Fast and slow waves of cell death are associated with ROS accumulation but not chromatin fragmentation. (A) ROS accumulation was measured in populations of wild-type (WT) and CN-deficient (cnb1 mutant) cells that contain or lack cytochrome c (−CYC) at various times after treatment with 60 μM α-factor. (B) Mutants lacking either cyclophilin D (cpr3), metacaspase (mca1), mitochondrial fission factors (fis1, dnm1, mdv1), cytochrome c (cyc1 cyc7), cytochrome-c heme lyase (cyc3), coenzyme-Q (coq1), complex-IV (cox6), complex-V (atp2), cell wall degradation factors (fus1, fus2, rvs161), low-affinity Ca²⁺ influx (fig1), membrane fusion factor (prm1), Rho-GAP of the CWI signaling pathway (lrg1), or factors involved in pheromone signaling (fus3, far1, bni1) were treated with 60 μM α-factor for 10 h in the presence or absence of CN inhibitor (2.5 μM FK506) and assayed for cell death. Wild-type cells treated with inhibitors of complex-III (1 μg/ml antimycin A; AM) or complex-V (1 μg/ml oligomycin; OM) were also analyzed. Fast cell death was estimated as the frequency of cell death occurring in the absence of FK506. Slow cell death was estimated as the net increase of cell death occurring in the presence of FK506. (C) Chromatin fragmentation was monitored in wild-type cells after 3-h treatment with 60 μM α-factor or 1 mM H₂O₂ as indicated. Similar results were obtained using CN-deficient cnb1 mutants and longer periods of treatment. (D) Time-lapse video microscopy was used to monitor the fate of mother cells and buds of sst1 mutant (WT) and sst1 fig1 double mutant (fig1) populations after treatment with 2 μM α-factor at RT. Dead cells were identified using PI instead of methylene blue in other experiments and the results for fig1 mothers and buds were combined. Data were fit to standard Sigmoid equations.

Fig1 is a member of the stargazin/VGCC-γ/claudin superfamily but promotes fast cell death independent of [Ca²⁺]c elevation. (A) Fig1 orthologues from yeast and 24 other fungi were identified by BLAST searches of genome databases and aligned using CLUSTALW algorithm. Sites of high, moderate, and poor sequence conservation are shaded black, dark gray, and light gray, respectively. White spaces indicate gaps. The positions of four predicted transmembrane segments and a conserved GΦΦGXC(n)C motif are indicated. (B) Cell death was measured in sst1 mutant and sst1 fig1 double mutant populations after 3-h treatment with 60 μM α-factor in the presence of varying amounts of BAPTA, a chelator of divalent metals such as Ca²⁺. Data were fit to standard sigmoid equations. (C) Cell death was measured in wild-type, pmc1 vcx1 double mutant, fig1 mutant, and fig1 pmc1 vcx1 triple mutant populations after 3.5-h treatment with 60 μM α-factor. (D and E) Luminescence of cytoplasmic aequorin was measured in the same strains at various times after treatment with 60 μM α-factor.

Working model of regulatory mechanisms controlling cell death in response to mating pheromones. Low concentrations of α-factor (αF) activate regulatory pathways leading to cell wall degradation (CW deg.) and stimulation of the high-affinity Ca²⁺ influx system (Cch1, Mid1) but not the Fig1-dependent low-affinity Ca²⁺ influx system. The resulting rise in [Ca²⁺]c activates CN, which prevents ROS accumulation by either inhibiting ROS production by the mitochondrial respiratory chain or stimulating ROS dissipation. The diminished cell wall strength may lead to increased ROS accumulation and intermediate lifespan of cells were it not for the activation of the CWI signaling pathway, which increases biosynthesis of new cell wall material by increasing the activities of chitin and glucan synthases (CHS and GLS). Because CN and CWI signaling pathways operate in wild-type cells, low concentrations of α-factor fail to induce significant amounts of cell death. High concentrations of α-factor activate the above processes but also induce Fig1-dependent Ca²⁺ influx, early ROS accumulation, and rapid cell death in a fraction of the population. Glucosamine (GlcN) supplement and deficiencies of Fus1, Fus2, or Rvs161 block all these responses and additionally prevent activation of CWI signaling. Fig1 deficiency does not affect cell wall degradation or CWI signaling.

Two waves of cell death during the response to α-factor are independently regulated. (A) Cell death was measured in populations of sst1 mutants and cnb1 sst1 double mutants lacking CN activity using methylene blue staining after 10 h of treatment with the indicated concentrations of α-factor. Dose–response curves for wild-type and cnb1 mutant populations were shifted to the right by ∼200-fold (unpublished data). (B and C) Populations of wild-type cells (WT) or CN-deficient cells (cnb1) lacking cytochrome c (−CYC) or experiencing 50 μM cyclosporin A (+CsA) were treated with 60 μM α-factor and monitored for cell death at the indicated times. Averages of three replicate experiments (±SD) are shown. Monophasic and biphasic patterns were evident in the data, of which the maxima, minima, and midpoints were estimated by nonlinear regression using the sum of one or two standard sigmoid equations. A fast wave of cell death was observed in ∼18–27% of all populations, and a slow wave of cell death was observed only in CN-deficient populations. The fast wave of cell death required sixfold higher concentrations of α-factor than CN-less death. Cytochrome c was important for the slow wave but not the fast wave.

Fast but not slow cell death requires dissolution of cell wall polymers. (A) Cell death was measured in wild-type, fig1 mutant, and cnb1 fig1 double mutant populations after 8.5-h treatment with 60 μM α-factor in the presence (gray bars) or absence (black bars) of 15 mM glucosamine. (B) Cell death was measured in single, double, and triple mutants at various times after exposure to 60 μM α-factor. The averages of three replicate experiments were plotted and fit to standard Sigmoid equations as described in Figure 1. (C) Contact-dependent lysis of wild-type, prm1 mutant, fig1 mutant, and fig1 prm1 double mutant prezygotes was measured as described previously (Jin et al., 2004).

CWI signaling inhibits fast cell death and an intermediate cell death. (A) Cell death was measured in wild-type, fus2 mutant, bck1 mutant, and bck1 fus2 double mutant populations after 3-h treatment with 60 μM α-factor. (B) Dead cells were measured in wild-type, bck1 mutant, fig1 mutant, and bck1 fig1 double mutant populations at various times after treatment with 60 μM α-factor. The average of three independent measurements were plotted (±SD) and fit to the sum of one or two standard sigmoid equations. (C) Percentages of ROS positive cells were measured over time in three replicate cultures of wild-type, bck1 mutant, fig1 mutant, and bck1 fig1 double mutant strains after treatment with 60 μM α-factor and plotted (mean ± SD). (D) Cell death was measured in sst1 mutant, sst1 bck1 double mutant, sst1 fig1 double mutant, and sst1 bck1 fig1 triple mutant populations after 3-h treatment with various concentrations of α-factor. Data were fit to the sum of one or two standard sigmoid equations.