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First published online 26 April 2005
doi: 10.1242/jcs.02337

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Identification of an upstream regulatory pathway controlling actin-mediated apoptosis in yeast

Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK

View larger version (77K): [in a new window]   Fig. 1. F-Actin is less dynamic in end3 and sla1118-511 cells. F-Actin in cells from low- and high-density cultures (log and early stationary phase) was visualized by fluorescent microscopy using rhodamine-phalloidin. In wild-type cells during log phase, actin patches polarize to areas of new cell growth (A) but, in end3 (C) and sla1118-511 (E) cells, cortical patches (while still polarized) were larger and less numerous. During early stationary phase, actin patches become depolarized and numerous in wild-type cells (B), and large F-actin aggregates were observed in a small proportion (<5%) of cells (arrowhead). By contrast, end3 (D) and sla1118-511 (F) F-actin appears as large aggregates in most (>95%) cells. Bar, 10 µm.

View larger version (102K): [in a new window]   Fig. 2. Actin dynamics are linked to respiratory function. (A) Wild-type, end3 and sla1118-511 cells were plated on media containing either glucose or the non-fermentable carbon source glycerol. Wild-type cells grew on both media, whereas both end3 and sla1118-511 cells were unable to grow on glycerol, indicating that these cells have a respiratory defect. (B,C) Mitochondrial morphology in log phase (B) and stationary phase (C) wild-type, end and sla1118-511 cells was assessed using a targeted GFP molecule. (D-F) Mitochondrial membranes were visualized by DiOC6 staining in wild-type (D), end3 (E) and sla1118-511 (F) cells at log phase. Bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 3. ROS accumulation in end3 and sla1118-511 cells. (A) ROS accumulation was assayed using H2DCFDA by flow cytometry in wild-type, end3 and sla1118-511 cells grown to early stationary phase. (B) Graphical representation of the proportion of wild-type, end3 and sla1118-511 cells showing a high build up of ROS. (C) The relative sensitivity to H2O2 was calculated in wild-type, end3 and sla1118-511 using a halo-assay approach. (D) ROS accumulation was assessed using H2DCFDA by flow cytometry in end3 and sla1118-511 cells, which were either rho0 strains or had been treated with 0.1 mg ml–1 of the mitochondrial inhibitor antimycin A. (E) Exposure of the early apoptotic marker PS was measured using FITC/annexin-V in response to the addition of H2O2 at sublethal levels. A representative data set is shown.

View larger version (47K): [in a new window]   Fig. 4. Actin dynamics are linked to mitochondrial membrane polarity in end3-1 cells. (A) The temperature-sensitive end3-1 allele displays a shift from the wild type to the end3 actin phenotype when shifted from 24°C to 37°C for 2 hours. (B) Cells expressing the end3-1 allele were plated on glucose- or glycerol-containing media at 24°C or 37°C; as positive and negative controls, wild-type, end3 and sla1118-511 cells were also plated. (C) Mitochondrial morphology was examined using targeted GFP in wild-type and end3-1 cells at 24°C and after 2 hours of growth at 37°C. (D) Mitochondria were visualized using DiOC6 in end3-1 cells shifted from 24°C or 37°C for 2 hours. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 5. Actin-stabilized end3-1 cells display markers of apoptosis. ROS accumulation using H2DCFDA (A) and caspase activation using FITC–VAD-fmk (B) were measured by flow cytometry in wild-type and end3-1 cells grown to log phase at 24°C and after 2 hours of growth at 37°C. (C) The viabilities were calculated of wild-type and end3-1 cultures grown to log phase at 24°C and after a shift to 37°C for 2 hours.

View larger version (45K): [in a new window]   Fig. 6. Overexpression of RSP5 can improve mitochondrial function, increase viability and restore actin dynamics in end3 cells. (A) Wild-type, end3 and end3[RSP5] cells were plated onto YP agar containing 2% glucose or 3% glycerol as the sole carbon source and grown for 3 days at 37°C. (B) Cultures from each of these strains were then assayed for ROS accumulation by flow cytometry using H2DCFDA. (C) Cell viability was assessed via a plating assay carried out in triplicate. (D) Wild-type, end3 and end3[RSP5] cells were grown to stationary phase and stained for F-actin with rhodamine-phalloidin. (E) The same strains were assayed for glycogen accumulation by iodine staining. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 7. Sla1p localization to the cortex in end3 cells is enhanced by RSP5 overexpression. (A) Wild-type, end3 and end3[RSP5] cells expressing an integrated Sla1p-GFP were examined by fluorescence microscopy. In wild-type cells, Sla1p-GFP appeared as punctate spots that appear largely within the buds of dividing cells. (B) In end3 cells, Sla1p appeared largely diffuse and cytoplasmic, and in a few punctate cortical structures. (C) In end3[RSP5] cells, the cytoplasmic staining was reduced and an increase in punctate cortical staining was observed in both mother and bud. Bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 8. PDE2 overexpression reduces ROS accumulation in end3 cells and restores viability. (A) Wild-type and end3 cells carrying either an empty Yep13 plasmid or Yep13 containing PDE2 (Yep13[PDE2]) were struck onto YPD plates containing 0 mM or 3 mM H2O2 and grown for 3 days at 30°C. (B) ROS accumulation was assessed in wild-type cultures containing YEp13, end3 cultures containing YEp13 and end3 cultures containing Yep13[PDE2] that had been grown overnight in YPD at 30°C. Cells were visualized after staining by ultraviolet fluorescence microscopy. (C) The proportion of viable cells was assessed in overnight wild-type cultures containing YEp13, wild-type cultures containing YEp13[PDE2], end3 cultures containing YEp13 and end3 cultures containing YEp13[PDE2] cultures. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 9. Reduced actin dynamics of end3 cells are increased by PDE2 overexpression. (A) F-Actin was examined in overnight cultures of end3 cells carrying either the YEp13 or YEp13 plasmid containing PDE2. (B) LatA-sensitivity halo assays were carried out on cells from wild-type cultures containing YEp13, wild-type cultures containing YEp13[PDE2], end3 cultures containing YEp13 and end3 cultures containing YEp13[PDE2]. (C) Glycogen accumulation was assessed in cells from wild-type cultures containing YEp13, end3 cultures containing YEp13 and end3 cultures containing YEp13[PDE2] cultures. (D) Heat-shock sensitivity was assessed in wild-type cultures containing YEp13, wild-type cultures containing YEp13[PDE2], end3 cultures containing YEp13 and end3 cultures containing YEp13[PDE2] cells. Bar, 10 µm.

View larger version (83K): [in a new window]   Fig. 10. CAP/Srv2p colocalization with F-actin in log- and stationary-phase wild-type and end3 cells. F-Actin (red) and CAP/Srv2p (green) were visualized by fluorescence microscopy in wild-type (A,C) and end3 (B,D) cells that had been grown to mid-log or stationary phase in liquid culture. Images were merged to observe areas of colocalization (yellow). Bars, 10 µm.

View larger version (19K): [in a new window]   Fig. 11. Model linking Ras/cAMP signalling to actin remodelling, ROS release and apoptosis. In response to environmental stresses such as nutrient depletion or heat shock, the actin cytoskeleton undergoes remodelling as part of the survival response. Evidence suggests that the Ras/cAMP pathway is important in this remodelling. Also important are the actin-regulatory activities of Sla1p, which is targeted to the cortex by both End3p and Rsp5p activities. Inappropriate activation of the Ras/cAMP pathway or reduced actin dynamics as a result of a loss of Sla1p function lead to high levels of ROS accumulation. The exposure to high levels of oxidative stress lead to a reduced lifespan and an increased likelihood of apoptosis. We suggest that there is a cross-talk mechanism between the actin cytoskeleton and Ras/cAMP signalling machinery that regulates this pathway. This model provides a mechanism by which a colony of unicellular organisms can filter out older or genetically unfit individuals in response to environmental change.

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