Gamma-amino butyric acid (GABA) release in the ciliated protozoon Paramecium occurs by neuronal-like exocytosis.

Paramecium primaurelia expresses a significant amount of gamma-amino butyric acid (GABA). Paramecia possess both glutamate decarboxylase (GAD)-like and vesicular GABA transporter (vGAT)-like proteins, indicating the ability to synthesize GABA from glutamate and to transport GABA into vesicles. Using antibodies raised against mammalian GAD and vGAT, bands with an apparent molecular weight of about 67 kDa and 57 kDa were detected. The presence of these bands indicated a similarity between the proteins in Paramecium and in mammals. VAMP, syntaxin and SNAP, putative proteins of the release machinery that form the so-called SNARE complex, are present in Paramecium. Most VAMP, syntaxin and SNAP fluorescence is localized in spots that vary in size and density and are primarily distributed near the plasma membrane. Antibodies raised against mammal VAMP-3, sintaxin-1 or SNAP-25 revealed protein immunoblot bands having molecular weights consistent with those observed in mammals. Moreover, P. primaurelia spontaneously releases GABA into the environment, and this neurotransmitter release significantly increases after membrane depolarization. The depolarization-induced GABA release was strongly reduced not only in the absence of extracellular Ca(2+) but also by pre-incubation with bafilomycin A1 or with botulinum toxin C1 serotype. It can be concluded that GABA occurs in Paramecium, where it is probably stored in vesicles capable of fusion with the cell membrane; accordingly, GABA can be released from Paramecium by stimulus-induced, neuronal-like exocytotic mechanisms.

Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity.

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.

Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite.

In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immuno-reactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes), and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.

Trichocyst membrane fate in living Paramecium primaurelia visualized by multimodal confocal imaging.

Trichocysts are secretory organelles located at the surface of several ciliates, docked at the plasma membrane. Their secretion is similar to other exocytic processes: the trichocyst membrane fuses with the plasma membrane, its content is released outside the cell, and the membrane is retrieved back into the cell. The fate of the trichocyst membrane in living Paramecium primaurelia was investigated by inducing massive synchronous exocytosis in the presence of fluorescein isothiocyanate-conjugated lectins or of cationized ferritin. The marker is trapped within the retrieved trichocyst membrane sac, and many regularly spaced, fluorescent ghosts are formed. As time proceeds, the number of labeled ghosts decreases, and few fluorescent vacuoles appear within the cell. The relationship between trichocyst ghosts and the vesicles of the phagosome-lysosome system was examined by labeling cells with Texas Red-conjugated bovine serum albumin, a fluorescent marker for phagocytosis. Starting from two confocal images of the same cell labeled with the two fluorescent probes, a new single image was generated by associating each image with a different red or green value. This multimodal analysis showed that trichocyst ghosts fuse with secondary lysosomes or are incorporated into digestive vacuoles. The vacuolar content is degraded and fluorescence is then found in the vesicles of the phagosome-lysosome system, and, at last, in small weakly labeled vesicles located on the cell surface.

Time-variant analysis of organelle and vesicle movement during phagocytosis in Paramecium primaurelia by means of fluorescence confocal laser scanning microscopy.

Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical microscope (CLSM) to display and analyze formation, movement, and fusion of vesicles during the phagocytosis of Paramecium primaurelia, in the x-y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intracellular movement of food vacuoles from their formation at the cytostome to their egestion at the cytoproct was visualized, and food vacuoles were selected in a specific digestion stage. Small pinocytic vesicles are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enlarge the membrane of the nascent food vacuoles or fuse with stage II food vacuoles, when the vacuoles of stage II increase their size, changing from an acidic to an alkaline status. A multimodal analysis of confocal fluorescence images and the false-color technique were used to visualize vesicle movement vs. time. Starting from three images of the same cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color corresponding to each sampling point in time. The composite image shows that vesicles move away from the food vacuole in a scattered manner exhibiting changes in direction.

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Studies on the structure of sperm heads of Eledone cirrhosa by means of CLSM linked to bioimage-oriented devices.

We have used a confocal laser scanning optical microscope imaging device and a bioimage-oriented workstation equipped for augmented reality to study the helical sperm head of the octopus Eledone cirrhosa. This approach allows us to study different complex organisational motifs due to the spatial arrangement of linear helical structures. We consider this helical specimen an enlarged copy of one of the most important biostructures governing cell functioning such as chromatin-DNA. Moreover, this very same sample is made of highly compacted chromatin that can be studied at higher resolution, i.e., by means of scanning force microscopy. Fluorescence optical sectioning has been used to enter the spatial organisation. Three-dimensional images of single, twisted, and folded fibers are shown.

Improvement in volume estimation from confocal sections after image deconvolution.

The confocal microscope can image a specimen in its natural environment forming a 3D image of the whole structure by scanning it and collecting light through a small aperture (pinhole), allowing in vivo and in vitro observations. So far, the confocal fluorescence microscope (CFM) is considered a true volume imager because of the role of the pinhole that rejects information coming from out-of-focus planes. Unfortunately, intrinsic imaging properties of the optical scheme presently employed yield a corrupted image that can hamper quantitative analysis of successive image planes. By a post-image collection restoration, it is possible to obtain an estimate, with respect to a given optimization criterium, of the true object, utilizing the impulse response of system or Point Spread Function (PSF). The PSF can be measured or predicted so as to have a mathematical and physical model of the image-formation process. Further modelling and recording noise as an additive Gaussian process has used the regularized Iterative Constrained Tykhonov Miller (ICTM) restoration algorithm for solving the inverse problem. This algorithm finds the best estimate iteratively searching among the possible positive solutions; in the Fourier domain, such an approach is relatively fast and elegant. In order to compare the effective improvement in the quantitative image information analysis, we measured the volume of reference objects before and after image restoration, using the isotropic Fakir method.

Image analysis of lysosomal activity during the early clonal life of Paramecium primaurelia.

Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.

Changes in the endoplasmic reticulum structure of Paramecium primaurelia in relation to different cellular physiological states.

The fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], a vital dye utilized to stain the endoplasmic reticulum (ER) of animal and plant cells, has been used to visualize the ER-type structures of Paramecium primaurelia under confocal laser scanning microscopy (CLSM). The morphology of the ER has been studied in paramecia in different physiological conditions. Cells are analysed in early and late logarithmic growth phases, in stationary and in death phases, during shift-up by refeeding after starvation and shift-down by using a starvation medium. In log-phase growing paramecia, the ER constitutes an anastomosing membrane system consisting of short tubules and flattened sacs forming a peripheral network, which is abundant in the cortical region around the trichocysts and the ciliary basal bodies. The tubular network and cytoplasmic membranes are reduced in stationary-phase cells; the original conditions are restored in starved cells after refeeding. The analysis of serial optical sections collected by CLSM at 0.5 microm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells.

Acid phosphatase activity in mating type I and mating type II cell lines of Paramecium primaurelia.

The cellular acid phosphatase content, a marker enzyme for lysosomal activity, in Paramecium primaurelia mating type I and mating type II cells was determined by optical laser scanning microscopy. The naphthol AS-TR phosphatase-hexazotized pararosaniline method was used to visualize acid phosphatase activity by the light microscopy. Cell lines of both mating types were tested during culture life, from the early log phase to the death phase. The amount of acid phosphatase was higher in mating type II than in mating type I until the onset of the stationary phase, and then the values reversed. Indeed, during the log phase of growth, mating type II cells formed a higher number of food vacuoles, so that, by taking up a higher amount of bacteria, they sooner became deprived of food. It is suggested that, by lacking nutrients, their synthesis activities and acid phosphatase content were reduced as compared with mating type I cells.

Tubulin posttranslational modifications induced by cadmium in the sponge Clathrina clathrus.

As sessile filter feeders, sponges are exposed to environmental stress due to pollutants of both anthropogenic and natural origins and are able to accumulate harmful substances. Thus, sponges are considered a good tool for the biomonitoring of coastal areas. In this study, we used biochemical and immunocytochemical analyses to provide new data on the cadmium-related changes in sponge cells. In particular, we analyzed the effects of different concentrations of cadmium on the microtubule network in the calcisponge Clathrina clathrus. Quantitative densitometry of the immunoblots showed that, while the levels of α- and ß-tubulin remained relatively constant in C. clathrus when exposed to 1 and 5 µM CdCl2, there were progressive shifts in the levels of some tubulin isoforms. Exposure for 24h to sublethal concentrations of cadmium reduced the level of tyrosinated α-tubulin and enhanced the levels of acetylated and detyrosinated α-tubulin relative to the levels in controls. Confocal microscopy analysis of immunolabeled tissue sections showed that the inhibitory effect of cadmium was associated with a decrease in the labeling of the cells with a monoclonal antibody that recognizes tyrosinated α-tubulin. By contrast, the reactivity with a monoclonal antibody that recognizes acetylated α-tubulin and with a polyclonal antibody specific for detyrosinated α-tubulin was enhanced at the same time points. Because the acetylation and detyrosination of α-tubulin occur on stable microtubules, the marked enhancement of α-tubulin acetylation and detyrosination in Cd(2+)-treated cells indicates that divalent Cd ions stabilize microtubules. The possibility that Cd(2+) may increase the stability of cytoplasmic microtubules was tested by exposing Cd(2+)-treated cells to a cold temperature (0°C). As shown, the microtubule bundles induced by Cd(2+), which were labeled by the monoclonal antibodies against acetylated and detyrosinated α-tubulin, were resistant to cold.

Toxicity of metal oxide nanoparticles in immune cells of the sea urchin.

The potential toxicity of stannum dioxide (SnO2), cerium dioxide (CeO2) and iron oxide (Fe3O4) nanoparticles (NPs) in the marine environment was investigated using the sea urchin, Paracentrotus lividus, as an in vivo model. We found that 5 days after force-feeding of NPs in aqueous solutions, the three NPs presented different toxicity degrees, depending on the considered biomarkers. We examined: 1) the presence of the NPs in the coelomic fluid and the uptake into the immune cells (coelomocytes); 2) the cholinesterase activity and the expression of the stress-related proteins HSC70 and GRP78; 3) the morphological changes affecting cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. By Environmental Scanning Electron Microscope (ESEM) analysis, coupled with Energy Dispersive X-ray Spectroscopy (EDS) we found that NPs were uptaken inside coelomocytes. The cholinesterases activity, a well known marker of blood intoxication in vertebrates, was greatly reduced in specimens exposed to NPs. We found that levels of stress proteins were down-regulated, matching the observed ER and lysosomes morphological alterations. In conclusion, this is the first study which utilizes the sea urchin as a model organism for biomonitoring the biological impact of NPs and supports the efficacy of the selected biomarkers.

Food ingestion and egestion in mating reactive populations of Paramecium primaurelia.

The study of food ingestion and egestion carried out on Paramecium primaurelia mating reactive cells shows that, after their transfer into a medium with suspended particles, the complementary mating type cells exhibit very significant differences in the food vacuole formation and egestion rate. Under the same external environmental conditions, the mating type II cells form and egest a higher number of food vacuoles when compared with mating type I cells. The higher rate of food vacuole formation shown by the mating type II cells is related to their faster growth rate.

Lectin-binding glycoconjugates in Paramecium primaurelia: changes with cellular age and starvation.

Lectins with different sugar specificities and binding to phagosome-lysosome systems as well as cell surface constituents were used to study glycoconjugate variation throughout culture and clonal life in Paramecium primaurelia, particularly during the transition period from logarithmic to stationary growth phase and in relation to clonal decline, respectively. These lectins include Griffonia simplicifolia agglutinin II (GS II), Ricinus communis agglutinin (RCA120). Arachis hypogea agglutinin (PNA), succinyl concanavalin A (succinyl-con A), and Triticum vulgaris agglutinin (WGA). The labeling obtained varies both according to the lectin used and to the culture and clonal age of the cells. Negative results were obtained in logarithmic growth phase cells and in clonal young cells by using lectin GS II. Conversely, lectins RCA120 and PNA bind to the cell surface, the oral region as well as cilia, and do not undergo modifications with culture or clonal age and after permeabilization. WGA binds to constituents of the cell surface, trichocyst tips, food vacuoles, the oral region, and cilia but the extent of labeling decreases as culture age increases; during clonal decline, cells show the same labeling pattern as starved cells. Finally, the lectin succinyl-con A shows a large amount of binding sites on the cell surface, on trichocyst tips, and in the oral region of logarithmic-phase cells, whereas the number of sites decreases in late stationary phase. The data obtained partly differ from those reported in the literature and the differences can be attributed to the culture conditions and species examined. Nevertheless, the assumption that a rearrangement of some glycoconjugates of membrane throughout culture and clonal life of Paramecium is confirmed.

Age-related changes in neutral lipid content of Paramecium primaurelia as revealed by nile red.

Cellular neutral lipid content of Paramecium primaurelia was measured during culture and clonal life using nile red (9-diethylamino-5H-benzo[alpha] phenoxazine-5-one), a dye utilized for lipid analysis in both mammalian cells and in ciliated protozoa. Lipid droplets in P. primaurelia are concentrated in the anterior pole of the cell; their number is maximum in early log phase cells and decreases in late log phase cells. The quantitative determination of neutral lipids was obtained measuring the fluorescence from the excitation and emission spectra of 480 nm and 540 nm, respectively. Neutral lipid content decreases linearly during the log phase of growth while the decline is minimum during the stationary phase. In the late log phase, the amount is 30% of that of the early log-phase cells. Though the cell size declines too, cell area and lipid content decreases are not correlated in the middle log phase, because the maximum lipid reduction is obtained when the cell size is relatively constant. The cellular lipid content also changes during the clonal life. Neutral lipids decrease discontinuously (r = -0.75, P < 0.05) as the fission age increases. No relationship was found between lipid content and food vacuole formation during both culture and clonal life.