Biomedical subjects
E Frixione
Publications and source records attributed to E Frixione.
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.
Recurring views on the structure and function of the cytoskeleton: a 300-year epic.
Some unnoticed or seldom remembered precedents of current views on biological motion and its structural bases are briefly outlined, followed by a concise recapitulation of how the present theory has been constructed in the last few decades. It is shown that the evolution of the concept of fibers as main constituents of living matter led to hypothesizing microscopic structures closely resembling microtubules in the 18th century. At the beginning of this period, fibers sliding over each other and driven by interposed moving elements were envisioned as the cause of muscle contraction. In the following century, an account of the mechanism of myofibril contraction visualized longitudinal displacements of myosin-containing submicroscopic rodlets. The existence of fibrils in the protoplasm of non-muscle cells, a subject of long debate in the second half of the 19th century, was virtually discarded as irrelevant or fallacious 100 years ago. The issue resurfaced in the early 1930s as a theoretical notion--the cytosquelette--nearly two decades before intracellular filamentous structures were first observed with electron microscopy. The role originally assumed for such fibrils as signal conductors is nowadays being reappraised, although under new interpretations with a much wider significance including modulation of gene expression, morphogenesis, and even consciousness. Since all of the above ancestral conceptions were eventually abandoned, the corresponding current views are, to a certain extent, recurrent.
CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells.
Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.
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 microM), 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.
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 < or = 5 microM) 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 10 min 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 where 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+]3 poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.
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.
Conditional inhibition of screening-pigment aggregation by lidocaine in crayfish photoreceptors and frog retinal pigment epithelium.
Lidocaine, at concentrations equal to or lower than those that inhibit fast axoplasmic transport, was found to interfere with the dark-adapting migration of the screening pigments along crayfish photoreceptors and within the cells of the frog retinal pigment epithelium (RPE). The effects of the anesthetic on pigment movements were studied in isolated eyes incubated under light or dark conditions in media of different ionic compositions. Treatment of crayfish eyes with 25 mmol l-1 lidocaine in normal Van Harreveld's saline arrested pigment migration to the dark-adapted position or caused migration towards the light-adapted position in the dark. Similar results were obtained with frog eyecups exposed to 5 mmol l-1 lidocaine in Ringer's solution. In each case, the inhibition of dark adaptation was reversible and dependent on the levels of Na+ and Ca2+ in the incubation medium. A dark-adapted position of both pigments was compatible with lidocaine treatment provided that low-Na+, or high-Ca2+ or Co(2+)-containing solutions were used. These results indicate that light-adapted and dark-adapted pigment positions in both types of retinal cells can occur in the absence of local nervous input. Further, the data suggest a direct effect of lidocaine upon the photoreceptors or RPE cells. The inhibition of pigment aggregation is interpreted to be a consequence of an anesthetic-induced increase in the permeability of the plasma membrane, which in turn affects the intracellular ionic balance that controls pigment position.
Light-adapting migration of the screening-pigment in crayfish photoreceptors is a two-stage movement comprising an all-or-nothing initial phase.
The light-adapting response of the screening-pigment in crayfish retinal photoreceptors, previously described as a monophasic movement, was found to consist of two stages with different properties: (1) a rapid initial expansion that once started proceeds for at least half of the full distance, and (2) a slower and more variable continuation of the movement. The two components were resolved in isolated eyes stimulated under conditions expected to restrict Na+ influx into the photoreceptors. Only the second stage of the response to light was inhibited when Na+ was substituted with choline, or if the normal saline contained amiloride, a diuretic that hinders Na+ entry across many cell membranes. Amiloride in a medium without Na+ delayed, but did not curb, the first stage, whereas the rest of the movement was markedly restrained. Partial replacement of Na+ with Li+ blocked the second stage without affecting the rapid initial shift triggered by light. Exposure of dark-adapted eyes to high Na+ levels or to ouabain in the presence of Na+ in the dark also elicited a two-staged pigment dispersion to the light-adapted position. Low Na+ concentrations or amiloride affected the latency, but not the rate or extent, of the first stage of migration in ouabain-treated eyes. Consistent though less significant results were obtained with cyanide and the Na+ ionophore monensin. These observations suggest a differential control of pigment position over two defined domains along the photoreceptors, probably to integrate a double mechanism of light-adaptation: an all-or-nothing partial shift of the pigment screen as a safety factor against overexposure, followed by a regulated adjustment according to stimulation intensity.
Modulation of crayfish retinal sensitivity by 5-hydroxytryptamine.
The responsiveness of crayfish retinal photoreceptors to light was enhanced by exposure to 5-hydroxytryptamine (5-HT), either following injection into whole animals or following topical application to isolated eyestalks or retinas. The effect was measured as an increment in the amplitude of the receptor potential, and was dose-dependent in the range 10(-6)-10(-3) mol l-1 (injected as a 0.1 microliter dose in intact animals). It was more pronounced at low levels of illumination and was reversibly blocked by methysergide. The enhancement was a consequence of a dual effect: (a) retraction of the proximal pigment granules within the photoreceptors, with a corresponding increase in the light-admittance function of the retina; and (b) a direct effect, facilitating a membrane conductance increase which mediated the generation of the receptor potential. A set of axons in the lamina ganglionaris with a 5-HT-like immunoreactivity was found in the vicinity of the photoreceptor axons. 5-HT antagonists were capable of blocking the physiological retraction of pigment granules in photoreceptors at night, suggesting that 5-HT acts as a modulator during the nocturnal phase of the circadian cycle in the crayfish retina.
Retinomotor movements in the frog retinal pigment epithelium: dependence of pigment migration on Na+ and Ca2+.
The ionic dependence of the screening-pigment migrations in the frog retinal epithelium (RPE) was quantitatively studied with eyecups incubated in media of different compositions. Typical migrations in response to light and darkness, equivalent to those observed in the intact animal, were fully accomplished and maintained for up to 6 hr by the isolated organ bathed in Ringer solution rich with O2. Pigment migration in either direction was completed under the appropriate illumination conditions at any time during the day, indicating that circadian influences, if present in the intact animal, can be overridden in the isolated organ by light or darkness alone. Pigment aggregation toward the dark-adapted position was inhibited by: (a) low external Ca2+, (b) high external Na+, and (c) drugs expected to increase the cytoplasmic levels of either Na+, or Ca2+, like ouabain, caffeine and the ionophore A23187. However, the inhibition caused by low Ca2+ did not occur if Na+ was also reduced in the incubation medium. On the other hand, an increase in the concentration of external Ca2+ or the addition of Co2+ to the normal Ringer facilitated pigment aggregation in the dark. Pigment dispersion to the light-adapted position was unaffected by any of the above conditions. This is the first report of full and stable pigment responses in the RPE of vertebrate eyes incubated under simple physiological conditions. The results seem to conciliate a discrepancy of previous reports on the Ca2+ dependence of RPE movements, and are compatible with current views on ionic mechanisms in analogous systems of intracellular transport.
A black flexible support with excellent optic qualities for microdissection of nerve tissues.
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The microtubular system of crayfish retinula cells and its changes in relation to screening-pigment migration.
The 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.
Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells.
The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep-etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low-Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule-microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules.
Photomechanical responses in crustacean retinula cells: the role of microtubules.
Light and darkness promote opposite migrations of screening pigment granules in the retinal photoreceptors of the crayfish and other arthropods, and microtubules have been implicated in this part of the adaptation process. This report considers the topographic relationships of the migratory pigment granules with the microtubular system of the cells. Scanning electron microscopy was used to obtain a three-dimensional visualization of the internal organization of cells in different states of adaptation. Many pigment granules were found directly associated with the microtubular array, although most of them are not adjacent to microtubules irrespective of the adaptation conditions. However, the granules appear associated with each other and form extensive clusters or possibly a single conglomerate. Therefore, interaction of a limited number of granules with the microtubules could affect the movement of the whole granule population. When the pigment complex withdraws, many microtubules are still seen in the deserted regions. It is concluded that the microtubules might represent a supporting framework for the translocation of the pigment, which appears to move as a coherent mass rather than as independent particles.
Photomechanical migrations of pigment granules along the retinula cells of the crayfish.
The light-dependent migrations of proximal pigment granules along the photoreceptors of the crayfish compound-eye were studied in isolated retinas and eyestalks. The extent and kinetics of movement in each direction were found quantitatively equivalent to those observed in the organ in situ. These and other features make these cells to appear as intrinsically independent pigmentary effectors, directly responsive to light. During dark adaptation (DA) the pigment migrates away from the cell nucleus and accumulates along the axon in two distinct steps. Each step constitutes half of the total distance of about 180 microns and proceeds at 0.30 micron/sec. Only prolonged metabolic impairment inhibited the first phase, while the second was blocked by hypoxia, cyanide, colchicine, and D2O. The maintenance of a full DA position was also shown to be highly dependent upon metabolism. Light incidence on DA eyes is followed by an apparently monophasic expansion of the pigment from the axon towards the perikaryl region at 0.38 micron/sec. This movement was not affected by any of the foregoing agents and seems to be a passive relaxation process. Cytochalasin B had no effect on either motion. The migration in either direction has an exponential time course and is temperature dependent. Electron microscopy revealed two separate patterns of cytoplasmic organization corresponding to the cell areas where the two phases of DA occur. In the region close to the nucleus the pigment appears irregularly scattered, whereas in the axon the granules are situated arond a thick longitudinal bundle of microtubules. These results suggest the existence of two different mechanisms of pigment granule translocation operating in two separate regions of the retinula cell.
The role of osmotic pressure in the germination of Nosema algerae spores.
Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.
Structural alteration of the plasma membrane in spores of the microsporidium Nosema algerae on germination.
The fine structure of the plasma membrane in spores of the microsporidium Nosema algerae, a pathogen of mosquitoes, was examined in the resting condition and after the spores were stimulated to germinate in vitro. Slow penetration of resin caused collapse of the germinated spores. Thin sections of germinated spores showed peculiar membrane infoldings that were never found in ungerminated samples. Analogous germination-dependent configurations of the plasma membrane were observed in freeze-fractured preparations of spores either fixed and impregnated with glycerol prior to freezing, or rapidly frozen with liquid propane while in the process of germination. In every case, the replicas presented germinated spores with indentations in the protoplasmic face of the plasma membrane, and apparently complementary blunt spines on the external face, that were absent in ungerminated spores. It suggests that these alterations of the plasma membrane result from a structural adjustment to a spontaneous contraction of the spore case after germination. We discuss this interpretation with regard to conflicting views on the nature of such morphological features.