Apical Invaginations Research Articles

Na+ and K+ transport and maturation stage ameloblast modulation.

Introduction: Enamel mineralization requires calcium transport into the extracellular matrix for the synthesis of hydroxyapatite (HA) crystals. Formation of HA releases protons into the matrix, which are then neutralized when ameloblasts modulate from cells with apical invaginations, the so-called ruffle-ended ameloblasts (RE), to smooth-ended ameloblasts (SE). Ameloblast modulation is associated with the translocation of the calcium exchanger Nckx4 to the apical border of RE, to remove Na+ from the enamel matrix in exchange for Ca2+ and K+. As enamel matures, Na+ and K+ in the matrix progressively decrease. However, the transporter to remove K+ from mineralizing enamel has not been identified. Methods: Expression of K+ exchangers and channels in secretory and maturation stage of enamel organs were compared following an RNA-seq analysis. Kcnj15, which encodes the Kir4.2 inwardly rectifying K+ channel, was found to be the most upregulated internalizing K+ transporter in maturation stage of enamel organs. Kir4.2 was immunolocalized in wt, Nckx4-/-, Wdr72-/-, and fluorosed ameloblasts. Regulation of Wdr72 expression by pH was characterized in vitro and in vivo. Results: Kir4.2 immunolocalized to the apical border of wild type (wt) mouse RE and cytosol of SE, a spatial distribution pattern shared by NCKX4. In Nckx4-/- ameloblasts, Kir4.2 also localized to the apical surface of RE and cytosol of SE. However, in fluorosed and Wdr72-/- ameloblasts, in which vesicle trafficking is disrupted, Kir4.2 remained in the cytosol. In vitro, Wdr72 was upregulated in LS8 cells cultured in medium with a pH 6.2, which is the pH of the enamel matrix underlying RE, as compared to pH 7.2 under SE. Conclusion: Taken together these results suggest that Kir4.2 participates in K+ uptake by maturation ameloblasts, and that K+ and Na+ uptake by Kir4.2 and Nckx4, respectively, may be regulated by pH through WDR72-mediated endocytosis and membrane trafficking.

Gut anatomy and ultrastructural features of the paunch epithelium in the Neotropical termite Serritermes serrifer (Blattaria, Isoptera, Serritermitidae)

Termite gut comprises a long tube divided into several compartments. The well-developed hindgut includes five distinct portions, in which microbial communities are harbored, being responsible for different physiological and digestive processes. An enlarged compartment of the hindgut, the paunch, comprises a sac–like structure whose morphological features differ among termite taxa. The Neotropical termite Serritermes serrifer is the unique inquiline lower termite, and its cryptic habit results in sampling difficulties. Thus, studies regarding this termite species are rare. Here we provide a morphological approach of the gut regions in pseudergates and soldiers of S. serrifer, highlighting the ultrastructure features of the paunch, especially what concerns its function and association with microbial communities. The general morphology of the gut resembles those reported for other “lower termites”, and comprises a short foregut with a small stomodeal valve, a midgut devoid of mixed segment, and a long–compartmentalized hindgut. Eight Malpighian tubules are attached at the mesenteric–proctodeal transition, some of them extending towards the rectum. The ultrastructure of the paunch epithelium reinforces an involvement in content uptake, especially due to the extensive apical invaginations associated with mitochondria. Bacteria are attached to the paunch cuticle by an electron–dense material or are freely distributed in the lumen. The paunch of S. serrifer shares morphological similarities to that of Rhinotermitidae, but cup–like depressions, a likely synapomorphy for the Reticulitermes + Coptotermes + Heterotermes group, were absent. It suggests that bacterial attachment in S. serrifer paunch, as well as the uptake of compounds and microbial metabolites, occur along the epithelium rather than in specific sites, reinforcing the close relationship with basal rhinotermitids, in which such structures are also absent. Moreover, such characters also resemble those reported for Termitidae species.

Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

Toxic puffers accumulate their defense substance (tetrodotoxin; TTX) through the food chain. Although the previous study suggests that 5,6,11-trideoxyTTX, a nontoxic TTX analog detected simultaneously with TTX in toxic puffers or their prey, acts as an olfactory chemoattractant for grass puffers, it is unclear whether toxic puffers are commonly attracted to 5,6,11-trideoxyTTX, and which types of olfactory sensory neurons (OSNs) detect 5,6,11-trideoxyTTX. Here, we demonstrated that green spotted puffer, a phylogenetically distant species from the grass puffer, is attracted to 5,6,11-trideoxyTTX. 5,6,11-TrideoxyTTX administration made green spotted puffers stay longer at the administered site, whereas a food odor (l-Arg) made them actively swim throughout the aquarium. Attractive responses were not observed when TTX or its vehicle was administered, nor when 5,6,11-trideoxyTTX was administered to anosmic fish. Furthermore, double immunohistochemistry with activity marker and crypt OSN marker antibodies labeled oval cells with apical invagination on the olfactory epithelium surface treated with 5,6,11-trideoxyTTX. These results suggest that 5,6,11-trideoxyTTX acts as an olfactory chemoattractant detected by crypt OSNs, and attraction to 5,6,11-trideoxyTTX odor appears to be a trait shared by toxic puffers for social communication or effective toxification.

Two unusual new genera of cavernicolous Hydrometridae (Insecta: Heteroptera) from eastern Brazil

Two new genera of cavernicolous Hydrometridae are described from limestone caves in northeastern Brazil: Spelaeometra n. gen., with type species S. gruta n. sp., from the Lapa D’Água do Zezé, Lapa do Nestor, Lapa do Saco Velho, and Lapa do Caboclo caves in Minas Gerais state; and Cephalometra n. gen., with type species C. pallida n. sp., from the Natividade cave in Tocantins state. Both new taxa are flightless, possess reduced eyes, and exhibit other reductional character states linked to life in underground ecosystems. The new taxa are interpreted to be highly aberrant members of the family Hydrometridae, based on the possession of eyes set well forward on the head capsule, the posterior pair of cephalic trichobothria arising from distinct tubercles, the presence of an apical invagination on the fourth antennal segment, and the structure of the male parameres. They are provisionally placed in the subfamily Hydrometrinae based on the apical articulation between the first and second antennal segments. On the basis of a preponderance of shared apomorphic character states these new genera appear most closely allied to Veliometra schuhi Andersen, which is also endemic to Brazil, although currently placed in a different subfamily. Color habitus photographs and line drawings of key characters are provided for both new taxa, accompanied by maps and photographs of the caves in which the species occur.

Neuromuscular structure of the larva to early ancestrula stages of the cyclostome bryozoan Crisia eburnea

AbstractFor the first time, the development of a cyclostome bryozoan has been studied with immunochemistry and confocal laser scanning microscopy, with emphasis on nerves and muscles. The larva is covered by multiciliated cells, which are latitudinally strongly elongated and show phalloidin‐stained cell junctions. We hypothesize that these cells contract at metamorphosis and squeeze the apical invagination and the adhesive sac out. Ectodermal, longitudinal muscle cells extend from the cells of the inner, conical cuticularized part of the apical invagination to the lower part of the corona, around the adhesive sac pore. These muscles are retained in the ancestrula. Scattered monociliated nerve cells are interspersed between the coronal ciliary cells. An equatorial nerve in the larva disappears at metamorphosis. The central, conical part of the cuticle becomes the terminal membrane of the ancestrula, and the underlying ectodermal and mesodermal cell layers differentiate into the polypide bud, forming a deep narrow invagination, differentiating into vestibule–atrium, mouth ring and pharynx–stomach–rectum. Tentacles develop from the ring of cells around the mouth, and a small ganglion with four nerves innervating each of the tentacles develops at the anal side of the mouth. These new findings yield further support for previous homology statements of bryozoan larvae and development.

Morphophysiological study of digestive system litter-feeding termite Cornitermes cumulans (Kollar, 1832).

Termites are the major decomposers of lignocellulosic biomass on Earth and are commonly considered as biological reactor models for lignocellulose degradation. Despite their biotechnological potential, few studies have focused on the morphophysiological aspects of the termite digestive system. We therefore analyze the morphology, ultrastructure and gut luminal pH of the digestive system in workers of the litter-feeding termite Cornitermes cumulans (Blattodea: Termitidae). Their digestive system is composed of salivary glands and an alimentary canal with a pH ranging from neutral to alkaline. The salivary glands have an acinar structure and present cells with secretory characteristics. The alimentary canal is differentiated into the foregut, midgut, mixed segment and hindgut, which comprises the ileum (p1), enteric valve (p2), paunch (p3), colon (p4) and rectum (p5) segments. The foregut has a well-developed chewing system. The midgut possesses a tubular peritrophic membrane and two cell types: digestive cells with secretory and absorptive features and several regenerative cells in mitosis, both cell types being organized into regenerative crypts. The mixed segment exhibits cells rich in glycogen granules. Hindgut p1, p4 and p5 segments have flattened cells with a few apical invaginations related to mitochondria and a thick cuticular lining. Conversely, the hindgut p3 segment contains large cuboid cells with extensive apical invaginations associated with numerous mitochondria. These new insights into the morphophysiology of the digestive system of C. cumulans reveal that it mobilizes lignocellulose components as a nutritional source by means of a highly compartmentalized organization with specialized segments and complex microenvironments.

Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes.

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

Coordination of signaling and tissue mechanics during morphogenesis of murine intestinal villi: a role for mitotic cell rounding.

Efficient digestion and absorption of nutrients by the intestine requires a very large apical surface area, a feature that is enhanced by the presence of villi, fingerlike epithelial projections that extend into the lumen. Prior to villus formation, the epithelium is a thick pseudostratified layer. In mice, villus formation begins at embryonic day (E)14.5, when clusters of mesenchymal cells form just beneath the thick epithelium. At this time, analysis of the flat lumenal surface reveals a regular pattern of short apical membrane invaginations that form in regions of the epithelium that lie in between the mesenchymal clusters. Apical invaginations begin in the proximal intestine and spread distally, deepening with time. Interestingly, mitotically rounded cells are frequently associated with these invaginations. These mitotic cells are located at the tips of the invaginating membrane (internalized within the epithelium), rather than adjacent to the apical surface. Further investigation of epithelial changes during membrane invagination reveals that epithelial cells located between mesenchymal clusters experience a circumferential compression, as epithelial cells above each cluster shorten and widen. Using a computational model, we examined whether such forces are sufficient to cause apical invaginations. Simulations and in vivo data reveal that proper apical membrane invagination involves intraepithelial compressive forces, mitotic cell rounding in the compressed regions and apico-basal contraction of the dividing cell. Together, these data establish a new model that explains how signaling events intersect with tissue forces to pattern apical membrane invaginations that define the villus boundaries.

Fine structure of the copulatory apparatus of the tapeworm Tetrabothrius erostris (Cestoda: Tetrabothriidea).

The organization and fine structure of the complex copulatory apparatus of Tetrabothrius erostris (Tetrabothriidea) is investigated by light and transmission electron microscopy. A diversity of microstructures was found on the surface of genital ducts. The apical surfaces of male gonadoducts possess tubular and blade-like microtriches that have specific structure in each section of the duct. The apical part of the tubular microtriches contains numerous constrictions in the proximal section of the sperm duct; blade-like microtriches of cirrus possess longitudinal striation in the apical part, and their basal part is reinforced with electron-dense strands. Two types of microtriches occur on the surface of cirrus, and their presence may be considered as systematic features. Prostate glands containing granules of medium electron density (up to 130 nm diameter) are localized in the cirrus sac. The genital atrium contains numerous non-ciliated receptors. Paramyosin-like fibers (up to 200 nm) were found in the muscle fibers surrounding the male atrium canal. Microtriches on the surface of the distal region of the male atrial canal are covered by a glycocalyx. Electron-dense, membrane-like structures (up to 40 nm) lie under the apical membrane of the genital atrium and vagina. These structures do not form a continuous layer; its edges turn down and sink into the apical invaginations of epithelium. Hypotheses on the possible ways of copulation in T. erostris based on the observed ultrastructure are discussed.

The venom apparatus in stenogastrine wasps: Subcellular features of the convoluted gland

In the wasp venom apparatus, the convoluted gland is the tract of the thin secretory unit, i.e. filament, contained in the muscular reservoir. Previous transmission electron microscope investigation on Stenogastrinae disclosed that the free filaments consist of distal and proximal tracts, from/to the venom reservoir, characterized by class 3 and 2 gland patterns, respectively. This study aims to extend the ultrastructural analysis to the convoluted tract, in order to provide a thorough, subcellular representation of the venom gland in these Asian wasps. Our findings showed that the convoluted gland is a continuation of the proximal tract, with secretory cells provided with a peculiar apical invagination, the extracellular cavity, collecting their products. This compartment holds a simple end-apparatus lined by large and ramified microvilli that contribute to the processing of the secretory product. A comparison between previous and present findings reveals a noticeable regionalization of the stenogastrine venom filaments and suggests that the secretory product acquires its ultimate composition in the convoluted tract.

Histological and Immunohistochemical Studies on the Furstenberg’s Rosette in Cows

Summary The purpose of this study was to investigate the light and electron microscopic structure of Furstenberg’s rosette region and to determine of uptake of macromolecules from follicle associated epithelium (FAE) of healthy cows in lactation period. In this study, 26 distal teat end taken from 10 animals in lactation period were used as material. Tissue samples were prepared for examination by light and transmission electron microscope. Indirect immunoperoxidase method was implemented to determine of cells containing IgA and IgG. Ferritin solution (25 mg/ml) was applied to distal teat end 1 h before slaughtered to show uptake of macromolecules in FAE. It was observed that Furstenberg’s rosette region consists of lymphoid and nonlymphoid areas. Solitary and aggregate lymphoid follicles were seen in lymphoid areas. It was attracted attention that upper surfaces of these follicles were covered with FAE. IgA positive cells were seen in centrum germinativum of lymphoid follicles in the lymphoid region and subepithelial areas. High endothelial venules (HEV), which is characteristic feature of lympho-epithelial tissues, were observed at connective tissue which is around of the lymphoid follicles. In thin sections, the teat end tissue have been previously applied to ferritin, ferritin particles were detected in apical invaginations and pinocytotic vesicles of membranous cells (M cells). It was determined that there is Furstenbeg’s rosette associated lymphoid tissue (FALT) in teat end and this region was generated spesific immune response against antigens after their uptaking and proccessing. Thus, we have reached conclusion that FALT may play a role mucosal immunity of teat end.

Unexpected Complexity in the Mechanisms That Target Assembly of the Spectrin Cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant beta-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP(2) interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP(2) and the spectrin PH domain codistribute with one another in cells but not with authentic wild type alphabeta-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP(2) binding activity.

An ultrastructural study of the cercarial excretory system in Bucephaloides gracilescens and Prosorhynchus squamatus.

The ultrastructure of the flame cells, capillaries, collecting tubes, excretory bladder, excretory atrium, caudal vesicle, lateral caudal ducts and excretory pores of cercariae of Bucephaloides gracilescens (Rudolphi, 1819) Hopkins, 1954 and Prosorhynchus squamatus Odhner, 1905 (Digenea: Bucephalidae) is described. Both species are essentially similar except for some details. The terminal parts of the protonephridia have all the structural features that are typical of trematodes. The collecting tubes in the cercarial body are composed of cells that are wrapped around the lumen. The main collecting tubes are joined to the excretory bladder syncytium by septate junctions. Features of P. squamatus excretory bladder epithelium indicate that it is involved in secretory activity, but this is not the case in B. gracilescens. In both species the luminal surface of the excretory bladder epithelium is increased by lamellae, and the basal plasma membrane forms invaginations. In the bladder syncytium of P. squamatus both apical lamellae and basal invaginations are more developed and mitochondria are also more numerous. The excretory atrium is lined by a syncytium with nucleated cytons located in the surrounding parenchyma. The atrium lining is not continuous with the body tegument and possesses specific secretory inclusions and a thick glycocalyx. Septate junctions connect the atrium syncytium to the excretory bladder epithelium at its anterior end and to the syncytial excretory epithelium lining the caudal vesicle and the lateral caudal ducts at its posterior. In the excretory pores the caudal duct syncytium is joined to the tegument by septate desmosomes.

Occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier (Hymenoptera, Meliponini)

The occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier, 1836 are presented. The Dufour gland is not present in workers. In virgin queens the gland cells show characteristics of low activity, which are described in the text. In physogastric queens the gland epithelium is higher and the cells more active than in virgin queens, showing numerous basal plasmic membrane invaginations impregnated by an electrondense material, increased apical invaginations and accumulation of substances that will be released to the gland lumen in the subcuticular space. Therefore, the data show that the Dufour gland is more developed in physogastric than in virgin queens, indicating a possible involvement of the Dufour gland in the reproduction of this species.

Spatial organization and isotubulin composition of microtubules in epidermal tendon cells ofArtemia franciscana

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.

Endocytosis by absorptive cells in the middle segment of the suckling rat small intestine.

In neonatal suckling rats, the absorptive cells of the duodenum, jejunum and ileum endocytose maternal milk macromolecules, including antibodies from the lumen. To determine whether the absorptive cells from the middle segment of small intestine have an apical endocytic membrane system that is the same as that of the duodenum, jejunum or ileum, we applied horseradish peroxidase by intraluminal injection, and examined the absorptive cells using light and transmission electron microscopy. In the middle segment of the small intestine, the absorptive cells had an apical endocytic membrane system including apical coated pits, apical invaginations, coated vesicles, vesicles, tubules, early endosomes, late endosomes and a large homogeneous electron-dense lysosome at the supranuclear region. This would suggest that the endocytic membrane system in the absorptive cells from the middle segment of the small intestine is specialized for quick and active intracellular digestion. The apical endocytic membrane system of the absorptive cells varied according to the segment of the small intestine, and the absorptive cells made their transition gradually from jejunal type to middle type, and then from middle type to ileal type at the suckling stage. The jejunal and ileal type of absorptive cells increased with growth, in contrast to the middle type.

Effect of temperature on apical membrane remodeling in ADH-stimulated toad urinary bladders.

Pretreatment and removal of vasopressin (ADH) in toad urinary bladder renal model tissues induces endocytosis at 25 degrees C. The objective of the current study is to determine if apical membrane remodeling, as well as transepithelial water flow, can be affected by lowering the temperature to 15 degrees C. Control toad urinary bladders in the presence of an osmotic gradient at either 25 degrees C or 15 degrees C when visualized by scanning electron microscopy (SEM) show a typical apical membrane surface with no apparent surface differences. ADH-treated tissues following 15-min stimulation at 25 degrees C or 15 degrees C revealed a propagation of apical microvilli on their surface membranes. After 15 min following removal of ADH, bladder tissues at 25 degrees C or 15 degrees C showed surface invaginations involving over 44% and 80% of granular cells, respectively. The rate of water flow in tissues at 15 degrees C remained elevated compared to tissues held at 25 degrees C. This was consistent with the observation that ADH-stimulated tissues following washout at 15 degrees C still had marked apical membrane surface involvement. However, at 30 min and 60 min postwashout, ADH-stimulated tissues at 15 degrees C recovered considerably, with a reduction in the number of shallow apical membrane invaginations involving fewer than 33% and 20% of granular cells respectively. This may indicate that the membrane undergoes continuous remodeling even at cold temperature conditions but with a different half-time. Control bladder tissues subjected to transmission electron microscopy (TEM) reveal a dense cytoplasmic profile with a scattered distribution of secretory granules, rough ER cisternae, mitochondria, and little or no vacuolation. In contrast, ADH-stimulated bladder tissues displayed a vacuolated cytoplasm, expanded rough ER cisternae, and ruffled basolateral membranes. These observations suggest that the apical membrane undergoes considerable reorganization following cessation of hormone action and that lowering the temperature reduces the rate of membrane remodeling and thus may provide a means to monitor the processes of endocytosis and the mechanisms responsible for water channel retrieval.

The ultrastructure of ionocytes from osmoregulatory integumental windows of Parastenocaris vicesima (Crustacea, Copepoda, Harpacticoida)

Species of Parastenocarididae with a few exceptions have integumental windows on the dorsal side of the cephalothorax and of four (male) or three (female) abdominal somites. By vital staining with silver nitrate and by ultrastructural observations it can be shown that these windows are sites of ion exchange. The cuticle of these windows is very thin and the epidermal cells underlying it show apical invaginations of the plasma membrane and abundant mitochondria. The cephalothoracal window could be shown by SEM to consist of two components. A central window for osmoregulation is surrounded ring-like by a second one of unknown function. It is shown for the first time that a species of Chappuisiidae (Harpacticoida), Chappuisius inopinus KIEFER 1938, also has windows as revealed by vital staining with silver nitrate. These windows are oval and lie dorsally on the thoracal somites bearing the peraeopods 3, 4, and 5 respectively.

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells. Implications for the prolonged serum half-life of proinsulin.

Recent studies have reported that elevated proinsulin levels are indicative of an increased cardiovascular risk. Renal proximal tubular cells represent a major site for the metabolism of insulin-like hormones after glomerular filtration into the tubular lumen. To determine the binding and degradation of proinsulin in comparison with insulin and insulin-like growth factor-1 (IGF-1), we have used a rabbit proximal tubular cell line (PT-1). As confirmed by electron microscopy. PT-1 cells exhibit bipolar differentiation, demonstrating apical microvilli and invaginations of the basolateral membrane. To allow selective incubation of both compartments, cells were grown on filter membranes. Performing equilibrium binding assays with 125I-labelled hormones, severalfold higher binding was found at the apical than at the basolateral cell membrane, with the capacity range IGF-1 > insulin > proinsulin. Half-maximal displacement of 125I-labelled insulin and IGF-1 was observed at 0.6 and 2 nM, respectively, while crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Half-maximal displacement of 125I-proinsulin binding was obtained at approx. 8 nM proinsulin and insulin, whereas IGF-1 was 10-fold less potent. The relative degradation of specifically bound tracer was lowest for proinsulin (apical 10%, basolateral: 13%). IGF-1 was degraded by 20% at the apical cell membrane, and up to 78% at the basolateral membrane. In contrast, almost the total amount of insulin bound was degraded at both membrane sites (apical 99%, basolateral: 83%). These results suggest separate insulin and IGF-1 receptors while proinsulin binds with high affinity to a third insulin-like receptor on the apical membrane of PT-1 cells.

Spatially dynamic intercellular adhesion junction is coupled to a microtubule-based motility system: evidence from an in vitro binding assay.

During spermatogenesis, spermatids change position in the seminiferous epithelium along an axis that is perpendicular to the seminiferous tubule wall. During this period, spermatids are attached to apical invaginations of Sertoli cells. In areas of this attachment, unique junction plaques occur in Sertoli cells. These plaques consist of regions of the plasma membrane involved with intercellular adhesion, a layer of actin filaments that are hexagonally packed, and an underlying cistern of endoplasmic reticulum (ER). It previously has been proposed that these junction plaques, and therefore the attached spermatids, are translocated, by motor proteins, along microtubule tracts in the Sertoli cell. If this is true, microtubules should bind to the junction plaque in a nucleotide dependent fashion. To verify this prediction, seminiferous epithelia of the rat were separated from tubule walls and then mechanically fragmented. These epithelial preparations were incubated, in both the presence and absence of 10 mM Mg(+)(+)ATP, with exogenous microtubules stabilized with taxol. Then unbound microtubules were separated from microtubules bound to larger epithelial components by centrifuging the samples through a step sucrose gradient. The fraction enriched for elongate spermatids was collected and processed for electron microscopy. The results indicate that the junction plaques remain attached to spermatids, the plaques are intact, and the cytoplasmic face of the ER binds microtubules in a nucleotide dependent fashion. The results are consistent with the presence of motor proteins on the ER component of the junction plaques and with the general hypothesis of microtubule-dependent spermatid translocation.