Mechanisms of apoptosis in Crustacea: What conditions induce versus suppress cell death?

Abstract

Arthropoda is the largest of all animal phyla and includes about 90% of extant species. Our knowledge about regulation of apoptosis in this phylum is largely based on findings for the fruit fly Drosophila melanogaster. Recent work with crustaceans shows that apoptotic proteins, and presumably mechanisms of cell death regulation, are more diverse in arthropods than appreciated based solely on the excellent work with fruit flies. Crustacean homologs exist for many major proteins in the apoptotic networks of mammals and D. melanogaster, but integration of these proteins into the physiology and pathophysiology of crustaceans is far from complete. Whether apoptosis in crustaceans is mainly transcriptionally regulated as in D. melanogaster (e.g., RHG 'killer' proteins), or rather is controlled by pro- and anti-apoptotic Bcl-2 family proteins as in vertebrates needs to be clarified. Some phenomena like the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) are apparently lacking in crustaceans and may represent a vertebrate invention. We speculate that differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for some species to survive harsh environmental insults. Pro-apoptotic stimuli described for crustaceans include UV radiation, environmental toxins, and a diatom-produced chemical that promotes apoptosis in offspring of a copepod. Mechanisms that serve to depress apoptosis include the inhibition of caspase activity by high potassium in energetically healthy cells, alterations in nucleotide abundance during energy-limited states like diapause and anoxia, resistance to opening of the calcium-induced MPTP, and viral accommodation during persistent viral infection. Characterization of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death.

Fig. 1

Overview of proteins that are possibly conserved in the apoptotic machineries of arthropods. Potential apoptotic players in crustaceans were identified by blasting a wide range of protein sequences with well known apoptotic functions found in the protein database of NCBI for D. melanogaster and H. sapiens against the NCBI database for non-redundant protein sequences (blast-p) and non-human, non-mouse EST’s (tblast-n). All proteins for which homologues in crustaceans were identified are printed and boxed in red. Crustacean homologues could be found for all proteins investigated with the exceptions of Smac/Diablo and the D. melanogaster RHG-proteins. The questions marks indicate apoptotic mechanisms for which data are completely lacking in crustaceans. For example, does Traf signaling activate JNK? Does JNK activate crustacean caspases, and if so, how (i.e., mediated through IAP inhibition, apoptosome activation, or direct interaction with caspases)? Does Apip inhibit recruitment of caspases to the apoptosome? Is mitochondrial outer membrane permeabilization (MOMP), as regulated by homologues of Bcl-2 family proteins, involved in signaling and amplifying apoptosis in crustaceans? For abbreviations please see text

Fig. 2

Phylogenetic tree obtained by neighbor-joining from multiple sequence alignments of the p20 and p10 subunit of crustacean, D. melanogaster (drICE), and H. sapiens caspases (casp-3, 7, and-9). The bootstrap values are added at each branch point and represent percentages of replicate trees in which the associated protein sequences clustered together (500 replicates). The tree is drawn to scale, with branch lengths displayed in the same units (i.e., number of amino acid substitutions per site) as the evolutionary distances used to infer the tree. Similar phylogenetic relationships were obtained based on maximum parsimony analysis. The tree resolves four major branches (I–IV) of which three are related. Analyses of phylogeny and molecular evolution were conducted using MEGA version 4.1 [149]. H arthropod subphylum Hexapoda; D Decapoda, M Maxillopoda, B Branchiopoda, all within arthropod subphylum Crustacea

Fig. 3

Phylogenetic tree obtained by neighbor-joining from multiple sequence alignments of Bcl-2 proteins for crustaceans and D. melanogaster (Drob-1 and Buffy). The bootstrap values are added at each branch point and represent percentages of replicate trees in which the associated protein sequences clustered together (500 replicates). The tree is drawn to scale, with branch lengths displayed in the same units (i.e., number of amino acid substitutions per site) as the evolutionary distances used to infer the tree. The same phylogenetic relationships were obtained based on maximum parsimony analysis of the corresponding cDNA sequences. The tree resolves three branches of which two are related. Analyses of phylogeny and molecular evolution were conducted using MEGA version 4.1 [149]. H arthropod subphylum Hexapoda; D Decapoda, M Maxillopoda, B Branchiopoda, all within arthropod subphylum Crustacea

Fig. 4

Sequence alignment of a deduced protein (Apaf-1) from D. pulex (FE416154.1) with H. sapiens Apaf-1 (NP_001151.1). The translated EST from D. pulex includes the Caspase Recruitment and Activation Domain (CARD, shaded in light), the highly conserved NACHT-NTPase domain (shaded in dark), and is highly conserved with the human protein. Importantly, no significant homologies to Dark, the Apaf-1 analog from D. melanogaster, were found (c.f., Table 1). ‘*’ = identical. ‘:’ = conserved substitutions. ‘.’ = semi-conserved substitution

Fig. 5

Evolutionary relationships between Apaf-1 Interacting Proteins (Apips) from H. sapiens, D. melanogaster and deduced proteins from crustacean ESTs. The phylogenetic tree was obtained by the neighbor-joining method. The bootstrap values are added at each branch point and represent percentages of replicate trees in which the associated protein sequences clustered together (500 replicates). The tree is drawn to scale, with branch lengths displayed in the same units (i.e., number of amino acid substitutions per site) as the evolutionary distances used to infer the tree. The tree resolves two major branches. Analyses of phylogeny and molecular evolution were conducted using MEGA version 4.1 [149]. H arthropod subphylum Hexapoda; D Decapoda, M Maxillopoda, B Branchiopoda, all within arthropod subphylum Crustacea

Fig. 6

Swelling assay upon addition of Ca²⁺ and HgCl₂ (in the presence of 1 mM phosphate) for mitochondria isolated from hepatopancreas of the ghost shrimp Lepidophthalmus louisianensis. Mitochondrial swelling (i.e., a decrease in absorbance) is not observed after addition of 1 mM CaCl₂ to energized mitochondria (succinate plus rotenone). Mitochondrial volume does increase after addition of 20 μM HgCl₂. b The absence of Ca²⁺-induced release of Ca²⁺ by mitochondria isolated from hepatopancreas of L. louisianensis. Mitochondria were incubated in exogenously added Ca²⁺ in the presence of the Ca²⁺ probe fluo-5 N, which reports the extra-mitochondrial Ca²⁺. Fluorescence expressed as a percentage of the total fluorescence when the dye is saturated with Ca²⁺ (% maximal fluorescence). Control samples contained no mitochondria. Energized mitochondria (succinate plus rotenone) reduced the concentration of exogenously added Ca²⁺ at all concentrations investigated, relative to controls. De-energized mitochondria (no succinate or rotenone, but with FCCP) were less effective in calcium uptake than energized mitochondria. Importantly, no release of Ca²⁺ was observed across the entire range of exogenously added Ca²⁺ by either the energized or de-energized mitochondria. Each bar represents mean ± SD of n = 4 experiments. * Significantly different from control (ANOVA, Tukey’s pair-wise comparison, P < 0.001) (Modified after [74])

Fig. 7

Influence of Ca²⁺ and KCl on caspase 9 (casp-9)-like activity in cytosolic extracts from diapause embryos of Artemia franciscana. Casp-9-like activity was measured as the increase in fluorescence due to cleavage of the fluorogenic substrate Z-LEHD-R110. As KCl concentrations are decreased, the addition of 5 mM calcium promotes an increase in activity of casp-9. At 0 mM KCl, the addition of 5 mM calcium leads to a 4.86 ± 0.19 (SD)-fold (n = 3) increase in activity. The activation is even more dramatic (67-fold) when expressed relative to the depressed activity at 150 mM KCl. The influence of Ca²⁺ and KCl was expressed as fold changes relative to control. Each value is expressed as the mean (SD) of n = 3–5 experiments. * Statistically different activity after calcium addition (P < 0.05) (Modified after [20])

Fig. 8

Impact of ATP, GTP, and ADP (in the presence of MgCl₂) on casp-9-like activity in cytosolic extracts from diapause embryos of Artemia franciscana. Casp-9-like activity was measured as the increase in fluorescence due to cleavage of the fluorogenic substrate Z-LEHD-R110. Addition of 2.5 mM MgCl₂ in the absence of nucleotides leads to a 1.7-fold increase in casp-9 activity in diapause extract. A biphasic pattern of activation followed by inhibition is observed for ATP and GTP. ADP is highly inhibitory at all concentrations investigated. Each value is expressed as the mean (SD) of n = 3–9 experiments. * Statistically different to 0 mM added nucleotides (P < 0.05) (Modified after [20])