Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.
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.
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 email@example.com firstname.lastname@example.org.Show MeSH
Mentions: After 6 hours of culture, wild-type N. crassa cells expressing the codon-optimized bioluminescent [Ca2+]c reporter aequorin (Nelson et al., 2004) were incubated with 20 µM staurosporine, and the luminescence was monitored over time. Staurosporine induced a well-defined signature of [Ca2+]c changes (Fig. 1A). The signature included two major Ca2+ peaks that we identified as ‘A’ and ‘B’, and a third broad increase in cytosolic Ca2+ (‘C’). Peak A occurred immediately upon addition of staurosporine and lasted for ∼20 minutes, and peak B, having the greatest amplitude, occurred after 35–40 minutes and lasted for ∼80 minutes. UCN-01, a natural stereoisomer of 7-hydroxystaurosporine currently in clinical trials for cancer treatment (Gani and Engh, 2010), also provoked an immediate peak of [Ca2+]c, although the overall Ca2+ signature was different from that caused by staurosporine (Fig. 1A).
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 email@example.com firstname.lastname@example.org.