Jacinta S. D’Souza

A homologue of the defender against the apoptotic death gene (dad1) in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death

We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1) from Chlamydomonas reinhardtii cells. Using polymerase chain reaction (PCR), we investigated its expression in the execution process of programmed cell death (PCD) in UV-C exposed dying C. reinhardtii cells. Reverse-transcriptase (RT)-PCR showed that C. reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C. reinhardtii cells. We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1) and the physiological changes that occur in C. reinhardtii cells upon exposure to 12 J/m2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors. The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation. The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215) from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2); this sequence was found to show 100% identity, both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues. The deduced amino acid sequence of the putative C. reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56% identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens, Sus scrofa, Gallus gallus, Rattus norvegicus and Mus musculus.

Co-expressed recombinant human Translin-Trax complex binds DNA

Trax, expressed alone aggregates into insoluble complexes, whereas upon co‐expression with Translin becomes readily soluble and forms a stable heteromeric complex (∼430 kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI‐TOF‐MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420 kDa) or a heptamer each of Trax and Translin (calculated MW 425 kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431 kDa). The complex binds single‐stranded/double‐stranded DNA. ssDNA gel‐shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin “chaperones” Trax and forms heteromeric complex that is DNA binding competent.

Programmed cell death is induced by hydrogen peroxide but not by excessive ionic stress of sodium chloride in the unicellular green alga Chlamydomonas reinhardtii

Eukaryotic microalgae serve as indicators of environmental change when exposed to severe seasonal fluctuations. Several environmental stress conditions are known to produce reactive oxygen species in cellular compartments, resulting in oxidative damage and apoptosis. The study of cell death in higher plants and animals has revealed the existence of an active ‘programmed cell death’ (PCD) process and similarities between such processes suggest an evolutionary origin. A study was undertaken to examine the morphological, biochemical and molecular responses of the unicellular green alga Chlamydomonas reinhardtii after exposure to oxidative (10 mM H2O2) and osmotic (200 mM NaCl and 360 mM sorbitol) stress. Concentrations of H2O2 (2–50 mM), NaCl and sorbitol (100–800 mM) were negatively correlated with growth. Biochemical analyses showed an increase in intracellular H2O2 production (2.2-fold with H2O2 and ~1.2–1.4-fold with NaCl and sorbitol) and activities of some antioxidant enzymes [super oxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)]. Alteration of mitochondrial membrane potential (MMP) was observed upon treatment with H2O2 and NaCl, but not with sorbitol, indicating that the ionic stress component of NaCl altered the MMP. In addition, H2O2 led to the activation of a caspase-3-like protein, increase in the cleavage of a poly(ADP) ribose polymerase-1 (PARP-1)-like enzyme and formation of DNA nicks and laddering. With NaCl and sorbitol, no caspase activation, nor oligonucleosomal DNA laddering was observed, indicating non-apoptotic death. However, genomic DNA of NaCl (800 mM)-stressed cells, but not those of sorbitol-treated cells showed complete shearing. We conclude that the ionic rather than the osmotic component of NaCl leads to necrosis. These results unequivocally suggest that the vegetative cells of C. reinhardtii respond differentially to various stress agents, leading to different death types in the same organism. Moreover, unlike most other organisms, when exposed to NaCl this alga does not undergo PCD.

Ca2+ dPKs from the protonema of the moss Funaria hygrometrica: Effect of indole-acetic acid and cultural parameters on the activity of a 44 kDa Ca2+ dPK

Using the in-gel kinase assay, four polypeptides of Mr 44, 48, 63, and 70 kDa showed calcium-dependent phosphorylation in the crude extract of the moss Funaria hygrometrica Hedw. Analysis by SDS-PAGE suggests that these phosphopolypeptides represent the calcium-dependent protein kinases (PK), which could undergo autophosphorylation and phosphorylate casein. Temporal changes in the activity of PK in cells grown under different conditions were detectable using the in-gel kinase assay. The 44 kDa Ca 2+ dPK showed an increase in activity in cells grown either in the presence of auxin, at pH 5, or when starved of nitrate. This PK is shown to share epitopes with calmodulin suggesting that it could be a PK with calmodulin-like domain. The in-gel kinase assay, its possible applications and the existence of multiple Ca 2+ dPKs in moss are discussed.

Quantifying ligand-receptor interactions for gorge-spanning acetylcholinesterase inhibitors for the treatment of Alzheimer's disease.

There is a need for continued development of acetylcholinesterase (AChE) inhibitors that could prolong the life of acetylcholine in the synaptic cleft and also prevent the aggregation of amyloid peptides associated with Alzheimer’s disease. The lack of a 3D-QSAR model which specifically deconvulates the type of interactions and quantifies them in terms of energies has motivated us to report a CoRIA model vis-à-vis the standard 3D-QSAR methods, CoMFA and CoMSIA. The CoRIA model was found to be statistically superior to the CoMFA and CoMSIA models and it could efficiently extract key residues involved in ligand recognition and binding to AChE. These interactions were quantified to gauge the magnitude of their contribution to the biological activity. In order to validate the CoRIA model, a pharmacophore map was first constructed and then used to virtually screen public databases, from which novel scaffolds were cherry picked that were not present in the training set. The biological activities of these novel molecules were then predicted by the CoRIA, CoMFA, and CoMSIA models. The hits identified were purchased and their biological activities were measured by the Ellman’s method for AChE inhibition. The predicted activities are in unison with the experimentally measured biological activities.

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved ‘9+2’ axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26–57%) and beat frequencies (by 8–16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.

Flagella-generated forces reveal gear-type motor in single cells of the green alga, Chlamydomonas reinhardtii

Optically trapped single cells of the biflagellated, green alga, Chlamydomonas reinhardtii, rotate. The rotational dynamics of trapped wild-type and mutant cells show that functional flagella play a decisive role: the entire flagellar apparatus (central microtubules, radial spokes, and dynein arms) is involved. Any aberration in this apparatus leads to non-functionality, indicating a gear-type mechanism. The translational and rotational motions of the wild-type and mutant cells do not differ significantly. Optical forces alone do not play a vital role in the rotational dynamics of this cellular motor, making them useful as probes of the internal dynamics without external influence.

Optical control of filamentation-induced damage to DNA by intense, ultrashort, near-infrared laser pulses

We report on damage to DNA in an aqueous medium induced by ultrashort pulses of intense laser light of 800 nm wavelength. Focusing of such pulses, using lenses of various focal lengths, induces plasma formation within the aqueous medium. Such plasma can have a spatial extent that is far in excess of the Rayleigh range. In the case of water, the resulting ionization and dissociation gives rise to in situ generation of low-energy electrons and OH-radicals. Interactions of these with plasmid DNA produce nicks in the DNA backbone: single strand breaks (SSBs) are induced as are, at higher laser intensities, double strand breaks (DSBs). Under physiological conditions, the latter are not readily amenable to repair. Systematic quantification of SSBs and DSBs at different values of incident laser energy and under different external focusing conditions reveals that damage occurs in two distinct regimes. Numerical aperture is the experimental handle that delineates the two regimes, permitting simple optical control over the extent of DNA damage.

Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella

BackgroundFlagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical ‘9 + 2’ cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain.ResultsWe found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus.ConclusionIt appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.

Dynamics of DNA and Portein-DNA Complexes Viewed Through Time-Domain Fluorescence

One of the recurring themes in modern biology, namely, dynamics along with structure forms the basis of function of biomolecular systems is getting increasing level of support from various experimental and molecular dynamics simulation techniques. In this chapter, the power of time-resolved fluorescence spectroscopy in bringing to light various intricate aspects of DNA dynamics and, in some cases, their correlation with function has been shown. We believe that this is a very fruitful approach in seeking explanations for complex activity o f DNA based systems especially in the light of the enormous level of structural information being gathered in recent times.