Eugenio Frixione

Ca2+-dependence of conoid extrusion in Toxoplasma gondii tachyzoites

The role of Ca2+ in conoid extrusion was investigated in isolated Toxoplasma gondii tachyzoites by treatment with Ca2+‐ionophores, Ca2+‐chelating agents and an inhibitor of the Ca2+‐ATPase at the endoplasmic reticulum. The results were evaluated by light phase‐contrast microscopy and electron microscopy. lonomycin (0.5‐1 μM) caused an immediate and sustained extrusion of the conoid in up to 80% of the tachyzoites, depending on the concentrations of ionophore and Ca2+ in the medium. However, over 50% of the tachyzoites extruded the conoid when treated with ionomycin in Ca2+‐free saline complemented with EGTA. The effect of ionomycin was reversible and could be induced a second time in about half of the responsive population. Similar results were obtained with A23187. Conoid extrusion induced by ionomycin in Ca2+‐free medium was almost completely abolished when the tachyzoites were previously loaded with a permeable compound known to chelate intracellular Ca2+ (BAPTA/AM; 25μM). On the other hand, exposure of tachyzoites to the Ca2+‐ATPase inhibitor thapsigargin (0.5‐1μM) produced significant extrusion of the conoid. Tachyzoites loaded with BAPTA/AM as well as those treated with ionomycin, i.e. with conoids paralyzed in opposite positions, had a diminished capacity to invade cultured epithelial cells. A substantial reduction in the response to stimulation by ionomycin was found also in parasites treated with cytochalasin‐D, a drug that depolymerizes actin‐filaments. The results suggest that Ca2+‐release from internal stores may act as a key signal to activate a mechanism of conoid extrusion probably mediated, at least in part, by actin‐filaments.

Ionic dependence of screening pigment migrations in crayfish retinal photoreceptors

SummaryThe ionic dependence of the screening pigment migrations in crayfish (Procambarus bouvieri) retinula cells was studied by incubation of isolated eyes in solutions of different compositions. The pigment aggregation that normally occurs in the dark was inhibited by high potassium in a concentration dependent manner. Inhibition of aggregation was observed also with high external sodium or when sodium was replaced by lithium. Pigment aggregation is favored in the presence of high calcium, cobalt ions or in sucrose solution. The pigment dispersion induced by light is partly inhibited by lithium and in sucrose solution. Neither movement was affected by high magnesium, EDTA or EGTA. Agents expected to increase the intracellular activity of sodium or calcium, as ouabain, caffeine, ruthenium red and the ionophore A23187, all inhibited pigment aggregation. These observations assert the view that the crustacean retinula cells are independent pigmentary effectors, and agree with the notion of an engagement between membrane events and pigment position. It is suggested that sodium influx induced by light results in calcium release from internal stores, which in turn triggers dispersion of the pigment granules within the retinula cells.

Kinematic analysis of Toxoplasma gondii motility

Toxoplasma gondii tachyzoites execute a complex and little understood combination of rapid movements to reach and penetrate human or other animals cells. In the present study, computer-assisted simulation was used to quantitatively analyze the motility of these parasites in three-dimensional space with spatial and temporal resolutions in the micrometer and subsecond ranges. A digital model based on electron-micrographs of a serially sectioned tachyzoite was animated according to a videomicrographed sequence of a characteristic repetitive movement. Keyframe animation defined over 150 frames by a total of 36 kinematic parameters for specific motions, of both the whole model and particular domains, resulted in a real-time life-like simulation of the videorecorded tachyzoite movement. The kinematic values indicate that a full revolution of the model is composed of three half-turns accomplished in nearly 5 s with two phases: a relatively slow 180 degrees tilting with regard to the substratum plane, followed by fast (over 200 degrees/s) spinning almost simultaneous with pivoting around the posterior end, each clockwise and for about 180 degrees. Maximal flexing of the body, as well as bowing and retraction of its anterior end, occur at midway during the tilting phase. An estimated 70 degrees. clockwise torsion of the body seems to precede the spinning-pivoting phase. The results suggest the operation of two basic forces in the motility of T. gondii tachyzoites: (1) a clockwise torque that causes torsion, spinning, and pivoting; and (2) a longitudinal pull that contracts, bends and tilts the parasite. We discuss the possibility that both of these forces might result from the action of an actin-myosin system enveloping the twisted framework of microtubules characteristic of these organisms.

Myosin II-actin interaction in MDCK cells: role in cell shape changes in response to Ca2+ variations.

Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+]e≤5μm) with a loss of transepithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal structure in place seems necessary to preserve the architecture of the tight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+]e. Recent work provided evidence of actin–myosin interactions at the filament ring, thus suggesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and causes an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10min of removal of [Ca2+]e; recovery began immediately after replacing calcium, although it took longer for completion. Conventional and confocal epifluorescence studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electron-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction when Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+]e variations were inhibited by cytochalasin-D (an actin-filament disrupting drug), by okadaic acid (an inhibitor of myosin light-chain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cells previously depleted of metabolic energy by 2,4-dinitrophenol and 2-deoxy-D-glucose preincubation. The actin–myosin mediated reversible structural transformation of MDCK cells in response to [Ca2+]e poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.

Calcium uptake by smooth endoplasmic reticulum of peeled retinal photoreceptors of the crayfish

SummaryThe localization and basic properties of Ca2+-accumulating sites in crayfish photoreceptors were studied with a novel preparation of peeled retinula cells in suspension. Peeled photoreceptors were obtained by gentle mechanical disruption of the retina, and incubated in media based on a Ca2+-EGTA buffer with ATP and oxalate. Electron microscopy of photoreceptors so treated showed the appearance of peculiar dense deposits inside vesicles of smooth endoplasmic reticulum (SER). EGTA-extraction and energy-dispersive X-ray microanalysis identified Ca as a major constituent of such deposits.45Ca2+ uptake experiments with peeled photoreceptors or with the crude particulate fraction of retinal homogenates revealed a rapid binding of radioactivity over the first 8 min, followed by a slower continued accumulation, which did not occur in the absence of ATP.45Ca2+ uptake is stimulated by an increase in the concentration of free Ca over the range 4×10−7 to 5×10−6M, and becomes inhibited at higher levels.45Ca2+ uptake is depressed when K+ is replaced by Na+ or Li+ as the main monovalent cation in the medium, but it is not affected by illumination nor by the presence of caffeine or ruthenium red. These findings attest that the SER has a Na+-sensitive capacity for regulating the intracellular concentration of Ca2+ in these photoreceptors, and support the hypothesis of its probable role in the control of pigment granule transport and other structural changes involved in light/dark adaptation.

Transformations in the cytoplasmic structure of crayfish retinula cells during light- and dark-adaptation

SummaryExisting descriptions of morphological correlates of light- and dark-adaptation in crayfish photoreceptors are primarily concerned with changes of the rhabdom or components derived from it, and little is known about mechanisms involved in screening pigment migration and other transformations that take place in the cytoplasm of the retinula cells. In the present study, electron microscopy was used to examine the effects of light and darkness on the cytoplasmic structure of the retinula cells.In the light-adapted state a multitude of pigment granules and many small vacuoles are dispersed throughout the cells (Figs. 1 a and 2a). On dark-adaptation the granules are withdrawn towards the axon and many of the small vacuoles in the cell body are replaced by large cisternae (Figs. 1b and 2b). The cisternae attain maximal development in the distal cytoplasm, close to the nucleus, and they do not show a preferential association with the rhabdom. Vacuoles of intermediate size and some pigment granules are seen in partly-adapted retinula cells (Fig. 3). A decrease in the density of the ground substance is observed in the cell body of dark-adapted specimens. At least part of the ground substance is constituted by a fuzzy filamentous material that in thin-sections appears surrounding and often interconnecting the pigment granules and other structures (Fig. 4). In thick-sections observed with stereo high-voltage electron microscopy, this material is revealed as a threedimensional fibrous matrix in which the pigment particles are suspended (Fig. 5). This matrix appears more compact where the granules aggregate, suggesting that it moves with the pigment.The transformation of small vacuoles into large cisternae, interpreted as equivalent to the growth of a subrhabdomal palisade in other compound eyes, is proposed to represent a structural adjustment related to the withdrawal of a cytoskeletal complex of pigment granules and their associated matrix during dark-adaptation (Fig. 6).

Monovalent Cations Induce Microsporidian Spore Germination In Vitro

ABSTRACT The relative capacity of Na+, K+ and Cl‐ to stimulate germination of spores of the microsporidian Nosema algerae, a pathogen of mosquitoes, was examined by ion substitution experiments. Sodium at 0.1 M was ineffective to produce the high percentage of germination that typically occurs with 0.1 M NaCl (the normal stimulation solution) if Cl‐ was substituted with the usually impermeant anions SO42‐, HPO42‐, or the organic acids oxalate, cacodylate, EGTA, MES and HEPES. However, substantial concentration‐ and pH‐dependent germination was seen with Na2SO4 in the 0.2‐0.8 M Na+ range. Similar results were obtained with solutions of K+ accompanied by impermeant anions. In contrast, the chloride salts of usually impermeant cations, like choline and triethanolamine, failed to germinate spores even at 0.8 M unless Na+ or K+ was independently added. The presence of 0.5 M choline chloride in the medium reduced the levels of Na2SO4 required to produce germination down to equivalence with those of Na+ in the normal stimulation solution. Monensin, a Na+ ionophore, facilitated the germination induced by a medium‐level stimulus (0.04 M NaCl) in sonicated samples. These findings indicate that N. algerae spores germinate in response to the alkali metal cations, while CI‐ plays a passive role by diffusing to maintain internal electroneutrality during cation influx. A possible mechanism of cation action in spore germination is suggested on the basis of these results and observations on other systems of intracellular motility.

The microtubular system of crayfish retinula cells and its changes in relation to screening-pigment migration

SummaryThe organization of the microtubular system in crayfish retinula cells and its changes in relation to the light-dependent migrations of the screening pigment were studied by electron microscopy. A massive column of microtubules extends longitudinally throughout each retinula cell and its axon. The column is formed by overlapping fascicles of microtubules that originate from the vicinity of the rhabdomeres at multiple levels along the rhabdom. The pigment granules and other organelles are in general aligned with these fascicles and peripheral to the microtubular column. Close associations between microtubules and pigment granules are frequent. The total number of microtubules decreases nucleofugally from an average of about 500 at the middle of the rhabdom, to 390 at the proximal end of the rhabdom, and 240 in the axon below the basement membrane. The longitudinal distribution of microtubules was found similar for cells with the screening pigment in opposite extreme positions. In cells with the pigment in an intermediate position the number of microtubules was found to be nearly doubled in each of the mentioned levels; however, this change was correlated with a parallel increase in the cross-sectional area of the cells during the intermediate state. Thus, the density of microtubules tends to remain fairly constant throughout the light/dark adaptation cycle. These observations suggest that the microtubular system of the crayfish retinula cells constitutes a relatively stationary framework during screening-pigment movements, and could possibly act as a supportive guiding track for pigment transport.

Contraction of epithelial (MDCK) cells in response to low extracellular calcium is dependent on extracellular sodium.

Like other cells of epithelial origin, MDCK cells respond with a reversible structural transformation to a diminution in the concentration of extracellular Ca2+. Upon deprivation of Ca2+ in the medium the cells undergo an active contraction mediated by the actin-myosin cytoskeleton, in parallel to detachment of the intercellular contacts and appearance of free spaces in the epithelium or monolayer (Castillo et al., 1998). We now present results indicating that the decrease of external Ca2+ plays an indirect and non-specific role in activating contraction, probably by allowing an influx of Na+. The omission of external Ca2+ had no effect when it was replaced by Mg2+, Ba2+ or Hg2+, and the addition of any of these divalent cations induced relaxation of cells previously contracted by exposure to low Ca2+. A null or weak response was observed also when Ca2+ was lowered in a solution where Na+ was replaced by choline or in the presence of amiloride (30 μM), which reduces the permeability of the plasma membrane to Na+. Restitution of Na+ or removal of amiloride were followed by contraction in the same cultures. Li+ proved an able substitute of Na+ as requisite for cell contraction in response to Ca2+ depletion. Monensin (0.1 mM) –an ionophore selective for Na+– and to a lesser extent ouabain (0.1 mM) –an inhibitor of Na+ extrusion across the plasma membrane– , both stimulated contraction in the presence of the normal level of external Ca2+. Decreasing by half the normal concentration of external K+ facilitated cell contraction, but typical responses were observed when K+ was increased to 40 mM by partial substitution for Na+. These findings attest that cell contraction in response to low Ca2+ is likely due to an increase in the permeability of the plasma membrane to Na+, though not to membrane depolarization as such. Evidences from other motile systems suggest that Na+ influx might in turn cause an elevation of cytoplasmic Ca2+, which activates the actin-myosin cytoskeleton.

Actin cytoskeleton role in the structural response of epithelial (MDCK) cells to low extracellular ca2

Kinetic and stereometric assessment of the mechanical responses of epithelial cells to variations in the concentration of extracellular Ca2+ was carried out in vivo at the single cell level. Continuous monitoring of individual MDCK cells in subconfluent cultures attested to an intense, immediately relaxable, and cytochalasin D-sensitive contraction, equivalent to that seen in confluent monolayers following depletion of external Ca2+ (<0.1 mM). Increasingly greater and less readily reversible contractions were performed upon repeated stimulation with short-term cycles of alternating normal (30 min) and low Ca2+ (30 min) media. Constriction of a narrow horizontal girdle corresponding in position to the major ring-like bundle of actin filaments eventually develops into a deep lateral furrow in intensely contracted cells. Substantial membrane infolding in the contracted state is indicated also by stereometric estimates of apparent bounding surface area. Irrespective of the contracted or relaxed cell condition, rhodamine–phalloidin labeling showed a marginal position of the ring-like bundle of microfilaments and other components of the actin cytoskeleton. These results suggest, contrary to prevalent views, that the actin–myosin system stays associated to the cortex and retains contractile capability in epithelial cells deprived of external Ca2+. Hence, the mechanical responses to variations of Ca2+ may be an overstrained expression of a physiological mechanism.