Effects of an extremely low frequency electromagnetic field on the cell division rate and plasma membrane of Paramecium tetraurelia

The eukaryotic protozoan, Paramecium, was examined as a model for effects of pulsated electromagnetic fields (PEMF) on cells. A 72-Hz PEMF similar to fields employed clinically increased cell division rates in Paramecium by 8.5%. Two calcium transport mutants of these organisms showed differential responses to the same field. Verapamil, a calcium channel blocker, abolished any effect of PEMFs on cell division rates. A fluorescent probe that is thought to sense changes in membrane potential also manifested an altered response in the PEMF-exposed cells whereas a fluorescent lipid bilayer fluidity probe produced evidence of decreased membrane fluidity in the exposed cells. An effect of PEMFs on ion transport mediated by either a direct or indirect effect on the cell membrane is suggested by these studies.     

Molecular identification of 26 syntaxin genes and their assignment to the different trafficking pathways in Paramecium

SNARE proteins have been classified as vesicular (v)- and target (t)-SNAREs and play a central role in the various membrane interactions in eukaryotic cells. Based on the Paramecium genome project, we have identified a multigene family of at least 26 members encoding the t-SNARE syntaxin (PtSyx) that can be grouped into 15 subfamilies. Paramecium syntaxins match the classical build-up of syntaxins, being 'tail-anchored' membrane proteins with an N-terminal cytoplasmic domain and a membrane-bound single C-terminal hydrophobic domain. The membrane anchor is preceded by a conserved SNARE domain of approximately 60 amino acids that is supposed to participate in SNARE complex assembly. In a phylogenetic analysis, most of the Paramecium syntaxin genes were found to cluster in groups together with those from other organisms in a pathway-specific manner, allowing an assignment to different compartments in a homology-dependent way. However, some of them seem to have no counterparts in metazoans. In another approach, we fused one representative member of each of the syntaxin isoforms to green fluorescent protein and assessed the in vivo localization, which was further supported by immunolocalization of some syntaxins. This allowed us to assign syntaxins to all important trafficking pathways in Paramecium.     

Induction of Phagocytosis and "Cannibalism" by the Giant Ameba, Chaos carolinensis

SYNOPSIS. It is evident from studies of cellular recognition resulting in phagocytosis by Chaos carolinensis that cilia from certain protozoa serving as prey, e.g. Tetrahymena pyriformis , influence phagocytic action. Glutaraldehyde-fixed C. carolinensis to which cilia, isolated from Tetrahymena , are attached by the benzidine reaction, are engulfed by living Chaos but not digested. Fixed Chaos without attached cilia are not engulfed. Adding increased numbers of cilia from Tetrahymena progressively inhibits the rate at which Chaos phagocytizes Paramecium caudatum , suggesting blockage by the cilia of contact sites normally available to initiate phagocytosis of Paramecium.     

TMA-DPH a fluorescent probe of membrane dynamics in living cells. How to use it in phagocytosis

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells--specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phagocytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Stimulation of Paramecium phagocytosis by phorbol ester and forskolin

Phorbol ester (PMA) exerted a dose- and time- dependent stimulating effect on phagocytosis in axenic Paramecium aurelia. When cells were exposed to 200-800 nM PMA in the presence of latex beads, the phagocytic coefficient was enhanced 2.25 to 3.14 times, during 10 min of continuous treatment and then rapidly declined. A similar effect was observed when the cells were exposed to a forskolin treatment, which resulted in nearly a twofold increase in phagocytic activity after a 10 min pulse. Both PMA and forskolin strongly stimulated phagocytosis (i.e. fivefold and threefold, respectively) in cells in which such activity had been completely inhibited by pre-exposure to the beta-receptor antagonist 1-propranolol.     

Purification of the temperature-specific surface antigen of Paramecium primaurelia with its glycosyl-phosphatidylinositol membrane anchor

The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.     

Uptake and rapid transfer of fluorescent ceramide analogues to acidosomes (late endosomes) in Paramecium.

The ciliated protozoan Paramecium incorporates sphingolipids into its cell membranes. However, it is still unclear if these sphingolipids are metabolically synthesized in the cell or if their precursors are taken up from exogenous materials. Here we studied the route of uptake of fluorescence-labeled analogues of ceramide. Fluorescent ceramide was taken up rapidly independent of phagosome formation. Cold treatment caused a decrease in uptake, while reduction in the amount of cytosolic ATP induced by NaN(3) and deoxyglucose resulted in accumulation without internalization of fluorescence at the plasma membrane. These results suggest that uptake of fluorescent ceramide occurs at the plasma membrane, that it is an ATP-dependent process, and that it is not a result of simple diffusion. At first intracellular fluorescence appeared principally in the posterior half of the cell and then spread throughout the cytosol. In particular, a high accumulation of fluorescence occurred in association with acidosomes (late endosome or multivesicular body-like vesicles) that bind to the surface of nascent and young phagosomes. Therefore, in the Paramecium cell a significant proportion of ceramide apparently enters the cell by endocytosis and is quickly relayed to acidosomes along the endocytic pathway before becoming part of the digestive vacuole (phagoacidosome) membrane.     

Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis

Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

Recent advances in the molecular genetics of Paramecium

Paramecium continues to be used to study motility, behavior, exocytosis, and the relationship between the germ and the somatic nuclei. Recent progress in molecular genetics is described. Toward cloning genes that correspond to mutant phenotypes, a method combining complementation with microinjected DNA and library sorting has been used successfully in cloning several novel genes crucial in membrane excitation and in trichocyst discharge. Paramecium transformation en masse has now been shown by using electroporation or bioballistics. Gene silencing has also been discovered in Paramecium, recently. Some 200 Paramecium genes, full length or partial, have already been cloned largely by homology. Generalizing the use of gene silencing and related reverse-genetic techniques would allow us to correlate these genes with their function in vivo.     

Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo

cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.     

Cycloheptaamylose-dansyl chloride complex as a fluorescent label of surface membranes in living ciliates

Labelling of surface membrane of living ciliates: Paramecium aurelia and Tetrahymena pyriformis with fluorescent compound--cycloheptaamylose-dansyl chloride complex (CDC) has been achieved. Fluorescence micrographs of the dried samples showed specific localization of CDC on the cell membrane without any intracellular penetration. On the contrary the ciliates which have been dead during labelling revealed a non-specific fluorescence of their whole bodies. Microspectrofluorimetric analysis of labelled Paramecium cells was performed with Leitz microspectrograph. Spectrum of fluorescence emission measured over the cell membrane level had maximum at 450 nm. Strikingly, the emission maximum of the cells dead at the moment of labelling was shifted 10 nm to a longer wavelength. The rate of photofading measured in this case was almost 3-fold higher than for the ciliates labelled as living ones. Fluorescence excitation spectra did not show any difference in the peak position. Thus CDC staining appears to be an useful method of supravital labelling of cell surface enabling also to distinguish--on the basis of spectral characteristics--the ciliates being alive from those dead at the moment of fluorochrome binding.     

Initial steps of infection of the ciliate Paramecium with bacteria Holospora sp

New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided. At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome. The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus. The authors subdivide the process of infection into 6 steps. Some strains of P. caudatum never take up infectious Holospora bacteria in the course of phagocytosis.     

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.     

Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.     

Characterization of the effects of propranolol on the physical state of platelet membrane

The direct membrane effect of propranolol was studied in vitro on washed platelet preparations. Propranolol has been reported to inhibit platelet aggregation by mechanisms unrelated to its beta-blocking activity. In the present study, the drug was found to enhance 1-anilino-8-naphthalene sulfonate binding to platelet membrane by increasing the number of binding sites. Steady-state anisotropy was studied by labeling the platelets with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Propranolol was observed to decrease the equivalent microviscosity of the membrane. When the infinitely slow decaying component of fluorescence anisotropy (r infinity), which is proportional to the square of lipid order parameter, was calculated from the anisotropy data, a decrease in these parameters was also indicated. A higher fusion activation energy for viscosity in the propranolol-treated platelets reflected a lesser degree of order of the hydrocarbon chains in the lipid bilayer.     

Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

A novel phosphatidylinositol 4,5-bisphosphate-binding domain targeting the Phg2 kinase to the membrane in Dictyostelium cells

Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P₂-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P₂-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P₂ during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P₂ disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P₂ metabolism regulates the dynamics and the function of Phg2.