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Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.

Gonçalves AP, Cordeiro JM, Monteiro J, Muñoz A, Correia-de-Sá P, Read ND, Videira A - J. Cell. Sci. (2014)

Bottom Line: Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca(2+) signature.Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca(2+) entry does not occur through the hitherto described high- and low-affinity Ca(2+) uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family.Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca(2+) release from internal stores following IP3 formation combines with the extracellular Ca(2+) influx.

View Article: PubMed Central - PubMed

Affiliation: IBMC-Instituto de Biologia Molecular e Celular - Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal apgoncalves@ibmc.up.pt avideira@ibmc.up.pt.

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PLC-2 is required for the Ca2+ signature and for cell death induced by staurosporine, and is involved in N. crassa hyphal development. (A) The sensitivity profiles of deletion strains for the four N. crassa phospholipase C genes were evaluated by spotting conidia onto GFS medium containing 2.5 µM staurosporine (STS). The GFS medium contained 2% sorbose to produce compact colonies. (B) The levels of apoptosis in wild-type and Δplc-2 cells were detected by staining with YO-PRO1, and the percentage of positive cells was measured by flow cytometry. *P<0.05 (for 50 µM STS versus DMSO for each strain); #P<0.05 (for STS-treated wild-type versus Δplc-2 cells). (C) [Ca2+]c was measured in aequorin-expressing Δplc-2 cells after treatment with 20 µM STS. (D,E) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in C. Data show the mean±s.e.m.; *P<0.05. (F,G) Growth of wild-type and Δplc-2 strains in solid medium. The panels show growth at 48 h post-inoculation (F) and hyphal extension rates over time as the mean±s.e.m. (G). (H) Growth of wild-type and Δplc-2 strains in liquid medium over 24 hours was obtained by measuring absorbance (Abs) at 450 nm. Data show the mean±s.e.m. (I) Representative micrographs of wild-type and Δplc-2 strains at 8 hours and 24 hours after inoculation in liquid Vogel's minimal medium. The percentage of germinated cells is indicated in the upper right corner (mean±s.e.m.). Note the presence of swollen conidia and several ungerminated conidia in 24-hour-cultures of Δplc-2 (arrows). *P<0.05. Scale bars: 40 µm.
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f04: PLC-2 is required for the Ca2+ signature and for cell death induced by staurosporine, and is involved in N. crassa hyphal development. (A) The sensitivity profiles of deletion strains for the four N. crassa phospholipase C genes were evaluated by spotting conidia onto GFS medium containing 2.5 µM staurosporine (STS). The GFS medium contained 2% sorbose to produce compact colonies. (B) The levels of apoptosis in wild-type and Δplc-2 cells were detected by staining with YO-PRO1, and the percentage of positive cells was measured by flow cytometry. *P<0.05 (for 50 µM STS versus DMSO for each strain); #P<0.05 (for STS-treated wild-type versus Δplc-2 cells). (C) [Ca2+]c was measured in aequorin-expressing Δplc-2 cells after treatment with 20 µM STS. (D,E) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in C. Data show the mean±s.e.m.; *P<0.05. (F,G) Growth of wild-type and Δplc-2 strains in solid medium. The panels show growth at 48 h post-inoculation (F) and hyphal extension rates over time as the mean±s.e.m. (G). (H) Growth of wild-type and Δplc-2 strains in liquid medium over 24 hours was obtained by measuring absorbance (Abs) at 450 nm. Data show the mean±s.e.m. (I) Representative micrographs of wild-type and Δplc-2 strains at 8 hours and 24 hours after inoculation in liquid Vogel's minimal medium. The percentage of germinated cells is indicated in the upper right corner (mean±s.e.m.). Note the presence of swollen conidia and several ungerminated conidia in 24-hour-cultures of Δplc-2 (arrows). *P<0.05. Scale bars: 40 µm.

Mentions: Staurosporine induces a well-defined Ca2+ signature. (A) Aequorin-expressing wild-type cells grown for 6 hours were incubated with 20 µM staurosporine (STS) or 20 µM UCN-01, and the timecourse emission of luminescence was monitored over 5 hours. The STS-induced Ca2+ signature contained two major Ca2+ transients (phases ‘A’ and ‘B’) and a third broad [Ca2+]c increase (‘C’) and represents an average of 30 independent experiments, each with three to six replicates. The ‘staurosporine-induced amplitude of response’ was calculated by subtracting the solvent DMSO control curve shown in this figure (this was also performed for the following Figs 2–6). [Ca2+]c measurements in Figs 1–6 are also presented with errors bars in supplementary material Fig. S1. (B) Cell death as a readout of membrane permeabilization was examined after staining with propidium iodide (PI). Data show the mean±s.e.m.; *P<0.05.


Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.

Gonçalves AP, Cordeiro JM, Monteiro J, Muñoz A, Correia-de-Sá P, Read ND, Videira A - J. Cell. Sci. (2014)

PLC-2 is required for the Ca2+ signature and for cell death induced by staurosporine, and is involved in N. crassa hyphal development. (A) The sensitivity profiles of deletion strains for the four N. crassa phospholipase C genes were evaluated by spotting conidia onto GFS medium containing 2.5 µM staurosporine (STS). The GFS medium contained 2% sorbose to produce compact colonies. (B) The levels of apoptosis in wild-type and Δplc-2 cells were detected by staining with YO-PRO1, and the percentage of positive cells was measured by flow cytometry. *P<0.05 (for 50 µM STS versus DMSO for each strain); #P<0.05 (for STS-treated wild-type versus Δplc-2 cells). (C) [Ca2+]c was measured in aequorin-expressing Δplc-2 cells after treatment with 20 µM STS. (D,E) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in C. Data show the mean±s.e.m.; *P<0.05. (F,G) Growth of wild-type and Δplc-2 strains in solid medium. The panels show growth at 48 h post-inoculation (F) and hyphal extension rates over time as the mean±s.e.m. (G). (H) Growth of wild-type and Δplc-2 strains in liquid medium over 24 hours was obtained by measuring absorbance (Abs) at 450 nm. Data show the mean±s.e.m. (I) Representative micrographs of wild-type and Δplc-2 strains at 8 hours and 24 hours after inoculation in liquid Vogel's minimal medium. The percentage of germinated cells is indicated in the upper right corner (mean±s.e.m.). Note the presence of swollen conidia and several ungerminated conidia in 24-hour-cultures of Δplc-2 (arrows). *P<0.05. Scale bars: 40 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150065&req=5

f04: PLC-2 is required for the Ca2+ signature and for cell death induced by staurosporine, and is involved in N. crassa hyphal development. (A) The sensitivity profiles of deletion strains for the four N. crassa phospholipase C genes were evaluated by spotting conidia onto GFS medium containing 2.5 µM staurosporine (STS). The GFS medium contained 2% sorbose to produce compact colonies. (B) The levels of apoptosis in wild-type and Δplc-2 cells were detected by staining with YO-PRO1, and the percentage of positive cells was measured by flow cytometry. *P<0.05 (for 50 µM STS versus DMSO for each strain); #P<0.05 (for STS-treated wild-type versus Δplc-2 cells). (C) [Ca2+]c was measured in aequorin-expressing Δplc-2 cells after treatment with 20 µM STS. (D,E) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in C. Data show the mean±s.e.m.; *P<0.05. (F,G) Growth of wild-type and Δplc-2 strains in solid medium. The panels show growth at 48 h post-inoculation (F) and hyphal extension rates over time as the mean±s.e.m. (G). (H) Growth of wild-type and Δplc-2 strains in liquid medium over 24 hours was obtained by measuring absorbance (Abs) at 450 nm. Data show the mean±s.e.m. (I) Representative micrographs of wild-type and Δplc-2 strains at 8 hours and 24 hours after inoculation in liquid Vogel's minimal medium. The percentage of germinated cells is indicated in the upper right corner (mean±s.e.m.). Note the presence of swollen conidia and several ungerminated conidia in 24-hour-cultures of Δplc-2 (arrows). *P<0.05. Scale bars: 40 µm.
Mentions: Staurosporine induces a well-defined Ca2+ signature. (A) Aequorin-expressing wild-type cells grown for 6 hours were incubated with 20 µM staurosporine (STS) or 20 µM UCN-01, and the timecourse emission of luminescence was monitored over 5 hours. The STS-induced Ca2+ signature contained two major Ca2+ transients (phases ‘A’ and ‘B’) and a third broad [Ca2+]c increase (‘C’) and represents an average of 30 independent experiments, each with three to six replicates. The ‘staurosporine-induced amplitude of response’ was calculated by subtracting the solvent DMSO control curve shown in this figure (this was also performed for the following Figs 2–6). [Ca2+]c measurements in Figs 1–6 are also presented with errors bars in supplementary material Fig. S1. (B) Cell death as a readout of membrane permeabilization was examined after staining with propidium iodide (PI). Data show the mean±s.e.m.; *P<0.05.

Bottom Line: Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca(2+) signature.Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca(2+) entry does not occur through the hitherto described high- and low-affinity Ca(2+) uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family.Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca(2+) release from internal stores following IP3 formation combines with the extracellular Ca(2+) influx.

View Article: PubMed Central - PubMed

Affiliation: IBMC-Instituto de Biologia Molecular e Celular - Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal apgoncalves@ibmc.up.pt avideira@ibmc.up.pt.

Show MeSH
Related in: MedlinePlus