Membrane particle distribution in the sternal epithelia of the terrestrial isopod Porcellio scaber latr. (Crustacea, oniscidea) during CaCO(3) deposit formation and resorption, a freeze-etch analysis.

The anterior sternal epithelium of terrestrial isopods transports cuticular Ca(2+) to and from large sternal CaCO(3) deposits. We analyzed the anterior and posterior sternal epithelium by the means of the freeze-etch technique and measured the size distribution and density of intramembrane particles (IMPs) during three different molting stages. At least three IMP size classes around 4.5, 7.7, and 9.4 nm can be distinguished on the P-face of the apical and basolateral plasma membrane. An additional size class of around 12.8 nm is restricted to the apical compartment. In the anterior sternal epithelium, the density of these large particles changes by a factor of 1.9 during the molt cycle, suggesting a role in CaCO(3) formation and/or resorption. The density of the smaller IMPs rises transiently by a factor of 1.3 in the posterior sternal epithelium only. The IMP density of the basolateral plasma membrane increases significantly by a factor of 1.4 and 1.3 in the anterior and posterior sternal epithelia, respectively. The results indicate that increases in the IMP density contribute to the differentiation to an increased transport activity during the cyclic enlargements of the plasma membrane surface area in the anterior sternal epithelium.     

Morphometric analysis of the calcium-transporting sternal epithelial cells of the terrestrial isopods Ligia oceanica, Ligidium hypnorum, and Porcellio scaber during molt

Isopods shed first the posterior and then the anterior half of the body. Before molt, most terrestrial species resorb CaCO3 from the posterior mineralized cuticle. The mineral is stored in anterior sternal deposits, which are used to calcify the new posterior cuticle after molt. For Porcellio scaber it is known that the anterior sternal epithelium has specific structural differentiations for epithelial transport. These differentiations include the plasma membrane surface areas, and the volume fraction of the mitochondria. We analyzed the ultrastructure of the sternal epithelium and used a morphometric approach to study the variations of these parameters between species living in different terrestrial environments. In Ligidium hypnorum, which lives in moist environments, the plasma membrane surface area and volume fraction of mitochondria are much larger than in the semiterrestrial Ligia oceanica. This is in accordance with the relatively larger CaCO3 deposits and shorter time intervals for their formation and resorption in L. hypnorum. For P. scaber, which is adapted to mesic habitats, most values are between those of L. oceanica and L. hypnorum. However, P. scaber has even larger CaCO3 deposits which are formed and degraded within similar time intervals as in L. hypnorum. This unexpected result is considered from the standpoint of more effective mechanisms being present for epithelial ion transport.     

Ultrastructural changes of the anterior and posterior sternal integument of the terrestrial isopod Porcellio scaber Latr. (Crustacea) during the moult cycle

The ultrastructure of the anterior (ASE) and posterior sternal epithelium (PSE) was investigated during the biphasic moult cycle. During early premoult the sternal epithelial cells increase in size, accumulate huge amounts of glycogen, and increase the abundance of cellular organelles. CaCO(3) deposit formation begins before the secretion of the epicuticle in the anterior sternal integument and continues through the secretion of the first exocuticular layers. The deposition of cuticle is delayed in the ASE until the CaCO(3) deposit is fully resorbed between the anterior and posterior moult. The development of the interstitial network (IN), which enormously increases the basolateral compartment of the plasma membrane, starts at the beginning of the exocuticle deposition. During CaCO(3) deposit formation and resorption the volume fraction of the mitochondria is much larger in the ASE than in the PSE, although the cuticle is secreted faster in the posterior integument. The results suggest that the exocuticular and epicuticular layers are permeable to calcium and probably also carbonate ions; that the IN is required during late premoult, when CaCO(3) deposition is accelerated, but not during early CaCO(3) deposition; and that active mechanisms contribute to transepithelial ion transport during CaCO(3) deposit formation and resorption.     

Architecture of the organic matrix in the sternal CaCO3 deposits of Porcellio scaber (Crustacea, Isopoda)

Before the molt terrestrial isopods resorb calcium from the posterior cuticle and store it in large deposits within the first four anterior sternites. In Porcellio scaber the deposits consist of three structurally distinct layers consisting of amorphous CaCO3 (ACC) and an organic matrix that consists of concentric and radial elements. It is thought that the organic matrix plays a role in the structural organization of deposits and in the stabilization of ACC, which is unstable in vitro. In this paper, we present a thorough analysis of the ultrastructure of the organic matrix in the CaCO3 deposits using high-resolution field-emission scanning electron microscopy. The spherules and the homogeneous layer contain an elaborate organic matrix with similar structural organization consisting of concentric reticules and radial strands. The decalcification experiments reveal an inhomogeneous solubility of ACC within the spherules probably caused by variations in the stabilizing properties of matrix components. The transition between the three layers can be explained by changes in the number of spherule nucleation sites.     

Molting and cuticle deposition in the subterranean trichoniscid Titanethes albus (Crustacea, Isopoda)

Terrestrial isopods are a suitable group for the study of cuticle synthesis and calcium dynamics because they molt frequently and have evolved means to store calcium during molt. Little data is currently available on molting in Synocheta and subterranean isopods. We studied the molting dynamics in the subterranean trichoniscid Titanethes albus under laboratory conditions and performed a microscopic investigation of sternal CaCO(3) deposits and the tergal epithelium during molt in this species. In accordance with its lower metabolic rate, molting in the laboratory is roughly 2-3 times less frequent in Titanethes albus than would be expected for an epigean isopod under similar conditions. Animals assumed characteristic postures following the molt of each body half and did not consume the posterior exuviae after posterior molt. The structure of sternal calcium deposits and the ultrastructural characteristics of the epidermis during cuticle formation in Titanethes albus are similar to those described in representatives of Ligiidae. During the deposition of the exocuticle, the apical plasma membrane of epidermal cells forms finger-like extensions and numerous invaginations. In the ecdysial space of individuals in late premolt we observed cellular extensions surrounded by bundles of tubules.     

Membrane Particle Distribution in the Sternal Epithelia of the Terrestrial Isopod Porcellio scaber Latr. (Crustacea, Oniscidea) during CaCO3 Deposit Formation and Resorption, a Freeze-Etch Analysis

The anterior sternal epithelium of terrestrial isopods transports cuticular Ca²⁺ to and from large sternal CaCO₃ deposits. We analyzed the anterior and posterior sternal epithelium by the means of the freeze-etch technique and measured the size distribution and density of intramembrane particles (IMPs) during three different molting stages. At least three IMP size classes around 4.5, 7.7, and 9.4 nm can be distinguished on the P-face of the apical and basolateral plasma membrane. An additional size class of around 12.8 nm is restricted to the apical compartment. In the anterior sternal epithelium, the density of these large particles changes by a factor of 1.9 during the molt cycle, suggesting a role in CaCO₃ formation and/or resorption. The density of the smaller IMPs rises transiently by a factor of 1.3 in the posterior sternal epithelium only. The IMP density of the basolateral plasma membrane increases significantly by a factor of 1.4 and 1.3 in the anterior and posterior sternal epithelia, respectively. The results indicate that increases in the IMP density contribute to the differentiation to an increased transport activity during the cyclic enlargements of the plasma membrane surface area in the anterior sternal epithelium.     

The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO₃) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na⁺ followed by Mg²⁺, Ca²⁺ and K⁺. The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO₃ precipitation and dissolution.     

Ultrastructure of CaCO3 deposits of terrestrial isopods (Crustacea, Oniscidea)

 The ultrastructure of the sternal CaCO₃ deposits of 3 species of the Diplochaeta and 15 of the Crinochaeta was investigated by means of scanning electron microscopy of fractured surfaces. In the Diplochaeta Li-gia italica and L. oceanica, the deposits consist exclusively of individual spherules with diameters between 0.2 and 1.4 μm. No material was observed within the spaces between the spherules. In Ligidium hypnorum, two structurally distinct regions exist. A proximal layer resembling the deposit of Ligia italica and L. oceanica and a distal layer in which the spherules appear to be fused with each other. In the species of the Crinochaeta, the CaCO₃ deposits comprise a spherular region which resembles the deposits of Ligidium hypnorum, and a homogeneous layer located between the spherular part of the deposit and the hypodermal cell layer. In some species the diameters of the spherules may be up to 3.1 μm. In the homogeneous layer and the distal spherular layer more calcium per volume can be stored than in the proximal spherular layer in which the spaces between the spherules are devoid of CaCO₃. This suggests that the multiple layered deposits are an adaptation to terrestrial life, as a consequence of the need for increased resorption of cuticular calcium. Accepted: 7 January 1997     

Evidence that mineralized spherules are involved in the formation of the superficial layer of the elasmoid scale in cichlids Cichlasoma octofasciatum and Hemichromis bimaculatus (Pisces,Teleostei): an epidermal active participation?

Summary The region between the epidermis and the surface of the overlapping part of scales has been studied in two cichlid teleosts using transmission electron microscopy. In a few specimens only, numerous mineralized spherules (1 m in diameter) are observed in the loose dermis and at the scale surface, and form a large part of the superficial outer limiting layer of the scale. In the loose dermis (stratum laxum) and close to the scale surface spherules are either free or included in dermal cells. When free, they are dispersed in the extracellular matrix of the dermis, among the fibrils of anchoring bundles, and fused with the scale surface. When included in cell vacuoles, they lie close to the lamina densa and to the scale surface. Steps in the formation of the mineralized spherules are only seen in the lamina densa of the basement membrane. The spherules contain needle-like mineral crystals radially orientated and an organic matrix of stippled material and dense granules, some of which form concentric lines around the centre of the spherules. The results suggest that mineralized spherules form in the lamina densa and pass through the dermis to the scale surface in which they are incorporated.     

Postnatal development of "synaptic" ribbons and spherules in the guinea pig pineal gland

Previous studies have shown that the functionally enigmatic pineal "synaptic" ribbons are structurally a heterogeneous group of organelles consisting of rodlike ribbons sensu stricto, spherules, and intermediate forms. As ribbons and spherules react differently under various experimental conditions, these organelles were studied qualitatively and quantitatively during the postnatal period in guinea pigs. It was found that the pinealocytes were highly differentiated at birth and contained all three forms of "synaptic" structures. Ribbons and intermediate forms were more abundant than spherules and exhibited a striking increase in number on postnatal days 1 and 2; this increase was followed by a distinct trough and by a second peak at days 12 and 13, after which their numbers declined to reach adult levels by day 20. The spherules were small in number at birth and did not show the large immediate postnatal increase observed for the ribbons and intermediate forms. Instead there was a steady numerical increase up to day 12 (absolute number) or day 15 (relative numbers), followed by a decrease to adult level by day 20. Whereas during the early postnatal period (days 1 to 3) the majority of pinealocytes were characterized by ribbons and intermediate forms, with increasing age spherule-bearing pinealocytes increased in number. As ribbons and spherules were usually not found in the same pinealocyte, the present findings are interpreted to mean that ribbons and spherules characterize different types of pinealocytes showing an inverse numerical development postnatally. Developmentally intermediate forms behave like ribbons.     

X-ray microprobe analysis of epithelial calcium transport

The sternal epithelium of Porcellio scaber was used as a novel model to study the subcellular elemental distribution in control and Ca(2+)-transporting stages in situ. The anterior sternal epithelium (ASE) is specialized for transport of cuticular Ca to sternal CaCO(3) deposits during premolt, and from these deposits during intramolt. The less specialized posterior sternal epithelium transports Ca(2+) to and from the cuticle. In the ASE cells basal [Na], [Cl], and [Mg] are higher than in the apical side. The basal [Na] increases from 105 to 173 mmol/kg dry mass between control and Ca(2+)-transporting stages, accompanied by a decrease in [Cl] and [K]. The [Mg] increases, suggesting transepithelial Mg(2+)-transport. Cytosolic [Ca] varied insignificantly between 4.5 and 5.7 mmol/kg dry mass, however, the number of Ca hot-spots with concentrations between 15 and 50 mmol/kg dry mass increased during transport. Mitochondrial [Ca] decreased in the ASE from 3.3 in the control to 1.0 in the late premolt and to 2.0 mmol/kg dry mass in the intramolt stage. The results suggest Na(+)-dependent mechanisms for transcellular Ca(2+)-transport and the presence of Ca(2+)-binding proteins. Organelles, probably the smooth endoplasmic reticulum, sequester Ca(2+) during intracellular Ca(2+)-transport. A role of mitochondria as a storage site for cuticular Ca is excluded.     

Morphological Diversity of the Rod Spherule: A Study of Serially Reconstructed Electron Micrographs

Purpose

Rod spherules are the site of the first synaptic contact in the retina’s rod pathway, linking rods to horizontal and bipolar cells. Rod spherules have been described and characterized through electron micrograph (EM) and other studies, but their morphological diversity related to retinal circuitry and their intracellular structures have not been quantified. Most rod spherules are connected to their soma by an axon, but spherules of rods on the surface of the Mus musculus outer plexiform layer often lack an axon and have a spherule structure that is morphologically distinct from rod spherules connected to their soma by an axon. Retraction of the rod axon and spherule is often observed in disease processes and aging, and the retracted rod spherule superficially resembles rod spherules lacking an axon. We hypothesized that retracted spherules take on an axonless spherule morphology, which may be easier to maintain in a diseased state. To test our hypothesis, we quantified the spatial organization and subcellular structures of rod spherules with and without axons. We then compared them to the retracted spherules in a disease model, mice that overexpress Dscam (Down syndrome cell adhesion molecule), to gain a better understanding of the rod synapse in health and disease.

Methods

We reconstructed serial EM images of wild type and Dscam^GoF (gain of function) rod spherules at a resolution of 7 nm in the X-Y axis and 60 nm in the Z axis. Rod spherules with and without axons, and retracted spherules in the Dscam^GoF retina, were reconstructed. The rod spherule intracellular organelles, the invaginating dendrites of rod bipolar cells and horizontal cell axon tips were also reconstructed for statistical analysis.

Results

Stereotypical rod (R1) spherules occupy the outer two-thirds of the outer plexiform layer (OPL), where they present as spherical terminals with large mitochondria. This spherule group is highly uniform and composed more than 90% of the rod spherule population. Rod spherules lacking an axon (R2) were also described and characterized. This rod spherule group consists of a specific spatial organization that is strictly located at the apical OPL-facing layer of the Outer Nuclear Layer (ONL). The R2 spherule displays a large bowl-shaped synaptic terminal that hugs the rod soma. Retracted spherules in the Dscam^GoF retina were also reconstructed to test if they are structurally similar to R2 spherules. The misplaced rod spherules in Dscam^GoF have a gross morphology that is similar to R2 spherules but have significant disruption in internal synapse organization.

Conclusion

We described a morphological diversity within Mus musculus rod spherules. This diversity is correlated with rod location in the ONL and contributes to the intracellular differences within spherules. Analysis of the Dscam^GoF retina indicated that their R2 spherules are not significantly different than wild type R2 spherules, but that their retracted rod spherules have abnormal synaptic organization.     

Change in the activity of Cl-,HCO3(-)-ATPase in microsome fraction during early development of the sea urchin, Hemicentrotus pulcherrimus

In sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-,HCO3(-)-ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-,HCO3(-)-ATPase. The activity of Cl-,HCO3(-)-ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-,HCO3(-)-ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.     

The role of matrix proteins in the control of nacreous layer deposition during pearl formation

To study the function of pearl oyster matrix proteins in nacreous layer biomineralization in vivo, we examined the deposition on pearl nuclei and the expression of matrix protein genes in the pearl sac during the early stage of pearl formation. We found that the process of pearl formation involves two consecutive stages: (i) irregular calcium carbonate (CaCO(3)) deposition on the bare nucleus and (ii) CaCO(3) deposition that becomes more and more regular until the mature nacreous layer has formed on the nucleus. The low-expression level of matrix proteins in the pearl sac during periods of irregular CaCO(3) deposition suggests that deposition may not be controlled by the organic matrix during this stage of the process. However, significant expression of matrix proteins in the pearl sac was detected by day 30-35 after implantation. On day 30, a thin layer of CaCO(3), which we believe was amorphous CaCO(3), covered large aragonites. By day 35, the nacreous layer had formed. The whole process is similar to that observed in shells, and the temporal expression of matrix protein genes indicated that their bioactivities were crucial for pearl development. Matrix proteins controlled the crystal phase, shape, size, nucleation and aggregation of CaCO(3) crystals.     

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton’s jelly stem cells (hWJSCs)

Human Wharton’s jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P?<?0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.     

Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes

Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.All positive-strand RNA viruses replicate their genomes in association with cellular membranes. The formation and activity of the membrane-bound replication complexes (RCs) can result in extensive alteration of membrane structures (11, 40, 48). Different viruses use different cytoplasmic membrane compartments as platforms for replication. Currently, there is only a limited understanding of how the virus-encoded and cellular proteins coordinate the formation of the replication-induced membrane structures. We address the mechanisms of membrane-bound replication with alphaviruses, particularly Semliki Forest virus (SFV). The alphaviruses comprise several human and animal pathogens, including the encephalitogenic alphaviruses (e.g., Western, Eastern, and Venezuelan equine encephalitis viruses) as well as the recently reemerging chikungunya virus, which belongs to the SFV clade of alphaviruses. During the past 5 years, chikungunya virus has caused more than 2 million infections and 500 deaths, and a new strain has spread throughout the areas surrounding the Indian Ocean (50). The alphaviruses use mosquitoes as intermediate hosts and transmission vectors, and at present no vaccines or antivirals are available to control these infections.The cytoplasmic replication of alphaviruses depends on the four viral nonstructural (ns) proteins, nsP1 to nsP4, which are all essential and act as a membrane-bound replication complex. The nsPs are translated from the viral positive-sense RNA genome as one large polyprotein. Cleavages catalyzed by the nsP2 moiety result in the release of the individual proteins. A large fraction of the synthesized nsPs is involved in genome replication and associates with membranes, but a sizable fraction dissociates and is distributed in different cellular compartments: nsP1 binds to the inner surface of the plasma membrane (PM); nsP2 is translocated into the nucleus; nsP3 seems to form aggregates in the cytoplasm; and most of the extra nsP4, the core RNA polymerase, is degraded by the proteasome. While the major enzymatic functions of the individual nsPs have been elucidated (21), little is known of how they function together in the replication machinery.As in other positive-strand RNA viruses, the RCs of alphaviruses are associated with altered intracellular membranes, which were first described in the late 1960s and early 1970s (13, 14, 18). In these early studies, it was shown that virus replication induces bulb-shaped membrane invaginations with a diameter of ∼50 nm, which were called spherules. The spherules were found on the limiting membranes of large cytoplasmic vacuoles, which were named virus-induced cytopathic vacuoles of type I (CPV-I). On rare occasions, the spherules were seen also at the PM. By electron microscopic (EM) autoradiography, it was also shown that the spherules both at the CPV-I and at the PM could be sites of RNA synthesis (18). Subsequently, Froshauer et al. (15) showed that CPV-I are positive for endosomal and lysosomal markers. Moreover, using EM, they showed that the inside of the spherule is connected to the cytoplasm by a pore from which electron-dense material (which the authors suggest to be the newly synthesized RNA) seems to diffuse into the cytoplasm.During the past decade, our group has addressed the biogenesis of the CPV-I. We demonstrated that the formation of the spherules did not require structural proteins (44) and, more recently, that all four nsPs were associated with the spherules together with newly formed RNA (labeled by bromouridine), strongly suggesting that they were the actual units of RNA replication (RCs) (28). We also suggested as one possibility that the spherules could first arise at the PM; subsequent endocytosis of the spherules could account for the formation of the CPV-I (28, 44). Of the four nsPs, only nsP1 has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the PM (45). NsP1 is a monotopic membrane protein; its affinity for membranes is dictated by an amphipathic alpha helix, located in the central region of the protein (4, 31). NsP1 has a specific affinity for negatively charged phospholipids, which could potentially account for its prevalent localization to the PM, where such lipids are enriched. Later we showed that the membrane binding of nsP1 through the amphipathic helix is essential for alphavirus replication (56).Several groups of positive-sense RNA viruses make spherules, which appear very similar to those made by the alphaviruses. However, for these virus groups, the spherules arise in different locations. For the well-characterized brome mosaic virus (BMV), a plant virus very distantly related to the alphaviruses, the spherules are seen in the endoplasmic reticulum (ER) adjacent to the nucleus (51). For the unrelated nodaviruses, the spherules are localized on mitochondrial surfaces (25). Recent models of the RCs of flaviviruses suggest that their replication complexes also resemble spherules (62). For the Flaviviridae, the RCs are found on the membranes of the secretory pathway.The aim of this study was to clarify the role of different membranes in the formation and maturation of alphavirus RCs, and particularly to test our hypothesis that the RCs (spherules) are formed at the PM and are internalized thereafter. By using confocal microscopy, live-cell imaging, and novel electron microscopic techniques, we demonstrate that the RCs of SFV undergo an unprecedented, highly dynamic trafficking between different cellular compartments. They are first detected at the PM, which serves as the major platform for spherule formation. A specific endocytic event results in the transfer of spherules to the limiting membrane of small cytoplasmic vesicles. Using pharmacological inhibitors, we have been able to block the internalization process, and we found that the exit of spherules from the PM is dependent on the activity of phosphatidylinositol3- kinase (PI3K). Following the intracellular dynamics associated with spherules in live cells, we show the contribution of actin and microtubule-based transport, as well as that of fusion events with preexisting acidic organelles, providing the first complete model for the biogenesis of the large static CPV-I, where spherules are found at later stages of infection.     

Otolith crystals (in Carapidae): growth and habit

The biomineralization of otoliths results mainly from the release of soluble Ca(2+), which is in turn precipitated as CaCO(3) crystals. In some Carapidae, sagittae sections have been shown to reveal a three-dimensional asymmetry with a nucleus close to the sulcal side, an unusual position. This study seeks to understand otolith formation in Carapus boraborensis. The unusual shape of the otolith is partly explained by the distribution of the epithelium cells, and particularly the sensory epithelium. Experimental evidence shows for the first time that aragonite growth takes place along the c-axis. These aragonite needles present two different habits. On the sulcal side is found the acicular form resulting from rapid growth during a short period of time. On the anti-sulcal side, the prismatic form seen there is due to a slower growth speed over longer periods. The otolith surface was observed each hour during a period of 24h in fishes reared in similar conditions. This allowed for the first time the direct observation on the otolith surface of the deposition of the two layers (L-zone and D-zone). In C. boraborensis, the organic-rich layer (D-zone) develops during the day, whereas the CaCO(3) layer (L-zone) seems to be deposited during the night.     

Uridine diphosphoglucose pyrophosphorylase activity and differentiation in the cellular slime mold Physarum polycephaluno

The specific activity of uridine 5'-triphosphate:alpha-d-glucose 1-phosphate uridyltransferase (EC 2.7.7.9) (also called uridine 5'-diphosphate [UDP]-glucose pyrophosphorylase) has been found to increase up to eightfold during spherule formation by the slime mold Physarum polycephalum. The enzyme accumulates during the first 8 to 9 h after initiation of spherule formation, declines to basal levels found in vegetative microplasmodia by 15 h, and is undetectable in completed spherules. Specific activities for UDP-glucose pyrophosphorylase in vegetative microplasmodia range from 15 to 30 nmol of UDP-glucose formed per min per mg of protein, whereas accumulated levels during spherule formation can attain a specific activity as high as 125 nmol of UDP-glucose formed per min per mg of protein. The scheduling and extent of accumulation are critically dependent on an early log-phase age of microplasmodia originally induced to form spherules. Spherule induction by 0.2 M or 0.5 M mannitol delays this schedule in a variable and unpredictable manner. Spherule-forming microplasmodia which have accumulated high levels of UDP-glucose pyrophosphorylase spontaneously excrete the enzyme when transferred to salts medium containing 0.2 M or 0.5 M mannitol. The excreted enzyme is subsequently destroyed or inactivated. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of UDP-glucose pyrophosphorylase requires concomitant protein synthesis and prior ribonucleic acid synthesis.     

Expression of Ca2+-ATPase and Na+/Ca2+-exchanger is upregulated during epithelial Ca2+ transport in hypodermal cells of the isopod Porcellio scaber

It is thought that a plasma membrane Ca(2+)-transport ATPase (PMCA) and a Na(+)/Ca(2+)-exchange (NCE) mechanism are involved in epithelial Ca(2+) transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca(2+)-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca(2+)-transporting condition to the stages of CaCO(3) deposit formation and degradation. During Ca(2+)-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.     

Conical tomography of a ribbon synapse: structural evidence for vesicle fusion

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ～470 vesicles that were hemi-fused and ～187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.