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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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Staurosporine-induced Ca2+ influx occurs through an uptake system distinct from the high- and low-affinity Ca2+ systems. (A,B) The Ca2+ signature in response to 20 µM staurosporine (STS) was compared in aequorin-expressing wild-type and Δcch-1 (A) or Δfig-1 (B) cells. The influence of pre-treatment with 4 mM BAPTA and 50 µM 2-APB on the Δcch-1 STS-induced Ca2+ signature is shown in A. (C,D) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in panels A,B. Data show the mean±s.e.m.; *P<0.05 (for Δcch-1 versus wild-type cells); #P<0.05 (for BAPTA and 2-APB pre-treated Δcch-1 cells versus Δcch-1 with STS alone). (E) The sensitivity of Δcch-1 and Δmid-1 strains was evaluated by spotting conidia onto GFS medium containing 2.5 µM STS.
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f05: Staurosporine-induced Ca2+ influx occurs through an uptake system distinct from the high- and low-affinity Ca2+ systems. (A,B) The Ca2+ signature in response to 20 µM staurosporine (STS) was compared in aequorin-expressing wild-type and Δcch-1 (A) or Δfig-1 (B) cells. The influence of pre-treatment with 4 mM BAPTA and 50 µM 2-APB on the Δcch-1 STS-induced Ca2+ signature is shown in A. (C,D) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in panels A,B. Data show the mean±s.e.m.; *P<0.05 (for Δcch-1 versus wild-type cells); #P<0.05 (for BAPTA and 2-APB pre-treated Δcch-1 cells versus Δcch-1 with STS alone). (E) The sensitivity of Δcch-1 and Δmid-1 strains was evaluated by spotting conidia onto GFS medium containing 2.5 µM STS.

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)

Staurosporine-induced Ca2+ influx occurs through an uptake system distinct from the high- and low-affinity Ca2+ systems. (A,B) The Ca2+ signature in response to 20 µM staurosporine (STS) was compared in aequorin-expressing wild-type and Δcch-1 (A) or Δfig-1 (B) cells. The influence of pre-treatment with 4 mM BAPTA and 50 µM 2-APB on the Δcch-1 STS-induced Ca2+ signature is shown in A. (C,D) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in panels A,B. Data show the mean±s.e.m.; *P<0.05 (for Δcch-1 versus wild-type cells); #P<0.05 (for BAPTA and 2-APB pre-treated Δcch-1 cells versus Δcch-1 with STS alone). (E) The sensitivity of Δcch-1 and Δmid-1 strains was evaluated by spotting conidia onto GFS medium containing 2.5 µM STS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f05: Staurosporine-induced Ca2+ influx occurs through an uptake system distinct from the high- and low-affinity Ca2+ systems. (A,B) The Ca2+ signature in response to 20 µM staurosporine (STS) was compared in aequorin-expressing wild-type and Δcch-1 (A) or Δfig-1 (B) cells. The influence of pre-treatment with 4 mM BAPTA and 50 µM 2-APB on the Δcch-1 STS-induced Ca2+ signature is shown in A. (C,D) Quantification (in arbitrary units, A.U.) of the [Ca2+]c transients A and B, respectively, which are shown in panels A,B. Data show the mean±s.e.m.; *P<0.05 (for Δcch-1 versus wild-type cells); #P<0.05 (for BAPTA and 2-APB pre-treated Δcch-1 cells versus Δcch-1 with STS alone). (E) The sensitivity of Δcch-1 and Δmid-1 strains was evaluated by spotting conidia onto GFS medium containing 2.5 µM STS.
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