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

Abstract

The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca2+ ([Ca2+]c) dynamics and a distinct Ca2+ signature that involves Ca2+ influx from the external medium and internal Ca2+ stores. We investigated the molecular basis of this Ca2+ response by using [Ca2+]c measurements combined with pharmacological and genetic approaches. 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 Ca2+ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca2+ entry does not occur through the hitherto described high- and low-affinity Ca2+ 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 Ca2+ release from internal stores following IP3 formation combines with the extracellular Ca2+ influx.

Hypotonic stress-induced calcium signaling in Saccharomyces cerevisiae involves TRP-like transporters on the endoplasmic reticulum membrane

“…Recently, in N. crassa staurosporine treatment was reported to trigger both Ca 2+ uptake from the extracellular milieu by a novel fungal influx system resembling a TRP channel, which also seems to be up-regulated in the absence of the HACS, and an IP 3 -mediated cytosolic recruitment of organelle-stored Ca 2+ [61]. Moreover, hyphal growth in N. crassa has been reported to rely on a tip-high [Ca 2+ ] i gradient that is internally derived by means of Ca 2+ channels activated by inositol-1,4,5-trisphosphate (IP 3 ) [62][63][64], though no IP 3 receptor seems to exist in fungi [65,66].…”

Translation Inhibition by Rocaglates Activates a Species-Specific Cell Death Program in the Emerging Fungal Pathogen Candida auris

“…Next, we determined whether rocaglate-induced cell death was mediated by calcium signaling, as changes in cytosolic calcium levels have been implicated in triggering cell death in response to chemical stress (47)(48)(49). To address this, we performed dose-response assays with the rocaglates against C. auris in medium with minimal levels of calcium (0.68 mM) and with high levels of calcium supplementation (80 mM) that did not affect growth alone.…”

Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

“…We showed recently that deletion of genes encoding phospholipase C enzymes, especially PLC-2, results in mutants resistant to staurosporine, while strains lacking the Ca 2+ channels CCH-1 and MID-1 are hypersensitive 12. Several other deletion strains that lack Ca 2+ -permeable channels, Ca 2+ -ATPases or other Ca 2+ binding molecules involved in cellular signaling showed a resistance phenotype to staurosporine that was dissimilar from the wild type strain (Fig.…”

“…Peak "A" occurs immediately after the addition of the drug and lasts approximately 20 minutes; peak "B" appears after 35-40 minutes and lasts approximately 80 minutes; phase "C" is a prolonged and continuous elevation of cytosolic Ca 2+ 12. In the absence of extracellular Ca 2+ , the Ca 2+ response to staurosporine was compromised, with peaks "A" and "B" showing substantial reduction (Fig.…”