Microtubule systems associated with the septate junctions of the gill cells of four gammarid amphipods

The microtubular systems associated with the septate junctions of the gill epithelial cells of four species of gammarid amphipod are described. The four species examined included two relatively stenohaline marine forms, Chaetogammarus marinus and Gammarus locusta; a highly euryhaline species, Gammarus duebeni, and a stenohaline freshwater species, Gammarus pulex. Of these amphipods, G. locusta and C. marinus maintain only a limited osmotic gradient between their haemolymph and the medium and have a poorly developed junctional microtubular system; G. pulex has haemolymph which is some 300 mOsmol hypertonic to freshwater and has a well ordered system of microtubules on both sides of fairly long septate junctions; G. duebeni from brackish water tend to have a somewhat shorter length of septate junctions lined by one or occasionally by a double row of microtubules. The most complex junctional microtubular systems are shown by specimens of the freshwater race of G. duebeni celticus which have been acclimated to seawater. These can take the form of multiple arrays in which some microtubules are linked to the plasma membrane by dense strands. It is suggested that these findings are consistent with the hypothesis that one role of these microtubules is to provide mechanical stability to enable the integrity of the septate junctions to be maintained during osmotic stress.     

Intercellular junctions in the hypodermis,salivary gland and gené's organ of the cattle tick,Boophilus microplus

Summary The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.The authors would like to thank Mr. R. Lamb for preparing the plates. M.W.J. Megaw was supported by an S.R.C. Studentship     

The role of cytoskeletal components in the maintenance of intercellular junctions in an insect

Summary Accessory glands of the cockroach are composed of secretory and supportive cells, the latter providing a skeleton-like framework of attentuated cytoplasmic processes into which the former are positioned. These two cell types are associated with one another laterally by adhaering, pleated septate, and gap junctions. Hemi-adhaerens junctions are also found on both luminal and basal surfaces of the gland; the former are associated with the cuticular lining of the lumen and the latter with extracellular matrix. The adhering and septate junctions are flanked by both filaments and microtubules; the former insert into the junctional membranes and are actin-like, binding both rhodamine-conjugated phalloidin and the S₁ subfragment of rabbit heavy meromyosin. The role of this cytoskeletal protein with the cellular junctions has been explored by treatment with a disruptive agent, cytochalasin D. Dissociation of actin leads to changes in septate junctions and in microtubular distribution. This suggests that the latter act as anchors for the actin filaments which, in turn, appear bound to certain of the intramembranous junctional components.Supported by a Conicet/Royal Society Visiting Fellowship     

Septate junctions in imaginal disks of Drosophila: a model for the redistribution of septa during cell rearrangement

The organization of septate junctions during morphogenesis of imaginal disks is described from freeze-fracture replicas and thin sections with a view to understanding junction modulation during rearrangements of cells in epithelia. The septate junctions of each epithelial cell of the disk are distributed in a number of discrete domains equal to the number of neighboring cells. Individual septa traverse domains of contact between pairs of adjacent cells, turn downwards at the lateral boundary of the domain and run parallel to the intersection with a third cell. This arrangement leaves small channels at three-cell intersections that are occupied by specialized structures termed "tricellular plugs." Cell rearrangement involves a progressive change in the width of contact domains between adjacent cells, until old contacts are broken and new ones established. It is proposed that the septate junction adjusts to the changing width of domains by the compaction or extension of existing septa. This redistribution of septa theoretically allows a transepithelial barrier to be maintained during cell rearrangements. The applicability of this model to other epithelial tissues is discussed.     

Accumulation of a microtubule-binding protein, pp170, at desmosomal plaques

The establishment of epithelial cell polarity correlates with the formation of specialized cell-cell junctions and striking changes in the organization of microtubules. A significant fraction of the microtubules in MDCK cells become stabilized, noncentrosomally organized, and arranged in longitudinal bundles in the apical-basal axis. This correlation suggests a functional link between cell-cell junction formation and control of microtubule organization. We have followed the distribution of pp170, a recently described microtubule-binding protein, during establishment of epithelial cell polarity. This protein shows the typical patchy distribution along microtubules in subconfluent fibroblasts and epithelial cells, often associated with the peripheral ends of a subpopulation of microtubules. In contrast to its localization in confluent fibroblasts (A72) and HeLa cells, however, pp170 accumulates in patches delineating the regions of cell-cell contacts in confluent polarizing epithelial cells (MDCK and Caco-2). Double immunolocalization with antibodies specific for cell-cell junction proteins, confocal microscopy, and immunoelectron microscopy on polarized MDCK cells suggest that pp170 accumulates at desmosomal plaques. Furthermore, microtubules and desmosomes are found in close contact. Maintenance of the desmosomal association of pp170 is dependent on intact microtubules in 3-d-old, but not in 1-d-old MDCK cell cultures. This suggests a regulated interaction between microtubules and desmosomes and a role for pp170 in the control of changes in the properties of microtubules induced by epithelial cell-cell junction formation.     

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

A junctional problem of apical proportions: epithelial tube-size control by septate junctions in the Drosophila tracheal system

The size of epithelial tubes is critical for the function of organs such as the lung, kidney and vascular system. However, the molecular mechanisms regulating tube size are largely unknown. Recent work in the Drosophila tracheal system reveals that septate junctions play a previously unsuspected role in tube-size control. Surprisingly, this tube-size function is distinct from the established diffusion barrier function of septate junctions, and involves regulation of cell shape rather than cell number. Possible tube-size functions of septate junctions include patterning of the apical extracellular matrix and regulation of conserved cell polarity genes such as Scribble and Discs Large.     

Effect of salinity on osmoregulatory patch epithelia in gills of the blue crab Callinectes sapidus

In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na+, K+-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.     

Septate junctions mediate the barrier to paracellular permeability in sea urchin embryos

In sea urchin embryos, blastula formation occurs between the seventh and tenth cleavage and is associated with changes in the permeability properties of the epithelium although the structures responsible for mediating these changes are not known. Tight junctions regulate the barrier to paracellular permeability in chordate epithelia; however, the sea urchin blastula epithelium lacks tight junctions and instead possesses septate junctions. Septate junctions are unique to non-chordate invertebrate cell layers and have a characteristic ladder-like appearance whereby adjacent cells are connected by septa. To determine the function of septate junctions in sea urchin embryos, the permeability characteristics of the embryonic sea urchin epithelia were assessed. First, the developmental stage at which a barrier to paracellular permeability arises was examined and found to be in place after the eighth cleavage division. The mature blastula epithelium is impermeable to macromolecules; however, brief depletion of divalent cations renders the epithelium permeable. The ability of the blastula epithelium to recover from depletion of divalent cations and re-establish a barrier to paracellular permeability using fluorescently labelled lectins was also examined. Finally, septate junction structure was examined in embryos in which the permeability status of the epithelium was known. The results provide evidence that septate junctions mediate the barrier to paracellular permeability in sea urchin embryos.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada     

日本沼虾生精细胞与支持细胞之间的连接关系

用透射电镜技术研究了日本沼虾精子发生过程中不同细胞之间的连接关系。结果表明,从精原细胞期到次级精母细胞期,在生精细胞之间存在间隙连接与分隔连接与分隔连接,并且两种连接相互邻接,桥粒仅在精原细胞之间发现;从精原细胞期到精细胞期,在生精细胞与支持细胞之间也存在相互邻接的间隙连接与分隔连接,两类细胞之间有大量桥粒,形成血淋巴－精巢屏障,这种屏障可保持生精细管内环境的稳定性;精子发生的不同时期,支持细胞之     

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions.     

Changes in the distribution of filament-containing septate junctions as coelenterate myoepithelial cells change shape

This paper describes the redistribution of septate junctions during an increase in diameter of myoepithelial cells from mesenteries of the sea anemone Metridium senile (L). Each septum was composed of a filament core, 9.5-10.2 nm in diameter, which had a double row of lateral projections from each side to the adjacent cell membrane. Septa were arranged in patches in which neighbouring septa lay parallel, 28-33 nm apart. When anaesthetized mesenteries were stretched, myoepithelial cell layers decreased from a mean of 32 to 8 micron thick; each cell shortened and its apical diameter increased. The integrity of the septate junctions was, however, maintained. The mean perimeter of septate junctions, corresponding to that of the cells, increased from 20 to 31 micron; mean depth decreased from 3.7 to 2.1 micron. There was no significant change in spacing between septa. Patches of septa, free to move in a fluid matrix of junction cell membranes, may form mobile attachment sites between cells, thus allowing those cells to change shape. Number and distribution density of microvilli decreased when cell diameter increased. This implies that the microvilli contribute membrane to the cell surface as its surface area increases. Gastrodermal cells are compared with epidermal cells that do not undergo dramatic changes in diameter.     

Cell Junctions in Arthropod Ion-transport Systems

The epithelial cells involved in the movement of ions and waterform a major subset of all epithelial cell types. Both the formand the functions of cell junctions present in these cells areessentially the same as those found elsewhere. Gap junctionsare believed to regulate intercellular communication; desmosomesand hemidesmosomes provide mechanical anchorage to other cellsand the extracellular matrix; septate junctions play roles inproviding cell to cell anchorage, and perhaps in sealing thelateral surfaces of adjacent cells together to prevent paracellularfluid and solute movement; tight junctions (of limited distributionin insects) are seals between adjacent cells. They form a barrierto the paracellular movement of solutes and water. Examination of the junctions in salivary glands and midgut provideinsight into the roles of these junctions in the developmentand function of ion transport systems. In Manduca sexta (Johannsen)the cells of the salivary gland are joined by pleated septateand gap junctions. Individual salivary cells have numerous foldsand canaliculi. The walls of the canaliculi consist of extensivelyfolded plasma membrane in intimate association with mitochondria.Gap junctions connect adjacent parts of the same cell acrossmembrane folds, effectively shortening diffusion distances inthe cells. Hemidesmosomes are present in the walls of developingcanaliculi. They are attached to pore filaments that occupythe lumen of the developing canaliculi. The hemidesmosomes andpore filaments may have a morphogenetic role as they disappearafter the canaliculi are formed. In Manduca sexta the midgut cells are joined by gap and septatejunctions. These junctions differ in morphology from their counterpartsin the salivary gland; physiological studies show the gobletcells are not coupled to neighboring tall columnar cells. Wehave shown the gap junctions joining them are typical of non-couplingjunctions. Preliminary studies suggest that the gap junctionschange form when the cells are coupled.     

Fine structure of the rectal pads in the desert locust Schistocerca gregaria with reference to the mechanism of water uptake

The rectal pads of Schistocerca gregaria are composed of three different cell types: epithelial, secondary and junctional cells. The rectal pads are interconnected by simple rectal cells and both are lined internally by a articular intima. The epithelial cells exhibit extensive infoldings of the apical plasma membranes that are closely associated with mitochondria. Their lateral plasma membranes are highly folded around large mitochondria and enclose intercellular channels and spaces. They are united by belt and spot desmosomes, septate junctions, gap junctions and scalariform junctions, but terminate in a basal syncytium without contacting the basal plasma membranes. The apical and basal cytoplasm contain coated vesicles, dense tubular elements, multivesicular bodies and lysosomes, suggesting receptor-mediated endocytosis of small peptide molecules into the epithelial cells. The apical membrane infoldings of the secondary cells are also associated with large mitochondria. Their basal plasma membranes are covered by connective cell processes and connected with them by spot desmosomes which may be involved in solute recycling. The presence of neurosecretory-like axons near the secondary cells suggests that they exert local control on the function of these cells. The ultrastructural details are examined in relation to their role in solute and water transport.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

Cell junctions in amphioxus (Cephalochordata): a thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent 'poorly developed' septate junctions.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system

Although the correct architecture of epithelial tubes is crucial for the function of organs such as the lung, kidney and vascular system, little is known about the molecular mechanisms that control tube size. We show that mutations in the ATPalpha alpha and nrv2 beta subunits of the Na+/K+ ATPase cause Drosophila tracheal tubes to have increased lengths and expanded diameters. ATPalpha and nrv2 mutations also disrupt stable formation of septate junctions, structures with some functional and molecular similarities to vertebrate tight junctions. The Nrv2 beta subunit isoforms have unique tube size and junctional functions because Nrv2, but not other Drosophila Na+/K+ ATPase beta subunits, can rescue nrv2 mutant phenotypes. Mutations in known septate junctions genes cause the same tracheal tube-size defects as ATPalpha and nrv2 mutations, indicating that septate junctions have a previously unidentified role in epithelial tube-size control. Double mutant analyses suggest that tube-size control by septate junctions is mediated by at least two discernable pathways, although the paracellular diffusion barrier function does not appear to involved because tube-size control and diffusion barrier function are genetically separable. Together, our results demonstrate that specific isoforms of the Na+/K+ ATPase play a crucial role in septate junction function and that septate junctions have multiple distinct functions that regulate paracellular transport and epithelial tube size.