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c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1.

Song Y, Luciani MF, Giusti C, Golstein P - Mol. Biol. Cell (2014)

Bottom Line: In these cases, c-di-GMP-induced cell death was rescued by complementation with exogenous DIF-1.Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites.This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université; Institut National de la Santé et de la Recherche Médicale, U1104; and Centre National de la Recherche Scientifique, UMR7280, 13288 Marseille, France.

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DIF-1–induced and c-di-GMP–induced pathways to cell death showed distinct features. (A) Starved DH1 wild-type cells showed vacuolization upon addition of 100 nM DIF-1 or 10 μM c-di-GMP. In contrast, DH1 talinB−, DhkMins, or iplA− mutant cells showed vacuolization upon addition of c-di-GMP but not of DIF-1. The vignettes show cells after 40 h in the presence or absence of inducers. The number at the upper right corner of each vignette shows the percentage of clumps, each comprising at least three vacuolized cells, determined at 16, 24, and 40 h, respectively, after addition of DIF-1 or c-di-GMP. Each of these percentage values was obtained by screening at least 100 clumps. For instance, at 16 h post–DIF-1 or post–c-di-GMP, there were no or only few clumps each with at least three vacuolized cells, whereas in the presence of both inducers together, every clump showed at least three vacuolized cells. For clarity, each individual percentage value is not accompanied by its confidence interval. Out of 100 clumps, for a measured percentage of 50%, the 95% confidence interval of the percentage would be ±9.8%, and this confidence interval would decrease for measured percentages deviating from 50%. Thus the 95% confidence interval of each of the measured percentages shown is ≤±9.8%. (B) Cells in LabTek chambers were starved and incubated for 24 h in the absence of inducer or in the presence of 100 nM DIF-1, 10 μM c-di-GMP, or both. HL5 medium was then added and incubation proceeded for 3 d, and the regrown cells were counted. Each column shows the mean of number of cells regrown in three cultures and the corresponding 95% confidence interval. Wild-type cells showed low regrowth with either inducer, whereas talinB− and iplA− mutant cells showed low regrowth with c-di-GMP but no significant difference with DIF-1 compared with control without inducer. Addition of both inducers further moderately reduced regrowth. (C) DimB nuclear translocation occurred upon addition of DIF-1 but not of c-di-GMP. DH1 cells transfected with GFP-DimB were subjected to starvation and 100 nM DIF-1 and/or 10 μM c-di-GMP for 10 min. The same fields were taken by phase contrast and fluorescence microscopy. From these pictures, the percentage of cells showing nuclear translocation of fluorescence was determined and is shown as numbers in each of the vignettes. Each of these percentage values was obtained by screening at least 100 cells, yielding 95% confidence intervals ≤±9.8%.
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Figure 1: DIF-1–induced and c-di-GMP–induced pathways to cell death showed distinct features. (A) Starved DH1 wild-type cells showed vacuolization upon addition of 100 nM DIF-1 or 10 μM c-di-GMP. In contrast, DH1 talinB−, DhkMins, or iplA− mutant cells showed vacuolization upon addition of c-di-GMP but not of DIF-1. The vignettes show cells after 40 h in the presence or absence of inducers. The number at the upper right corner of each vignette shows the percentage of clumps, each comprising at least three vacuolized cells, determined at 16, 24, and 40 h, respectively, after addition of DIF-1 or c-di-GMP. Each of these percentage values was obtained by screening at least 100 clumps. For instance, at 16 h post–DIF-1 or post–c-di-GMP, there were no or only few clumps each with at least three vacuolized cells, whereas in the presence of both inducers together, every clump showed at least three vacuolized cells. For clarity, each individual percentage value is not accompanied by its confidence interval. Out of 100 clumps, for a measured percentage of 50%, the 95% confidence interval of the percentage would be ±9.8%, and this confidence interval would decrease for measured percentages deviating from 50%. Thus the 95% confidence interval of each of the measured percentages shown is ≤±9.8%. (B) Cells in LabTek chambers were starved and incubated for 24 h in the absence of inducer or in the presence of 100 nM DIF-1, 10 μM c-di-GMP, or both. HL5 medium was then added and incubation proceeded for 3 d, and the regrown cells were counted. Each column shows the mean of number of cells regrown in three cultures and the corresponding 95% confidence interval. Wild-type cells showed low regrowth with either inducer, whereas talinB− and iplA− mutant cells showed low regrowth with c-di-GMP but no significant difference with DIF-1 compared with control without inducer. Addition of both inducers further moderately reduced regrowth. (C) DimB nuclear translocation occurred upon addition of DIF-1 but not of c-di-GMP. DH1 cells transfected with GFP-DimB were subjected to starvation and 100 nM DIF-1 and/or 10 μM c-di-GMP for 10 min. The same fields were taken by phase contrast and fluorescence microscopy. From these pictures, the percentage of cells showing nuclear translocation of fluorescence was determined and is shown as numbers in each of the vignettes. Each of these percentage values was obtained by screening at least 100 cells, yielding 95% confidence intervals ≤±9.8%.

Mentions: Induction in vitro by DIF-1 or by c-di-GMP led to cell death with similar subcellular lesions, such as vacuolization and synthesis of cellulose cell encasings (Levraud et al., 2003; Giusti et al., 2009; unpublished data). However, in Dictyostelium cells of the DH1 strain, whereas vacuolization induced by DIF-1 was prevented by the talB (Giusti et al., 2009), iplA (Lam et al., 2008), and DhkMins (Giusti et al., 2010) mutations (Figure 1A, second column), vacuolization induced by exogenous c-di-GMP was not prevented by these mutations (Figure 1A, third column). Consistently, these mutations prevented cell death (defined here as the inability of cells to regrow upon addition of rich medium) induced by DIF-1 but not that induced by exogenous c-di-GMP (Figure 1B; unpublished data). In this article, exogenous means experimentally added to the cells under test, and endogenous means produced by (some of) the cells under test and acting on the same or other cells. Similarly, cyclosporin A inhibited vacuolization and cell death induced by DIF-1 (Lam et al., 2008) but not by c-di-GMP (Supplemental Figure S1). In addition, as in other Dictyostelium strains (Huang et al., 2006; Zhukovskaya et al., 2006), in DH1 cells, the bZip transcription factor DimB translocated from the cytosol to the nucleus in a matter of minutes after addition of DIF-1 (with or without c-di-GMP; Figure 1C) but not after addition of c-di-GMP (Figure 1C). Taken together, these differences indicated that at least parts of the pathways to cell death induced by exogenous DIF-1 and exogenous c-di-GMP were distinct.


c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1.

Song Y, Luciani MF, Giusti C, Golstein P - Mol. Biol. Cell (2014)

DIF-1–induced and c-di-GMP–induced pathways to cell death showed distinct features. (A) Starved DH1 wild-type cells showed vacuolization upon addition of 100 nM DIF-1 or 10 μM c-di-GMP. In contrast, DH1 talinB−, DhkMins, or iplA− mutant cells showed vacuolization upon addition of c-di-GMP but not of DIF-1. The vignettes show cells after 40 h in the presence or absence of inducers. The number at the upper right corner of each vignette shows the percentage of clumps, each comprising at least three vacuolized cells, determined at 16, 24, and 40 h, respectively, after addition of DIF-1 or c-di-GMP. Each of these percentage values was obtained by screening at least 100 clumps. For instance, at 16 h post–DIF-1 or post–c-di-GMP, there were no or only few clumps each with at least three vacuolized cells, whereas in the presence of both inducers together, every clump showed at least three vacuolized cells. For clarity, each individual percentage value is not accompanied by its confidence interval. Out of 100 clumps, for a measured percentage of 50%, the 95% confidence interval of the percentage would be ±9.8%, and this confidence interval would decrease for measured percentages deviating from 50%. Thus the 95% confidence interval of each of the measured percentages shown is ≤±9.8%. (B) Cells in LabTek chambers were starved and incubated for 24 h in the absence of inducer or in the presence of 100 nM DIF-1, 10 μM c-di-GMP, or both. HL5 medium was then added and incubation proceeded for 3 d, and the regrown cells were counted. Each column shows the mean of number of cells regrown in three cultures and the corresponding 95% confidence interval. Wild-type cells showed low regrowth with either inducer, whereas talinB− and iplA− mutant cells showed low regrowth with c-di-GMP but no significant difference with DIF-1 compared with control without inducer. Addition of both inducers further moderately reduced regrowth. (C) DimB nuclear translocation occurred upon addition of DIF-1 but not of c-di-GMP. DH1 cells transfected with GFP-DimB were subjected to starvation and 100 nM DIF-1 and/or 10 μM c-di-GMP for 10 min. The same fields were taken by phase contrast and fluorescence microscopy. From these pictures, the percentage of cells showing nuclear translocation of fluorescence was determined and is shown as numbers in each of the vignettes. Each of these percentage values was obtained by screening at least 100 cells, yielding 95% confidence intervals ≤±9.8%.
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Related In: Results  -  Collection

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Figure 1: DIF-1–induced and c-di-GMP–induced pathways to cell death showed distinct features. (A) Starved DH1 wild-type cells showed vacuolization upon addition of 100 nM DIF-1 or 10 μM c-di-GMP. In contrast, DH1 talinB−, DhkMins, or iplA− mutant cells showed vacuolization upon addition of c-di-GMP but not of DIF-1. The vignettes show cells after 40 h in the presence or absence of inducers. The number at the upper right corner of each vignette shows the percentage of clumps, each comprising at least three vacuolized cells, determined at 16, 24, and 40 h, respectively, after addition of DIF-1 or c-di-GMP. Each of these percentage values was obtained by screening at least 100 clumps. For instance, at 16 h post–DIF-1 or post–c-di-GMP, there were no or only few clumps each with at least three vacuolized cells, whereas in the presence of both inducers together, every clump showed at least three vacuolized cells. For clarity, each individual percentage value is not accompanied by its confidence interval. Out of 100 clumps, for a measured percentage of 50%, the 95% confidence interval of the percentage would be ±9.8%, and this confidence interval would decrease for measured percentages deviating from 50%. Thus the 95% confidence interval of each of the measured percentages shown is ≤±9.8%. (B) Cells in LabTek chambers were starved and incubated for 24 h in the absence of inducer or in the presence of 100 nM DIF-1, 10 μM c-di-GMP, or both. HL5 medium was then added and incubation proceeded for 3 d, and the regrown cells were counted. Each column shows the mean of number of cells regrown in three cultures and the corresponding 95% confidence interval. Wild-type cells showed low regrowth with either inducer, whereas talinB− and iplA− mutant cells showed low regrowth with c-di-GMP but no significant difference with DIF-1 compared with control without inducer. Addition of both inducers further moderately reduced regrowth. (C) DimB nuclear translocation occurred upon addition of DIF-1 but not of c-di-GMP. DH1 cells transfected with GFP-DimB were subjected to starvation and 100 nM DIF-1 and/or 10 μM c-di-GMP for 10 min. The same fields were taken by phase contrast and fluorescence microscopy. From these pictures, the percentage of cells showing nuclear translocation of fluorescence was determined and is shown as numbers in each of the vignettes. Each of these percentage values was obtained by screening at least 100 cells, yielding 95% confidence intervals ≤±9.8%.
Mentions: Induction in vitro by DIF-1 or by c-di-GMP led to cell death with similar subcellular lesions, such as vacuolization and synthesis of cellulose cell encasings (Levraud et al., 2003; Giusti et al., 2009; unpublished data). However, in Dictyostelium cells of the DH1 strain, whereas vacuolization induced by DIF-1 was prevented by the talB (Giusti et al., 2009), iplA (Lam et al., 2008), and DhkMins (Giusti et al., 2010) mutations (Figure 1A, second column), vacuolization induced by exogenous c-di-GMP was not prevented by these mutations (Figure 1A, third column). Consistently, these mutations prevented cell death (defined here as the inability of cells to regrow upon addition of rich medium) induced by DIF-1 but not that induced by exogenous c-di-GMP (Figure 1B; unpublished data). In this article, exogenous means experimentally added to the cells under test, and endogenous means produced by (some of) the cells under test and acting on the same or other cells. Similarly, cyclosporin A inhibited vacuolization and cell death induced by DIF-1 (Lam et al., 2008) but not by c-di-GMP (Supplemental Figure S1). In addition, as in other Dictyostelium strains (Huang et al., 2006; Zhukovskaya et al., 2006), in DH1 cells, the bZip transcription factor DimB translocated from the cytosol to the nucleus in a matter of minutes after addition of DIF-1 (with or without c-di-GMP; Figure 1C) but not after addition of c-di-GMP (Figure 1C). Taken together, these differences indicated that at least parts of the pathways to cell death induced by exogenous DIF-1 and exogenous c-di-GMP were distinct.

Bottom Line: In these cases, c-di-GMP-induced cell death was rescued by complementation with exogenous DIF-1.Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites.This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université; Institut National de la Santé et de la Recherche Médicale, U1104; and Centre National de la Recherche Scientifique, UMR7280, 13288 Marseille, France.

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Related in: MedlinePlus