Environmentally induced plasticity of programmed DNA elimination boosts somatic variability in Paramecium tetraurelia.

Can ecological changes impact somatic genome development? Efforts to resolve this question could reveal a direct link between environmental changes and somatic variability, potentially illuminating our understanding of how variation can surface from a single genotype under stress. Here, we tackle this question by leveraging the biological properties of ciliates. When Paramecium tetraurelia reproduces sexually, its polyploid somatic genome regenerates from the germline genome through a developmental process that involves the removal of thousands of ORF-interrupting sequences known as internal eliminated sequences (IESs). We show that exposure to nonstandard culture temperatures impacts the efficiency of this process of programmed DNA elimination, prompting the emergence of hundreds of incompletely excised IESs in the newly developed somatic genome. These alternative DNA isoforms display a patterned genomic topography, impact gene expression, and might be inherited transgenerationally. On this basis, we conclude that environmentally induced developmental thermoplasticity contributes to genotypic diversification in Paramecium.

Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair.

DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

A genetic dissection of the photophobic response of Paramecium tetraurelia.

Paramecium tetraurelia displayed two behavioral responses upon the initiation of a light stimulus at 7 x 10(4) lux. The cells exhibited a photophobic response in the form of behavioral avoiding reactions, followed by an increase in forward swimming velocity that was significantly higher than prior to the light stimulus activation. It was determined that an intensity of approximately 6.5 x 10(3) lux was required to initiate a moderate avoidance behavioral response. Following the avoiding response, a gradual increase in speed occurred as the intensity increased, indicating that increased swimming speeds are dependent on the light intensity. Two mutants, pawnA and Dancer, were utilized since they affect known Ca(2+)-currents of the cell. The use of pawnA cells, which lack voltage-dependent Ca(2+) channel activity, showed that the two responses to light could be genetically separated, in that the cells showed no avoiding reactions, but did increase their swimming speed. The Dancer cells, which display exaggerated Ca(2+) channel activity, exhibited similar initial avoiding responses as the wild type cells, however did not increase their swimming speed as the intensity of the light was increased. This phenotype as replicated in wildtype cells that had been placed in 25 µM 8-Br-cGMP. These data demonstrate that the photophobic light response of Paramecium tetraurelia can be genetically dissected as a means of elucidating the molecular mechanisms of the light response.

A circadian clock regulates sensitivity to cadmium in Paramecium tetraurelia.

The heavy metal cadmium is a dangerous environmental toxicant that can be lethal to humans and other organisms. This paper demonstrates that cadmium is lethal to the ciliated protozoan Paramecium tetraurelia and that a circadian clock modulates the sensitivity of the cells to cadmium. Various concentrations of cadmium were shown to increase the number of behavioral responses, decrease the swimming speed of cells, and generate large vacuole formation in cells prior to death. Cells were grown in either 12-h light/12-h dark or constant dark conditions exhibited a toxic response to 500 microM CdCl(2); the sensitivity of the response was found to vary with a 24-h periodicity. Cells were most sensitive to cadmium at circadian time 0 (CT0), while they were least sensitive in the early evening (CT12). This rhythm persisted even when the cells were grown in constant dark. The oscillation in cadmium sensitivity was shown to be temperature-compensated; cells grown at 18 degrees C and 28 degrees C had a similar 24-h oscillation. Finally, phase shifting experiments demonstrated a phase-dependent response to light. These data establish the criteria required for a circadian clock and demonstrate that P. tetraurelia possesses a circadian-influenced regulatory component of the cadmium toxic response. The Paramecium system is shown to be an excellent model system for the study of the effects of biological rhythms on heavy metal toxicity.

A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila.

Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.

Chemosensory signal transduction in paramecium.

Paramecia are ciliated single-cell eukaryotic organisms that can respond to chemical cues in their environment. Glutamate is among those cues, which attract cells. We describe briefly here the following attributes of glutamate chemoresponse: 1) Cells are attracted to L-glutamate relative to KCl at high concentrations of glutamate. 2) There are at least two specific, relatively low affinity glutamate binding sites on the cell surface. Glutamate can be displaced from only one of the binding sites by inosine monophosphate (IMP), and quisqualate displaces glutamate from the second site, which is likely to be the glutamate receptor involved in attraction to glutamate. 3) IMP is a repellent and does not act synergistically with glutamate, whereas guanosine monophosphate (GMP) does. 4) Similarly, glutathione is an attractant, but glutamate and glutathione appear to use different transduction pathways. 5) Glutamate hyperpolarizes the cell. The ionic mechanism is not yet verified, but is likely to involve a K conductance. 6) Glutamate induces a rapid and robust increase in cAMP in the cell. Protein kinase A (PKA) is possibly involved in the transduction pathway because kinase inhibitors such as H7 and H8 inhibit glutamate response, but do not affect responses to other attractants, such as acetate and ammonium. Activation of PKA by the rapid rise in cAMP may sustain the hyperpolarization phosphorylation and activation of the plasma membrane calcium pump. 7) Candidate glutamate binding proteins are being identified among the cell surface proteins with the use of affinity chromatography.

Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in paramecium.

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the alpha- and beta- tubulin subunits are modified by only 1-5 and 2-9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

Influence of extremely low frequency electromagnetic fields on the swimming behavior of ciliates.

Different species of ciliates (Paramecium biaurelia, Loxodes striatus, Tetrahymena thermophila) have been taken as model systems to study the effects of extremely low-frequency electromagnetic fields (50 Hz, 0.5-2.0 mT) on the cellular level. A dose-dependent increase in the mean swimming velocity and a decrease in the linearity of cell tracks were observed in all wild-type cells. In contrast, field-exposure did not increase the number of directional turns of the Paramecium tetraurelia pawn mutant (d4-500r), which is characterized by defective Ca2+-channels. The described changes indicate a direct effect of low frequency electromagnetic fields on the transport mechanisms of the cell membrane for ions controlling the motile activity of cilia.

Effect of SERCA pump inhibitors on chemoresponses in Paramecium.

Inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-dependent ATPase) calcium pumps were used to investigate the involvement of internal Ca2+ stores in the GTP response in Paramecium. External application of these inhibitors was found to dramatically alter the typical behavioral and electrophysiological responses of Paramecium to extracellular chemical stimulation. In particular, 2,5-di-tert-butylhydroquinone (BHQ) strongly inhibited the backward swimming response of paramecia to externally applied GTP, though it did not inhibit the associated whirling response. BHQ also prolonged the normally brief electro-physiological response of these cells to GTP. BHQ completely blocked the behavioral and electrophysiological responses of Paramecium to extracellular Ba2+, but had no measurable effect on the behavioral or electrophysiological responses of these cells to another depolarizing stimulus, elevated external K+ concentration. These results suggest the involvement of nonciliary Ca2+ ions in the GTP and Ba2+ responses.

Mechanochemical aspects of axonemal dynein activity studied by in vitro microtubule translocation.

We have determined the relationship between microtubule length and translocation velocity from recordings of bovine brain microtubules translocating over a Paramecium 22S dynein substratum in an in vitro assay chamber. For comparison with untreated samples, the 22S dynein has been subjected to detergent and/or to pretreatments that induce phosphorylation of an associated 29 kDa light chain. Control and treated dyneins have been used at the same densities in the translocation assays. In any given condition, translocation velocity (v) shows an initial increase with microtubule length (L) and then reaches a plateau. This situation may be represented by a hyperbola of the general form v = aL/(L+b), which is formally analogous to the Briggs-Haldane relationship, which we have used to interpret our data. The results indicate that the maximum translocation velocity Vo(= a) is increased by pretreatment, whereas the length constant KL(= b), which corresponds to Km, does not change with pretreatment, implying that the mechanochemical properties of the pretreated dyneins differ from those of control dyneins. The conclusion that KL is constant for defined in vitro assays rules out the possibility that the velocity changes seen are caused by changes in geometry in the translocation assays or by the numbers of dyneins or dynein heads needed to produce maximal translocational velocity. From our analysis, we determine that f, the fraction of cycle time during which the dynein is in the force-generating state, is small--roughly 0.01, comparable to the f determined previously for heavy meromyosin. The practical limits of these mechanochemical changes imply that the maximum possible ciliary beat frequency is about 120 Hz, and that in the physiological range of 5-60 Hz, beat frequency could be controlled by varying the numbers of phosphorylated outer arm dyneins along an axonemal microtubule.

The dynein genes of Paramecium tetraurelia: the structure and expression of the ciliary beta and cytoplasmic heavy chains.

The genes encoding two Paramecium dynein heavy chains, DHC-6 and DHC-8, have been cloned and sequenced. Sequence-specific antibodies demonstrate that DHC-6 encodes ciliary outer arm beta-chain and DHC-8 encodes a cytoplasmic dynein heavy chain. Therefore, this study is the first opportunity to compare the primary structures and expression of two heavy chains representing the two functional classes of dynein expressed in the same cell. Deciliation of paramecia results in the accumulation of mRNA from DHC-6, but not DHC-8. Nuclear run-on transcription experiments demonstrate that this increase in the steady state concentration of DHC-6 mRNA is a consequence of a rapid induction of transcription in response to deciliation. This is the first demonstration that dynein, like other axonemal components, is transcriptionally regulated during reciliation. Analyses of the sequences of the two Paramecium dyneins and the dynein heavy chains from other organisms indicate that the heavy chain can be divided into three regions: 1) the sequence of the central catalytic domain is conserved among all dyneins; 2) the tail domain sequence, consisting of the N-terminal 1200 residues, differentiates between axonemal and cytoplasmic dyneins; and 3) the N-terminal 200 residues are the most divergent and appear to classify the isoforms. The organization of the heavy chain predicts that the variable tail domain may be sufficient to target the dynein to the appropriate place in the cell.

Subplasmalemmal Ca2+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum.

Isolated subplasmalemmal Ca2+ stores ('alveolar sacs') from Paramecium tetraurelia cells sequester 45Ca2+ depending on ATP concentration. 45Ca2+ uptake is sensitive to SERCA-type Ca(2+)-ATPase inhibitors. They cause a slow release of 45Ca2+, as does caffeine. Of some importance are also the negative results we obtained with ryanodine, inositol 1,4,5-trisphosphate (InsP3), cyclic adenosinediphosphoribose (cADPR), 3',5'-cyclic guanosine monophosphate (cGMP, +/- beta-nicotinamide-adenine dinucleotide) or with increased [Ca2+]. These data were corroborated by experiments in vivo, including microinjection studies. Again ryanodine, InsP3, cADPR or cGMP did not trigger exocytosis, the trigger effect of SERCA inhibitors was sluggish, whereas caffeine induced exocytosis in a dose-dependent fashion. We then tested 45Ca2+ release also with isolated cell cortices (cell fragments containing cell membranes with stores and secretory organelles still attached). Under conditions which initiate exocytosis in vitro (depending on [ATP], reduction of [Mg2+] in presence of Ca2+; c.f. Lumpert et al. 1990, Biochem. J. 269, 639) we observed significant 45Ca2+ release with cortices as with isolated alveolar sacs. Our interpretation is as follows. (a) Alveolar sacs have a SERCA-type Ca(2+)-pump. (b) They have some sensitivity to caffeine, but none to ryanodine, InsP3 or cADPR. (c) There might be a direct functional coupling of these subplasmalemmal Ca2+ stores to the plasmalemma to which they are connected via feet-like structures; also like the SR, activation of this store is modulated by Mg2+ and ATP.

Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

Regulation of peptide-calmodulin complexes by protein kinase C in vivo.

We used the freshwater protozoan Paramecium tetraurelia to investigate the potential regulation by protein kinase C of calmodulin interactions with binding peptides in intact cells. In these organisms, an action potential results in membrane depolarization and a period of backward swimming; repolarization and a return to forward swimming requires the presence of normal calmodulin. We postulated that injection of high-affinity calmodulin binding peptides might interfere with repolarization and thus prolong the period of membrane depolarization. Synthetic peptides spanning the protein kinase C phosphorylation site/calmodulin-binding domains of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (also known as F52 or MacMARCKS) were injected into cells; these caused a 2- to 3-fold increase in the duration of backward swimming. Similar changes were seen with two other calmodulin-binding peptides. This behavioral response could be prevented by coinjecting calmodulin. Activation of Paramecium protein kinase C with an active phorbol ester completely reversed (within 3 min) the behavioral effects of the normal MARCKS and MARCKS-related protein peptides. Injection of a nonphosphorylatable peptide, in which alanines were substituted for serines, resulted in the usual behavioral response; however, this was not reversed by phorbol ester treatment. The corresponding aspartate-substituted peptide, which has a 10-fold lower affinity for calmodulin, did not prolong backward swimming. These data suggest that these peptides can form complexes with calmodulin at the calcium concentrations that prevail in intact Paramecium cells and that such complexes can be disrupted by protein kinase C phosphorylation of the peptides.