.5 in Ste5ND
mutant cells, a value similar to that normally observed for Kss1 (nH
= 1.3) (). Thus under conditions where it is no longer modulated by Ste5, the dynamic and dose-dependent behavior of Fus3 resembles that of Kss1.
We also considered the possibility that Ste5ND
might affect the activity of other signaling components upstream of Fus3, such as Ste11 or Ste7. To test this we examined whether Ste5ND
alters the activity of Kss1, which likewise requires Ste11 and Ste7. In accordance with the model, Ste5ND
(in the absence of Fus3 expression) had no effect on Kss1 activation (Fig. S3
). Another upstream mechanism of regulation is pheromone degradation. The pheromone protease Bar1 is up-regulated upon prolonged pathway activation, and different degrees of induction by Fus3 and Kss1 might somehow underlie the differences observed for the two kinases. To address this concern, and as a further test of our computational model, we measured Fus3 and Kss1 activation in a bar1
Δ mutant strain (Fig. S4A
). As anticipated, differences in the dynamic and dose-dependent behaviors of Fus3 and Kss1 remain unchanged. Strikingly, by altering only the input signal profile the equations and parameters that govern kinase activation in wild-type cells could also be used to accurately describe kinase activation in the bar1
Δ mutant cells (Fig. S4B
Finally, we examined the physiological importance of the elongated-and chemotropic-growth behaviors in mating. Previous investigations employed the “pheromone-confusion” assay, which compares overall mating efficiency in the presence or absence of added pheromone. The underlying assumption is that exogenous pheromone will obscure natural pheromone gradients, and thereby diminish the ability of cells to detect a potential mating partner (Dorer et al., 1995
; Nern and Arkowitz, 1998
; Strickfaden and Pryciak, 2007
; Valtz et al., 1995
). One drawback to this method is that addition of excess exogenous pheromone is also likely to obscure or otherwise interfere with pheromone clearance by Bar1 protease, receptor occupancy, as well as adaptation/desensitization responses that further impinge on mating efficiency. As an alternative approach, we measured mating efficiency under conditions where potential mating partners are rare (10:1 ratio) and spatially segregated (plated on solid growth medium). As shown in , the Ste5ND
mutants mate substantially better than wild-type cells, presumably because they can better detect and respond to a distant mating partner. Conversely, Ste5ND
mutants mate more poorly under conditions where potential mating partners are more abundant (1:10 ratio) but still dispersed, presumably because they can no longer orient towards the closest of multiple potential mating partners. This reduction in mating efficiency occurs despite the increase
in pheromone sensitivity.
Physiological analysis of the role of Ste5 in mating
From these data we conclude that Ste5 confers the slow and ultrasensitive responses characteristic of Fus3. If Fus3 cannot bind to Ste5 the cells no longer undergo chemotropic growth and exhibit an altered ability to mate efficiently with partners at a distance.