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Activation of a TRP-Like Channel and Intracellular Ca2+ Dynamics During ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3817–3829 doi:10.1242/jcs.152058 RESEARCH ARTICLE Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death A. Pedro Gonc¸alves1,2,*,`, J. Miguel Cordeiro2,*, Joa˜o Monteiro1, Alberto Mun˜oz3, Paulo Correia-de-Sa´ 2, Nick D. Read3 and Arnaldo Videira1,2,` ABSTRACT (Clapham, 2007). Cells mobilize Ca2+ to the cytosol by the opening of channels in the plasma membrane (for Ca2+ influx The model organism Neurospora crassa undergoes programmed from the external medium) or in the membranes of organelles cell death when exposed to staurosporine. Here, we show that that act as intracellular stores. In fungi, Ca2+ uptake from staurosporine causes defined changes in cytosolic free Ca2+ 2+ 2+ the extracellular milieu can occur by different mechanisms, ([Ca ]c) dynamics and a distinct Ca signature that involves 2+ 2+ 2+ depending on the stimulus. The high-affinity Ca uptake system Ca influx from the external medium and internal Ca stores. We 2+ 2+ (HACS) is the best described fungal Ca uptake system. It investigated the molecular basis of this Ca response by using comprises the channel Cch1 and the regulatory proteins Mid1 and 2+ [Ca ]c measurements combined with pharmacological and genetic Ecm7 (Martin et al., 2011; Muller et al., 2001). The exact role of approaches. Phospholipase C was identified as a pivotal player Mid1 is unclear, as the protein has also been reported to behave as during cell death, because modulation of the phospholipase C a non-selective stretch-activated cation channel protein in some signaling pathway and deletion of PLC-2, which we show to be systems (Cavinder et al., 2011; Kanzaki et al., 1999; Lew et al., involved in hyphal development, results in an inability to trigger the 2008). The HACS seems to be particularly active in minimal characteristic staurosporine-induced Ca2+ signature. Using Dcch-1, medium, whereas the low-affinity Ca2+ system (LACS) is more Dfig-1 and Dyvc-1 mutants and a range of inhibitors, we show that active in nutrient-rich medium (Muller et al., 2001). So far, the extracellular Ca2+ entry does not occur through the hitherto only known member of the LACS system is the Fig1 channel, described high- and low-affinity Ca2+ uptake systems, but through which is involved in mating (Cavinder and Trail, 2012; Muller the opening of plasma membrane channels with properties et al., 2003). A glucose-induced Ca2+ influx system was recently resembling the transient receptor potential (TRP) family. Partial proposed (Groppi et al., 2011) but its molecular components are blockage of the response to staurosporine after inhibition of a unknown. putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that In tip-growing organisms such as the fungus Neurospora 2+ 2+ 2+ Ca release from internal stores following IP3 formation combines crassa, cytosolic free Ca ([Ca ]c) is important for polarized with the extracellular Ca2+ influx. growth. Hyphal growth in N. crassa has been reported to rely on a 2+ tip-high [Ca ]c gradient that is not maintained by extracellular KEY WORDS: Cell death, Inositol-1,4,5-trisphosphate, Ca2+ influx (Lew, 1999), but is internally derived by means Phospholipase C, Staurosporine, Transient receptor potential 2+ of inositol-1,4,5-trisphosphate (IP3)-activated Ca channels channel (Silverman-Gavrila and Lew, 2001; Silverman-Gavrila and Lew, 2002; Silverman-Gavrila and Lew, 2003). Evidence INTRODUCTION indicates that IP3 is generated by a stretch-activated tip- Calcium (Ca2+) is an essential intracellular messenger in all localized phospholipase C that senses tension due to hyphal organisms, from prokaryotes to humans. The ion binds to a expansion and converts phosphatidylinositol-4,5-bisphosphate diverse range of proteins, commonly at EF-hand or EF-hand-like (PIP2)toIP3 and diacylglycerol (DAG) (Silverman-Gavrila and domains, and promotes conformational and electrostatic Lew, 2002; Silverman-Gavrila and Lew, 2003). IP3 promotes the alterations in these proteins. These interactions modulate their release of Ca2+ through a large conductance channel, associated activity and contribute to a cascade of signaling events that with the vacuolar membrane (Cornelius et al., 1989; Silverman- ultimately defines a Ca2+-mediated cellular response to a stimulus Gavrila and Lew, 2002), and a small conductance channel, associated with endoplasmic reticulum (ER)- and Golgi-derived vesicles that have been proposed to accumulate near the hyphal 1IBMC-Instituto de Biologia Molecular e Celular - Universidade do Porto, Rua do 2 tip (Silverman-Gavrila and Lew, 2002). Only the latter is believed Campo Alegre 823, 4150-180 Porto, Portugal. ICBAS-Instituto de Cieˆncias 2+ Biome´dicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo to be involved in the generation of the tip-high [Ca ]c gradient Ferreira 228, 4050-313 Porto, Portugal. 3Manchester Fungal Infection Group, (Silverman-Gavrila and Lew, 2002; Torralba et al., 2001). Institute of Inflammation and Repair, CTF Building, Grafton Street, University of However, a recognizable IP receptor has not yet been Manchester, Manchester M13 9NT, UK. 3 *These authors contributed equally to this work identified in fungi (Borkovich et al., 2004; Zelter et al., 2004). 2+ ` The existence of a continuous tip-high [Ca ]c gradient in Authors for correspondence ([email protected]; [email protected]) growing hyphae has recently been challenged (Kim et al., 2012b). 2+ This is an Open Access article distributed under the terms of the Creative Commons Attribution In this study, the imaging of [Ca ]c, using a genetically encoded License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution 2+ and reproduction in any medium provided that the original work is properly attributed. Ca reporter expressed in Fusarium and Magnaporthe, 2+ demonstrated that [Ca ]c spikes with an irregular frequency Received 24 February 2014; Accepted 23 June 2014 occur in growing hyphal tips, with the result that the tip-focused Journal of Cell Science 3817 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 3817–3829 doi:10.1242/jcs.152058 Fig. 1. Staurosporine induces a well-defined Ca2+ signature. (A) Aequorin-expressing wild-type cells grown for 6 hours were incubated with 20 mM staurosporine (STS) or 20 mM UCN-01, and the timecourse emission of luminescence was monitored over 5 hours. The STS-induced Ca2+ signature 2+ 2+ contained two major Ca transients (phases ‘A’ and ‘B’) and a third broad [Ca ]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 2+ figure (this was also performed for the following Figs 2–6). [Ca ]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 mean6s.e.m.; *P,0.05. gradient appears and disappears in hyphae extending at constant RESULTS rates. Staurosporine induces a well-defined Ca2+ signature The phospholipase C pathway is the main modulator of transient After 6 hours of culture, wild-type N. crassa cells expressing 2+ 2+ receptor potential (TRP) channels, which are permeable to Ca . the codon-optimized bioluminescent [Ca ]c reporter aequorin The TRP channel family comprises several subfamilies: classical (Nelson et al., 2004) were incubated with 20 mM staurosporine, (TRPC), vanilloid (TRPV), melastatin (TRPM), polycystin (TRPP), and the luminescence was monitored over time. Staurosporine 2+ mucolipin (TRPML) and ankyrin (TRPA) (Rohacs, 2013). In N. induced a well-defined signature of [Ca ]c changes (Fig. 1A). crassa, the only known TRP-type channel is YVC-1 (Zelter et al., The signature included two major Ca2+ peaks that we identified as 2004). In Saccharomyces cerevisiae, the homolog of YVC-1 is ‘A’ and ‘B’, and a third broad increase in cytosolic Ca2+ (‘C’). localized in the vacuolar membrane and is involved in the release of Peak A occurred immediately upon addition of staurosporine Ca2+ into the cytosol (Palmer et al., 2001) after activation by stretch and lasted for ,20 minutes, and peak B, having the (Su et al., 2009) and PIP2 (Dong et al., 2010). Recently, an in silico greatest amplitude, occurred after 35–40 minutes and lasted genomic comparison of fungal pathogens identified additional TRP for ,80 minutes. UCN-01, a natural stereoisomer of 7- channel homologs (Prole and Taylor, 2012). hydroxystaurosporine currently in clinical trials for cancer The alkaloid staurosporine was initially isolated from treatment (Gani and Engh, 2010), also provoked an immediate 2+ 2+ Streptomyces staurosporeus during a screen for protein kinase peak of [Ca ]c, although the overall Ca signature was different C inhibitors (Omura et al., 1977) and was later shown to behave from that caused by staurosporine (Fig. 1A). as a broad kinase inhibitor (Karaman et al., 2008). It is largely The levels of staurosporine-induced cell death, as measured by known to trigger cell death in mammalian models. Although staining with propidium iodide, a reporter of plasma membrane staurosporine exhibits potent anticancer activity, the lack of permeabilization, were only significantly increased after 3 hours of selectivity and concomitant side effects makes it too
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