Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase

Abstract

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.

Hydrogen peroxide induces apoptotic-like cell death inMicrocystis aeruginosa(Chroococcales, Cyanobacteria) in a dose-dependent manner

“…Similar results are reported for Anabaena sp. (Ning et al 2002), Microcystis aeruginosa (Bouchard & Purdie 2011), Dunaliella viridis Teodoresco (Jimenez et al 2009) and Skeletonema costatum (Greville) Cleve (Chung et al 2005). However, we found only diffuse low molecular weight bands on agarose gels at 48 h following 250 mM H 2 O 2 exposure, not DNA laddering.…”

“…Various types of PCD, with overlapping morphological and physiological hallmarks, have been described in unicellular organisms (Madeo et al 2002;Herker et al 2004;Kroemer et al 2009;Reape & McCabe 2010). Other cell death types termed 'paraptosis', 'aponecrosis' and 'autophagy', which are fundamentally different from apoptosis, have also been described (Jimenez et al 2008(Jimenez et al , 2009. However, few studies have dealt with PCD in cyanobacteria.…”

Differential nitric oxide synthesis and host apoptotic events correlate with bleaching susceptibility in reef corals

“…This either indicates that caspase-like enzyme activity is almost non-existent in this species or it suggests that the enzymes that are present are so dissimilar to those in A. millepora or P. damicornis that they are unable to cleave the same substrate. The latter is highly improbable, as DEVDase activity has been successfully quantified in Hydra (Lasi et al 2010), unicellular chlorophyte algae (Jimenez et al 2009), and sponges (Wiens et al 2003). We can therefore conclude that baseline caspase-like enzyme activity in M. digitata is negligible.…”

Temporal variation of caspase 3-like protein activity in cultures of the harmful dinoflagellates Karenia brevis and Karenia mikimotoi

“…However, several other species of microalgae have been proposed to undergo programmed cell death (PCD), necrosis (Dunn et al, 2002) as well as a range of intermediate cell death modes (Franklin and Berges, 2004;Franklin et al, 2006;Jiménez et al, 2009). Because it results in the complete loss of the organism, the occurrence of PCD in unicellular cells seems counterintuitive.…”

“…Although caspases have never been identified in microalgae, antibodies raised against human caspases have been shown to crossreact with extracts of, among others, marine cyanobacteria and chlorophytes (Segovia et al, 2003;Berman-Frank et al, 2004;Moharikar et al, 2006;Zuppini et al, 2007). Additionally, caspase activity assays performed using artificial substrates (designed to be specific for mammalian caspases) showed increases in caspase-like protein activities associated with the occurrence of death in a variety of microalgal species under stress conditions (Berman-Frank et al, 2004;Segovia and Berges, 2005;Ross et al, 2006;Zuppini et al, 2007;Bidle and Bender, 2008;Darehshouri et al, 2008;Affenzeller et al, 2009;Bouchard and Yamasaki, 2009;Jiménez et al, 2009). Finally, the use of mammalian caspase inhibitors has been shown to prevent the occurrence of death in diatoms, dinoflagellates and chlorophytes (Vardi et al, 1999;Bidle and Bender, 2008;Segovia and Berges, 2009).…”