Jacinta S. D'Souza

APOPTOTIC-LIKE CELL DEATH PATHWAY IS INDUCED IN UNICELLULAR CHLOROPHYTE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELLS FOLLOWING UV IRRADIATION: DETECTION AND FUNCTIONAL ANALYSES1

Chlamydomonas reinhardtii (Ehrenberg) cells exhibited cell death process akin to that of apoptosis when exposed to ultraviolet (UV)‐C irradiation (1–100 J/m2). We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes, flipping of phosphatidylserine, and DNA fragmentation detected by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and oligonucleosomal DNA laddering assay. Interestingly, fluorescence imaging of DNA changes in UV‐C exposed cells, following PicoGreen staining, revealed that extra‐nuclear DNA disintegrates before that of nuclear changes, where the latter extensively diffuses out of the nuclear compartment, spreading into the whole cell and reaching the periphery of dying cells. Antibodies against a mammalian caspase‐3 shared epitopes with a protein of 28 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, growth experiments indicate that spent medium recovered from UV‐C exposed cells exhibit a protective effect against cell killing of fresh cultures of C. reinhardtii cells by UV irradiation. The protective effect of UV‐spent medium is not a general growth promotional response on normal cells, but rather, is specific to UV‐exposed cells. We propose a model that C. reinhardtii cells exposed to UV elicit apoptotic‐like changes, which in turn lead to an adaptive response in neighboring cells against fresh rounds of UV exposure, thereby promoting survival of the cell population.

Effect of intense, ultrashort laser pulses on DNA plasmids in their native state: strand breakages induced by in situ electrons and radicals

Single strand breaks are induced in DNA plasmids, pBR322 and pUC19, in aqueous media exposed to strong fields generated using ultrashort laser pulses (820 nm wavelength, 45 fs pulse duration, 1 kHz repetition rate) at intensities of 1-12  TW cm(-2). The strong fields generate, in situ, electrons and radicals that induce transformation of supercoiled DNA into relaxed DNA, the extent of which is quantified. Introduction of electron and radical scavengers inhibits DNA damage; results indicate that OH radicals are the primary (but not sole) cause of DNA damage.

Menadione-induced caspase-dependent programmed cell death in the green chlorophyte Chlamydomonas reinhardtii

Menadione, a quinone that undergoes redox cycles leading to the formation of superoxide radicals, induces programmed cell death (PCD) in animals and plants. In this study, we investigated whether the unicellular green alga Chlamydomonas reinhardtii P.A.Dangeard is capable of executing PCD upon exposure to menadione stress. We report here, the morphological, molecular, and biochemical changes after menadione exposure of C. reinhardtii cells. The effect of menadione on cell death has been shown to be dose‐dependent; 5–100 μM menadione causes 20%–46% cell death, respectively. It appears that growth is inhibited with the concomitant degradation of the photosynthetic pigments and by a decrease in the photosynthetic capacity. Being an oxidative stress, we found an H2O2 burst within 15 min of menadione exposure, followed by an increase in antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and ascorbate peroxidase [APX]) activities. In parallel, RT‐PCR was performed for transcript analyses of Mn‐SOD, CAT, and APX. Our results clearly revealed that expression of these genes were up‐regulated upon menadione exposure. Furthermore, classical hallmarks of PCD such as alteration of mitochondrial membrane potential, significant increase in caspase‐3‐like DEVDase activity, cleavage of poly (ADP) ribose polymerase (PARP)‐1‐like enzyme, and DNA fragmentation as detected by terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) assay and oligosomal DNA fragmentation were observed. Moreover, antibodies against a mammalian active caspase‐3 shared epitopes with a caspase‐3‐like protein of ~17 kDa; its pattern of expression and activity correlated with the onset of cell death. To the best of our knowledge, this is the first report on menadione‐induced PCD through a mitochondrian‐caspase protease pathway in an algal species.

Optically-controllable, micron-sized motor based on live cells

We demonstrate rotation of live Chlamydomonas reinhardtii cells in an optical trap; the speed and direction of rotation are amenable to control by varying the optical trapping force. Cells rotate with a frequency of 60-100 rpm; functional flagella are shown to play a decisive role in rotation. The rotating cells generate torque (typically ~7500-12000 pN nm) that is much larger than that generated chemically by a dynein head in vitro (40 pN nm). The total force associated with a rotating live cell (~10 pN) suggests that activity of only a small fraction (~5%) of dynein molecules per beat cycle is sufficient to generate flagellar motion.

Purification and characterization of a Ca2+-dependent/calmodulin-stimulated protein kinase from moss chloronema cells

We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the mossFunaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 (AM free Ca2+, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Underin vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.

KCl induces a caspase-independent programmed cell death in the unicellular green chlorophyte Chlamydomonas reinhardtii (Chlorophyceae)

Abstract: Programmed cell death (PCD) plays an important role in mediating adaptation responses under adverse conditions such as high salinity. To understand the molecular mechanism of adaptation of algal cells to salt, the freshwater alga Chlamydomonas reinhardtii was challenged with 200 mM KCl. In the present study, vegetative cells of C. reinhardtii undergo cell death when exposed to 200 mM KCl, this death being dose-dependent, with 100–800 mM KCl causing 16–64% cell death. Within half an hour of KCl exposure, a ~1.9-fold rise in the intracellular H2O2 content followed an increase in antioxidant enzyme (superoxide dismutats, catalase, and ascorbate peroxidase) activities and their transcript levels. Furthermore, apoptotic hallmarks such as disruption of mitochondrial membrane potential, DNA nicks, apoptosis-inducing factor (AIF) release into the cytoplasm, and genomic DNA fragmentation were observed. Interestingly, KCl stress did not stimulate caspase-3-like protease activity. Additionally, cells undergoing PCD showed characteristic shrinkage with an accumulation of lipids and vacuoles, along with degraded chloroplast. These results illustrate that KCl induces reactive oxygen species production that leads to AIF release from mitochondria, causing a caspase-independent cell death in C. reinhardtii.