Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii

Abstract

Background and aims: Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied.

Methods: Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy.

Key results: MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death.

Conclusions: In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of the cells, algal PCD may take different forms and proceed through different pathways.

Fig. 1.

Effect of MP on cell death in C. reinhardtii. (A) Cell-death kinetics in 1 to 30 μm MP-treated cell suspension (approx. 3 × 106 cells mL⁻¹). Cell death was scored at 24-h intervals (0–120 h) after MP application following FDA staining of the living cells, and was calculated as a percentage of dead cells to total number of cells. (B) MP-induced cell death in algal suspension grown in fresh (FM) or in conditioned (CM) medium. Fresh algal cells were grown in conditioned medium collected from cultures treated for 24 h with 1 μm MP and cell death was scored after an additional 24-h treatment with 1 μm MP. Fresh cells without further MP treatment were also grown in conditioned medium from MP-treated and non-MP-treated cells. FM-no MP, Control non-treated cells grown in fresh medium; CM-no MP, cells grown in conditioned medium collected from non-MP-treated cells; CM + MP, cells grown in conditioned medium collected from MP-treated cells; FM + MP, MP-treated cells grown in fresh medium; CM + MP; +MP, cells grown in conditioned medium collected from MP-treated cells and exposed to additional treatment with MP. Error bars indicate ± s.e.m.

Fig. 2.

Temporal pattern of the effect of caspase inhibitors on MP-induced cell death in C. reinhardtii. The cells were treated with 1 μm MP and combinations of MP and inhibitors. (A) Cell death in response to 10 µm of the broad-range caspase inhibitor Z-Asp-CH2-DCB and 1 µm of caspase-3 inhibitor Ac-DEVD-CHO. (B) Cell death in response to 10 µm of caspase-1 inhibitor Ac-YVAD-CMK. Cell death was scored at the indicated time points after application of the chemicals following FDA staining of the living cells, and was calculated as a percentage of dead cells to total number of cells. Error bars indicate ± s.e.m. Cell-death effect of the chemicals at the individual time points of analyses is compared by LSD (P ≤ 0·05) as follows: (A) 0·2 h, 9·14; 0·5 h, 10·62; 2·0 h, 7·16; 12·0 h, 8·54; 24·0 h, 11·82; (B) 0·2 h, 6·17; 0·5 h, 7·65; 2·0 h, 4·73; 12·0 h, 8·82; 24·0 h, 11·41.

Fig. 3.

Kinetics of YVADase activity in MP-treated C. reinhardtii. Enzymatic activity was assayed in a time span of 0–24 h at consecutive time points after administration of the chemicals (1 µm MP, 10 µm Ac-YVAD-CMK and their combination). Control cells were left untreated. The fluorescence was measured and the relative caspase activity (normalized by cell count – 3 × 106 cells mL⁻¹) was calculated as F(t₁) – F(t₀) (for details refer to Materials and methods). Error bars indicate ± s.e.m.

Fig. 4.

Fluorescent field images of C. reinhardtii. The living cells were stained with FDA and the dead cells were distinguished by chlorophyll fluorescence: (A) untreated control cells; (B) cells treated with 1-μm MP; (C) cells treated with 1-μm MP + 10 µm of the caspase-1 inhibitor Ac-YVAD-CMK. The images were collected 24 h after administration of the chemicals and cell counting was carried out at ×200 magnification using a fluorescent microscope (Nikon Eclipse; Vienna, Austria; TS 100, filter B-2A, exciter 450–490, DM 505, BA 520) equipped with Nikon DXM 1200 digital camera. Thick arrows indicate dead cells; thin arrows indicate living cells. Scale bars = 50 µm.

Fig. 5.

CLSM images representing the cellular morphology of MP-treated C. reinhardtii cells: (A) FDA-positive living cell showing intact protoplast; (B) FDA staining of a living (diffuse nuclei visible) and a dead cell (FDA negative); (C) PI staining of the cells in (B) – note the PI-positive compacted nucleus and the homogenous content of the dead cell; (D) fluorescent image showing a multitude of lysosome-like LT-positive vesicles (white stars); (E) transmission light image of (D) in which the lytic vesicles (red stars) appear as dark round organelles – note retraction of the protoplast (black arrows) from the cell wall; (F) LT-stained living cell – no LT-positive vesicles are detected; (G) chlorophyll fluorescence emitted from the intact cup-shaped chloroplast of a living cell; (H) chlorophyll fluorescence emitted from a disintegrated chloroplast in a dead cell with large vacuoles; (I) PI-stained vacuolated dead cell with shrunken protoplast and compacted nucleus; (J) PI-stained dead cell with compacted nucleus and nearly empty cell-walled corpse (white arrow) containing only the remains of the digested protoplast; (K) PI-stained dead cells expressing features of different cell-death types – the upper cells show necrotic morphology with shrunken protoplast and condensed nucleus while the lower cells show an empty cell-walled corpse which is a feature of vacuolar cell death (white arrow); (L) PI-stained dead cell showing a feature of necrotic cell death – the remains of a largely unprocessed protoplast and nucleus. Images (A–C) and (G–K) were collected 24 h after treatment with 1 μm MP; images (D–F) were collected 2 h after treatment with 1 μm MP; image (L) was taken 24 h after treatment with 5 μm MP. Cellular morphology was examined using a TCS SP2 AOBS CLSM (Leica-Microsystems GmbH, Mannheim, Germany) mounted on an inverted Leica DM IRE2 microscope. Three different lasers (405, 488 and 561 nm) were employed for excitation and three emission channels for fluorescence imaging and one separate channel for non-confocal transmission imaging. Overlays and orthogonal projections were made using the Leica Confocal software. Abbreviations: chl, chloroplast; dc, dead cell; lc, living cell; nu, nucleus; p, protoplast; pyr, pyrenoid; cw, cell wall; v, vacuole. Scale bars = 5 µm.

Fig. 6.

DNA laddering in MP-treated C. reinhardtii. DNA was isolated from a 1 μm MP-treated suspension of C. reinhardtii cells and the analysis were performed at consecutive time points of 10 min, 30 min, 1, 2, 12 and 24 h after treatment with MP. DNA was subjected to agarose gel electrophoresis using equal amounts of 6 µg DNA per lane as described in Materials and methods. Lane 1: M, marker; lane 2: C, non-treated control; lanes 3–8, time points after MP application.

Fig. 7.

Comet assay of DNA integrity in MP-treated C. reinhardtii: (A) fluorescent images of comets (scale bar = 50 µm); (B) percentage of comet-positive nuclei. Chlamydomonas reinhardtii cells in suspension were treated with 1 μm MP. Samples were taken and analysed for comet DNA at consecutive time points at 10 min, 30 min, 2 h and 24 h. Comets were visually categorized in five classes as described in Materials and methods. Error bars indicate ± s.e.m.