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First published online 10 February 2009
doi: 10.1242/jcs.042424

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Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast

¹ Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
² School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD
³ Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
⁴ Department of Biotecnologie e Bioscienze University Milano-Bicocca, Piazza della Scienza 2, 20126, Milano, Italy

View larger version (54K): [in this window] [in a new window]   Fig. 1. Actin aggregation and mitochondrial DNA loss in whi2 cells. Wild-type (A,B) and whi2 (C,D) cells were grown for 24 hours to diauxic shift in YPAD media before being processed for F-actin and DNA staining using rhodamine-phalloidin and DAPI, respectively. Fragmented genomic DNA is highlighted by arrows (D). As a control experiment whi2 cells containing either an empty pYes2 expression vector, or pYes2 + WHI2 were grown for 24 hours to diauxic shift and processed for F-actin staining (E). Scale bars: 10 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 2. whi2 cells possess dysfunctional mitochondria, accumulate ROS and exhibit markers of apoptosis. We grew wild-type and whi2 cells that expressed a mitochondria-targeted GFP protein for 18 hours to late log phase and 24 hours to diauxic shift in YPAD media and visualised by fluorescence microscopy. As a control experiment WHI2 was re-introduced into whi2 cells on a pYes2 plasmid, and mitochondria were visualised after 24 hours of growth to diauxic shift. Mitochondria were visualised using a targeted GFP protein as above (A). Mitochondrial membrane potential was assessed in wild-type and whi2 cells grown to diauxic shift using the fluorescent indicator dye DiOC6. The accumulation of DiOC6 at the mitochondria is dependent upon the presence of a membrane potential. Cells were visualised by fluoresence microscopy (B); however as whi2 cells have reduced mitochondrial membrane potential and so fail to take up much dye the cells are also shown as bright field and long exposure fluorescence images (B). DiOC6 uptake was also assessed within wild-type and whi2 cells cultured for 24 hours to diauxic shift using flow cytometry as described in Materials and Methods (C). ROS accumulation was assayed by flow cytometry in wild-type and whi2 cells grown to diauxic shift using the dye H2DCF-DA (D). The same cells were co-stained with propidium iodide and H2DCF-DA to assess the level of necrosis in whi2 cultures (E). As a control experiment, whi2 cells containing either an empty pYes2 expression vector or pYes2 + WHI2 were grown for 24 hours to diauxic shift and ROS production assessed using H2DCF-DA (F). The effect of deletion of the WHI2 gene on culture viability after growth to diauxic shift was assessed. The effect of re-introducing the WHI2 gene on culture viability was also determined (G). Apoptotic yeast cells often exhibit a loss of vacuolar membrane integrity, which can be visualised using a GFP-Pep4 fusion protein. In wild-type cells GFP-Pep4 is retained in the vacuole whereas in cells that have lost vacuolar membrane integrity it is found distributed throughout the cell (H). Scale bars: 10 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Inhibition of actin aggregation prevents apoptosis in whi2 cells. Cells lacking Whi2p were grown for 24 hours in YPAD to diauxic shift with or without the presence of the actin monomer-binding drug latrunculin-A. These cells were then prepared for F-actin staining (A) or ROS assessment with H2DCF-DA (B) and visualised using fluorescence microscopy. ROS accumulation was also assayed in these cells using flow cytometry (C). The effect of latrunculin-A presence on whi2 culture viability was assessed after 24 hours of growth (D). Mitochondrial morphology was examined in whi2 cells grown with and without latrunculin-A using a targeted GFP molecule (E). Scale bars: 10 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 4. ROS production and mitochondrial dysfunction occurs as a result of elevated cAMP-PKA activity in whi2 cells. We examined the effect of overexpressing the high affinity phosphodiesterase PDE2 (A), and deletion of the PKA subunit TPK3 (B), on whi2 sensitivity to 2 mM H2O2 when grown on YPAD agar plates. The effect of PDE2 overexpression and TPK3 deletion on ROS accumulation was also assessed by flow cytometry using H2DCF-DA (C). Further experiments investigated the impact of TPK3 deletion on the accumulation of actin aggregates (D) and mitochondrial morphology (E) observed in whi2 cells grown to diauxic shift. Scale bars: 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Analysis of Ras2p localisation and activation in whi2 cells. GFP-Ras2p was localised in wild-type cells during log and diauxic phases of growth in wild-type and whi2 cells. Arrows indicate the location of the vacuole in these cells (A). GFP-Ras2p and Cox4p-RFP were colocalised in whi2 cells grown for 24 hours to the diauxic phase (B). GTP-bound active Ras proteins were detected using a probe consisting of three Raf 1-binding domains fused to GFP. 3xRBD-GFP localisation was examined in wild-type, whi2 and control ras2 cells at log and diauxic shift phases of culture (C). 3xRBD-GFP and Cox4p-RFP were colocalised in whi2 cells grown for 24 hours to the diauxic phase (D). Total protein was isolated from wild-type, whi2, ras2 and RAS2val19 strains at the time points indicated and western blots probed for Ras2p (E). The integral plasma membrane protein Pma1p was localised in wild-type and whi2 cells grown for 24 hours to diauxic shift (F). Scale bars: 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 6. The effect of whi2 and cAMP elevation on cell death within S. cerevisiae colonies. We wished to assess whether the loss of Whi2p function had an impact on the patterns of cell death observed in emerging colonies. Colonies were grown from single cells on YPAD containing phloxine B, which accumulates within dead cells and stains them red. Colonies formed from wild type, whi2 and whi2tpk3 were observed from above and in cross section after dissection. Arrows refer to zones of reduced phloxine B uptake (A). ROS accumulation and cell viability within these colonies were assessed, as described in Materials and Methods (B).

View larger version (26K): [in this window] [in a new window]   Fig. 7. Model depicting the regulation of Ras activity in wild-type and whi2 cells. In wild-type cells Whi2p functions to integrate nutritional sensing with maintenance of the dynamic nature of the cytoskeleton. Functional actin dynamics are required for the appropriate tracking and degradation of Ras2p, which in turn facilitates the shutdown of cAMP-PKA signalling and an appropriate cellular response. By contrast, the loss of Whi2p function leads to the loss of coordination between nutritional sensing and actin regulation. The stabilisation of F-actin structures results in the failure to correctly traffic Ras2p to the vacuole. Ras2p in turn becomes localised to the mitochondrial surface in an active form. The failure to shut down Ras signalling leads to mitochondrial dysfunction, the accumulation of damaging ROS and cell death.

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