Structurization of Cortical Layer of Loach Yolk Cell after Wounding as a “Minimal” Model of Morphogenesis

Structural rearrangements of the yolk cell surface were studied in loach embryos using SEM and TEM, which take place within 30 min after a point-like puncture at the late blastula stage. The effects of sucking off or addition of a part of yolk, lowered temperature, and absence of Ca²⁺ on structurization were studied. Around the area of puncture, the yolk granules were submerged, the number of vesicles increased, and numerous membrane folds were formed. The folds were aggregated to form two sharply distinct types of structures: a group of rounded evaginations around the site of puncture and a system of radial folds in the periphery. Small radial folds are aggregated in radial strands, several dozens folds in each. Sucking off a part of yolk accelerated the above processes, while addition of yolk, cooling, and absence of Ca²⁺ in the incubation medium slowed down or suppressed these processes. The observed structurization can be considered as self-organization at the level of the yolk cell cortical level, largely similar to that during normal morphogenesis at the level of multicellular sheets. Hence, the membrane dynamics in the yolk cell wall after its damage can be considered as one of simplified (minimal) models of morphogenesis. A study of this model makes it possible to narrow down the circle of factors essential for self-organization of morphogenetic processes.     

Kinetics of the reaction of yolk cell surface in the loach to puncture and mechanic deformation

Circumferential and radial components of the yolk cell surface movements were measured in the loach embryos at the late blastula stage within 40-50 min after puncture or indentation by an obliquely directed glass rod. The yolk cell surface was preliminarily marked by coal particles. It was shown that even closely located regions of the surface differed markedly in the rate and direction of their movements. In the vicinity of puncture, the yolk cell surface at first contracted in both circumferential and radial directions and then widened, but did not reach the initial values. In more remote areas, this surface continued to contract in the circumferential direction, but was extended in the radial direction. The degree of its contraction along different radii was unequal. The reaction to oblique indentation was anisotropic: the closest area of the yolk cell surface, located along the direction of indentation, contracted in both circumferential and radial directions and formed a fold "leaking" onto the rod, while the opposite area contracted in the circumferential direction, but extended in the radial direction. A conclusion was drawn that the yolk cell surface is a multivariant mechanosensitive system. Its active responses to mechanical influences obey the same patterns as multicellular embryonic tissues.     

Kinetics of the reaction of yolk cell surface in the loach to puncture and mechanic deformation

Circumferential and radial components of the yolk cell surface movements were measured in the loach embryos at the late blastula stage within 40–50 min after puncture or indentation by an obliquely directed glass rod. The yolk cell surface was preliminarily marked by coal particles. It was shown that even closely located regions of the surface differed markedly in the rate and direction of their movements. In the vicinity of puncture, the yolk cell surface at first contracted in both circumferential and radial directions and then widened, but did not reach the initial values. In more remote areas, this surface continued to contract in the circumferential direction, but was extended in the radial direction. The degree of its contraction along different radii was unequal. The reaction to oblique indentation was anisotropic: the closest area of the yolk cell surface, located along the direction of indentation, contracted in both circumferential and radial directions and formed a fold “leaking” onto the rod, while the opposite area contracted in the circumferential direction, but extended in the radial direction. A conclusion was drawn that the yolk cell surface is a multivariant mechanosensitive system. Its active responses to mechanical influences obey the same patterns as multicellular embryonic tissues.     

Alterations of wheat-germ agglutinin binding pattern on cell surface of blood platelets after thrombin stimulation

An attempt was made to demonstrate wheat-germ agglutinin (WGA) binding sites on platelet surfaces after thrombin stimulation, by means of a post-embedding cytochemical technique using colloidal gold as marker at an ultrastructural level. In unstimulated platelets washed with EDTA, an intense uniform labeling of WGA-gold complexes was found on the surface membrane. When washed platelets were stimulated by thrombin in the absence of Ca2+, only a release reaction was induced. WGA labeling on the surface membranes of these platelets decreased dramatically. However, the labeling intensity of WGA-gold complexes on the surface membrane of aggregated platelets induced by thrombin in the presence of Ca2+ increased significantly compared to that of thrombin-stimulated platelets in the absence of Ca2+. In contrast to the uniform labeling on the surface membranes of unstimulated platelets, clusters of gold label were often found on the surface membrane of the aggregated platelets, although there was no significant quantitative difference in the labeling intensity between these two groups. Thus, we present direct morphological evidence demonstrating qualitative and quantitative alterations of WGA labeling on the surface membrane of platelets after thrombin stimulation. The possibility is considered that WGA-binding glycoproteins in the surface membrane are involved in the aggregation response after thrombin stimulation.     

Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C

One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.     

The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). II. The role of calcium

The integumental pigment cells (erythrophores) of the squirrel fish, Holocentrus ascensionis, are specialized for rapid radial transport of the pigment granules contained within their cytoplasm. Pigment granules in isolated denervated erythrophores alternate spontaneously between a centrally aggregated state and a radially dispersed state. In the absence of external calcium, pigment aggregation does not occur spontaneously and cannot be induced by the aggregating agents epinephrine or high concentration of external K+. Pigment aggregation is also impaired in the presence of D600 or papaverine, compounds reported to antagonize calcium influx into the cell. Pigment aggregation can be induced by experimental elevation of the concentration of cytoplasmic free Ca2+, with a Ca-EGTA buffer system in conjunction with ionophore A23187. The threshold concentration of Ca2+ required to produce this effect is 5 X 10(-6) M. These results suggest that cytoplasmic free Ca2+ is involved in mediating pigment aggregation and that some, if not all, the Ca2+ is supplied by influx from the extracellular space.     

Calcium-activated membrane depolarization via modulation of chloride efflux from parietal cells during gastrin stimulation

In the isolated parietal cell the following observations were made: gastrin led to an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) even in the absence of medium Ca2+ and to transient membrane depolarization in both the absence of the medium Ca2+ and the fura-2-loaded cell. The incorporated Ca2+ chelator BAPTA inhibited the gastrin-induced membrane depolarization. The magnitude of depolarization caused by gastrin was unchanged on removal of medium Na+. Furosemide but not tetraethylammonium inhibited the gastrin-induced depolarization. The results suggest that the Ca2+ released from the store(s) induces membrane depolarization, possibly via modulation of a Cl- efflux across the luminal surface during gastrin stimulation.     

Ca2+ movement in smooth muscle cells studied with one- and two-dimensional diffusion models.

Although many of the processes involved in the regulation of Ca2+ in smooth muscle have been studied separately, it is still not well known how they are integrated into an overall regulatory system. To examine this question and to study the time course and spatial distribution of Ca2+ in cells after activation, one- and two-dimensional diffusion models of the cell that included the major processes thought to be involved in Ca regulation were developed. The models included terms describing Ca influx, buffering, plasma membrane extrusion, and release and reuptake by the sarcoplasmic reticulum. When possible these processes were described with known parameters. Simulations with the models indicated that the sarcoplasmic reticulum Ca pump is probably primarily responsible for the removal of cytoplasmic Ca2+ after cell activation. The plasma membrane Ca-ATPase and Na/Ca exchange appeared more likely to be involved in the long term regulation of Ca2+. Pumping processes in general had little influence on the rate of rise of Ca transients. The models also showed that spatial inhomogeneities in Ca2+ probably occur in cells during the spread of the Ca signal following activation and during the subsequent return of Ca2+ to its resting level.     

Insulin action in isolated fat cells. II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells.

The effects of ommission of Ca2+ and Mg2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca2+ + Mg2+)-free incubation medium incorporation of L-[14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[14C]leucine incorporation only in the presence of added CaCl2 or MgCl2. Incubation of the cells in the (Ca2+ + Mg2+)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 125I-labelled insulin to the fat cells was reduced in the absence of Ca2+ and Mg2+ but was not abolished, even in the presence of EDTA. Ca2+ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg2+, Sr2+ and Ba2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

A new model for proton pumping in animal cells: the role of pyrophosphate

The H+-PPase activity was characterized in membrane fractions of ovary and eggs of Rhodnius prolixus. This activity is totally dependent on Mg2+, independent of K+ and strongly inhibited by NaF, IDP and Ca2+. The membrane proteins of eggs were analyzed by western blot using antibodies to the H+-PPase from Arabidopsis thaliana. The immunostain was associated with a single 65-kDa polypeptide. This polypeptide was immunolocalized in yolk granule membranes by optical and transmission electron microscopy. We describe the acidification of yolk granules in the presence of PPi and ATP. This acidification is inhibited in the presence of NAF, Ca2+ and antibodies against H+-PPase. These data show for the first time in animal cells that acidification of yolk granules involves an H+-PPase as well as H+-ATPase.     

Presenilin 1 in migration and morphogenesis in the central nervous system

Morphogenesis of the central nervous system relies in large part upon the correct migration of neuronal cells from birthplace to final position. Two general modes of migration govern CNS morphogenesis: radial, which is mostly glia-guided and topologically relatively simple; and tangential, which often involves complex movement of neurons in more than one direction. We describe the consequences of loss of function of presenilin 1 on these fundamental processes. Previous studies of the central nervous system in presenilin 1 homozygote mutant embryos identified a premature neuronal differentiation that is transient and localized, with cortical dysplasia at later stages. We document widespread effects on CNS morphogenesis that appear strongly linked to defective neuronal migration. Loss of presenilin 1 function perturbs both radial and tangential migration in cerebral cortex, and several tangential migratory pathways in the brainstem. The inability of cells to execute their migratory trajectories affects cortical lamination, formation of the facial branchiomotor nucleus, the spread of cerebellar granule cell precursors to form the external granule layer and development of the pontine nuclei. Finally, overall morphogenesis of the mid-hindbrain region is abnormal, resulting in incomplete midline fusion of the cerebellum and overgrowth of the caudal midbrain. These observations indicate that in the absence of presenilin 1 function, the ability of a cell to move can be severely impaired regardless of its mode of migration, and, at a grosser level, brain morphogenesis is perturbed. Our results demonstrate that presenilin 1 plays a much more important role in brain development than has been assumed, consistent with a pleiotropic involvement of this molecule in cellular signaling.     

A role for N-cadherin in mesodermal morphogenesis during gastrulation

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.     

Alteration of UV-sensitivity of mice peritoneal macrophage plasma membranes by calcium ions

Effect of Ca2+ ions on UV-induced mice peritoneal macrophage plasma membrane damage has been studied. Drop of the extracellular Ca2+ concentration has been found to result in a reduced expression of this damage. On the contrary, a raised intra- and extra-cellular Ca2+ level is associated with a higher number of cells with damaged plasma membranes. These findings make it possible to suggest that this change in the plasma membrane photosensitivity might be a result of alterations in the membrane lipid matrix electrical stability owing to UV-induced lipid photo-peroxidation. This study has also shown that free radical peroxidation of membrane lipids plays a significant part in UV-induced cell damage.     

Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artificial membrane systems

Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.     

Ontogeny of vitamin D action on the morphology and calcium transport properties of the chick embryonic yolk sac

The ontogeny of the calcium transport properties and hormonal modulation of the yolk sac membrane in amniote embryos is presently poorly understood. We investigated the role of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on plasma calcium values, yolk sac morphology and the ability of the yolk sac membrane to transport 45Ca from yolk to embryo. 1,25-(OH)2D3 treatment caused significant hypercalcaemia in 9-, 12- and 15-day embryos. Additionally, this hormone caused a hypertrophy of the endodermal cell layer that comprises the bulk of the yolk sac membrane. Both of these effects were the most dramatic in the 15-day embryo, the oldest age tested. 45Ca added to the yolk was transported into the blood rapidly across the yolk sac membrane. 1,25-(OH)2D3 significantly enhanced this transport in all age groups. [14C]Inulin was also taken across the yolk sac membrane, but at a slower rate than 45Ca; this transport was unaffected by 1,25-(OH)2D3. Thus, the yolk sac responds to 1,25-(OH)2D3 treatment both morphologically and functionally. The mechanism for transport appears to be a specific one, rather than a simple enhancement of non-specific endocytosis.     

Assessment of IgE-receptor function through measurement of hydrolysis of membrane inositol phospholipids. New insights on the phenomena of biphasic antigen concentration-response curves and desensitization

Previous studies have shown that hydrolysis of membrane inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells depended on the rate and extent of the aggregation of receptors of IgE. This response was used as an experimental probe to study the role of IgE receptors in initiating stimulatory and inhibitory processes within the cell. The response was amplified markedly by increasing the concentration of external Ca2+ from 0 to 1 mM, but the concentration required to support half-maximal response varied from less than 0.1 mM for the most potent cross-linking reagent, DNP24BSA (24 molecules of DNP attached to 1 molecule of BSA) to 0.5 mM for the least potent reagent, aggregated OVA. The dependency of phosphoinositide hydrolysis on external Ca2+ was reduced to zero once hydrolysis of inositol phospholipids was underway but secretion of histamine remained totally dependent on the presence of 0.5 to 1 mM external Ca2+. The stimulatory response persisted as long as receptors remained aggregated but it was modulated by a biochemical process, possibly the activation of protein kinase C, that targeted specifically aggregated receptors, or an associated protein. For example, when cells had become desensitized to high concentrations of one Ag, a normal response could be evoked with a second Ag. Also cells that had become desensitized could be reactivated by permeabilizing the cells. Interestingly, bell-shaped Ag dose-response curves, which were characteristic for both the phosphoinositide and secretory responses, were transformed to sigmoid-shaped curves once cells were permeabilized and dialyzed.     

Alpha 1- and beta-adrenergic regulation of intracellular Ca2+ levels in brown adipocytes

In order to monitor changes in cytosolic Ca2+ levels, brown-fat cells were incubated with the fluorescent Ca2+-indicator fura-2 and the fluorescence intensity ratio followed. The addition of norepinephrine led to a rapid and persistent increase in the cytosolic Ca2+ level, which was dose-dependent with a maximal effect at about 1 microM. The response was diminished in the absence of extracellular Ca2+ and was inhibited more efficiently by phentolamine and prazosin than by propranolol or yohimbine, indicating alpha 1-adrenergic mediation. Accordingly, selective alpha 1-adrenergic stimulation also increased the cytosolic Ca2+ level. However, selective beta-adrenergic stimulation, as well as the adenylate cyclase activator forskolin, were also able to increase the cytosolic Ca2+ level in these cells to a certain extent. It was concluded that the major part of the increase in cytosolic Ca2+ was mediated, as in other cell types, via alpha 1-adrenergic receptors, but that Ca2+ levels were also positively modulated by a cAMP-mediated process. These observations are discussed in relation to known alpha 1/beta synergisms in brown adipose tissue.     

Mechanisms of excitation-contraction coupling and calcium liberation in striated muscles of vertebrates

In vertebrate skeletal muscle, the main part of excitation-contraction coupling occurs at the level of the triad, where membranes of T-system and of junctional SR are facing each other. From place to place, the junctional gap is bridged by "feet" structures which include the SR Ca2+ channel. Half of them are closely apposed to tubular intramembranous structures assumed to be DHP-sensitive voltage-sensors which are similar to tubular Ca2+ channels and act by controlling Ca2+ release from SR. During a twitch, the release of Ca2+ activator from SR is controlled both by voltage-sensors via the feet structures and by a tubular Na+ current via a Na+-induced Ca2+ release mechanism. During long-duration mechanical responses, additional mechanisms are involved: a Ca2+-induced Ca2+ release which can be activated by ICa; the release of Ca2+ from membrane, controlled by the operation of a Na+/Ca2+ exchanger and/or new arrangements of surface membrane charges. An IP3-mediated Ca2+ release could be involved too. All these mechanisms can be regulated by intracellular biochemical or ionic processes.     

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity: role of intracellular calcium

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity to isolated hepatocytes was studied. MPTP was more toxic to hepatocytes than its major metabolite, 1-methyl-4-phenylpyridine (MPP+); this may, in part, be explained by the lesser permeability of the hepatocyte plasma membrane to the cation compared to its parent compound, MPTP. Loss of cell viability was preceded by plasma membrane bleb formation and disturbance of intracellular Ca2+ homeostasis. MPTP caused a rapid depletion of the mitochondrial Ca2+ pool which was followed by a marked and sustained elevation of cytosolic free Ca2+ concentration. This increase of cytosolic Ca2+ level appeared to be associated with the impairment of the cell's Ca2+ extrusion system since the plasma membrane Ca2+-ATPase was markedly inhibited in MPTP-treated hepatocytes. Preincubation of hepatocytes with inhibitors of monoamine oxidase type B, but not A, protected the cells from MPTP-induced cytotoxicity. Moreover, the monoamine oxidase B inhibitor, pargyline, prevented the rise in cytosolic free Ca2+ concentration and partially protected the plasma membrane Ca2+-ATPase from inhibition by MPTP. As observed with MPTP, MPP+ caused an extensive loss of mitochondrial Ca2+ and significantly decreased the rate of Ca2+ efflux from hepatocytes. However, MPP+ was without effect on the plasma membrane Ca2+-ATPase. In conclusion, our studies demonstrate that MPTP caused a substantial elevation of cytosolic Ca2+ which preceded loss of cell viability and we propose that calcium ions are of major importance in the mechanism of MPTP- and MPP+-induced toxicity in hepatocytes.