Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

A freeze fracture and thin section study of intestinal cell membranes and intercellular junctions of a nematode, Ascaris

The microvillar and lumenal plasma membrane P-face of Ascaris intestinal cells is shown to be covered by relatively large (13 nm) particles at a fairly high density (1000/μm²), while the E-face has virtually none. The P-face of the lateral cell membranes, those separating the cells, have fewer and smaller (8 nm) particles. The intestinal cells are also shown to be connected by an apical complex of smooth septate and tricellular junctions similar to those found between some insect midgut cells. A periodic layer of tannic acid staining material is found on the cytoplasmic sides of the smooth septate junction, and when the intercellular space is filled with lanthanum, smoothly curved, 10 nm wide septal walls can be seen. Below the belt of septate junctions are a large number of gap junctions. These have closely packed arrays of particles on the P-face with some particle aggregates adhering to the closely packed pit arrays on the E-face.     

X-ray microanalysis with transmission electron microscopy determined presence and movement of tracer (lanthanum chloride) at blood-neuron barrier of Drosophila melanogaster (Diptera : Drosophilidae) larva

In studying the larval Drosophila (Diptera : Drosophilidae) blood-brain barrier, it was important to determine if even minute amounts of tracer ultimately seeped through the septate junctions between perineurial cells to reach the neuronal region. Concurrent TEM with X-ray microanalysis was undertaken to resolve that issue. Ultrathin sections of Drosophila nervous tissue in LR White embedment were exposed to ionic tracer (lanthanum chloride) and assayed for presence or absence of lanthanum extracellular to the perineurium and glia making up the nerve sheath. Tracer filled the distal interseptal lattice of pleated sheet-septate junctions, but was contained prior to reaching the proximal paracellular space. No detectable tracer passed through septate junctions to enter the glial-neuronal domain. Based on our present data and the research of others, septate junctions in immature Drosophila are multifunctional structures that enforce spatial relationships between cells, seal intercellular spaces, and control cell proliferation in the epithelia. Septate junctions in Drosophila with the (dlg) gene also exhibit protein homologies to the Z0–1 human tight junction component.     

Diffusion barriers in silkmoth sensory epithelia: application of lanthanum tracer to olfactory sensilla of Antheraea polyphemus and Bombyx mori

The distribution of diffusion barriers in silkmoth olfactory sensilla has been investigated with ionic lanthanum. The tracer was applied from the apical side of the sensory epithelium by first pinching off the hair tips and then dipping the antennal branches into the La(NO(3))(3) solution. The tracer neither passed the apical septate junctions between the dendrite and the thecogen cell nor those between thecogen, trichogen, and tormogen cells, nor the tight contact between the apical membrane of the tormogen cell and the cuticle. After perfusing the hemolymph space with La(NO(3))(3) solution, the tracer was found in the clefts between the thecogen, trichogen, tormogen, and epidermis cells, but not in those between the receptor cells and the thecogen cell, or between the axon and the glial envelope. Lanthanum neither entered the receptor-lymph space nor the subcuticular space. Therefore, (i) receptor-lymph space, subcuticular space, and hemolymph space are isolated from each other, and (ii) the cleft between thecogen and sensory cell is separated from the hemolymph as well as from the receptor-lymph spaces. Furthermore, the results indicate that pleated septate junctions form the diffusion barriers in silkmoth olfactory sensilla.     

Pheromone receptors in Bombyx mori and Antheraea pernyi

Summary The cellular organization of freeze-substituted antennal sensilla trichodea, which contain the sex pheromone receptors, was studied in male silkmoths of two species (Bombyx mori, Bombycidae; Antheraea pernyi, Saturniidae). The cellular architecture of these sensilla is complex, but very similar in both species. A three-dimensional reconstruction of a sensillum trichodeum of B. mori is presented. Two receptor cells (in A. pernyi 1–3) and three auxiliary cells are present. Of the latter, only the thecogen cell forms a true sheath around the receptor cells. A unique thecogen-receptor cell junction extends over the entire area of contact. Septate junctions occur between all sensillar cells apically, and in the region of the axonal origin basally. Gap junctions are also found between all cells except the receptor cells. The trichogen and tormogen cells show many structural indications of secretory activity and are thought to secrete the receptor lymph. Their apical membrane bordering the receptor-lymph space is enlarged by microvilli and microlamellae, but only those of the trichogen cell show regularly arranged membrane particles (portasomes), indicating secretory specialization among the auxiliary cells. Epidermal cells are found as slender pillars between sensilla, but extend apically along the non-sensillar cuticle and basally along the basal lamina.     

The organization and isolating function of insect rectal sheath cells: a freeze-fracture study.

In termites and roaches the well-defined rectal papillae each comprise a layer of columnar principal cells specialized for active transport and a layer of basal cells. The whole cell group is entirely surrounded by several series of flattened 'sheath cells' (formerly termed 'junctional cells') which abut onto the basal components of the papilla. The sheath cells secrete a specialized sclerified cuticle which forms the framework of the papilla. Their regularly pleated apical membrane is closely apposed to the cuticle and contains parallel and closely spaced rows of intramembranous particles. at this level, no subcuticular space is present and hence the space associated with the apical surface of the principal cells is defined as an isolated compartment. Typical septate junctions are present between the sheath and basal cells; however those linking adjacent sheath cells are structurally unusual: they extend to the basal surface rather than being restricted to the apical zone, are frequently interrupted and in replicas are represented by relatively short and irregularly oriented particle rows. Moreover, lateral sheath cell contacts display two further peculiarities: absence of an apical desmosomal ring and paucity of gap junctions. Structural observations suggest that the sheath cells isolate the principal cells from communication with the hemolymph, consequently enhancing their efficiency in water and ionic regulation. Comparable cells have been described in a number of insects, but the 'isolation' system presents varying degrees of complexity, for which an evolutionary scheme is proposed.     

The structure of the chambered nautilus siphuncle: The siphuncular epithelium

The siphuncle of the chambered nautilus (Nautilus macromphalus) is composed of a layer of columnar epithelial cells resting on a vascularized connective tissue base. The siphuncular epithelium taken from chambers that have not yet begun to be emptied of cameral liquid has a dense apical brush border. The great number of apical cell junctions (zonula adherens) compared to the number of nuclei suggests extensive interdigitation of these cells. The perinuclear cytoplasm of these preemptying cells is rich in rough endoplasmic reticulum. The siphuncular epithelium of both emptying and “old” siphuncle (which has already completed emptying its chamber) both show little rough endoplasmic reticulum but do contain extensive systems of mitochondria-lined infoldings of the basolateral plasma membranes. Active transport of NaCl into the extracellular space of this tubular system probably entrains the water transport involved in the chamber-emptying process. Both emptying and old siphuncular epithelium also show large basal infoldings (canaliculi) continuous with the hemocoel, which appear to be filled with hemocyanin. The apical cell junctions of emptying and old siphuncular epithelium contain septate desmosomes that may help to prevent back-flow of cameral liquid into the chambers.     

A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.     

The formation of intercellular junctions in insect stem cell progeny (cockroach intestinal epithelium)

Summary The stages in the development of intercellular junctions have been followed in the mesenteric caecal cells of the cockroach midgut, where two types of mature cell, the columnar and the secretory, exist. Nests of undifferentiated replacement cells occur at intervals along the basal lamina, consisting of central, dividing cells and peripheral semi-lunar cells; the former act as proliferative stem cells to give rise to either pre-columnar or pre-secretory cells. The semi-lunar cells are pre-columnar and produce an attenuated process which gradually projects up to the luminal surface, producing microvilli and a dense extracellular substance en route. Intercellular gap junctions appear between these maturing columnar cell borders first, while septate junctions differentiate later; these are assembled from two different sets of intramembranous particle which become organized into either plaques or rows in parallel alignment, possibly mediated by actin filaments and microtubules. The pre-secretory cells, which are much fewer in number, remain associated only with the basal lamina and never reach the lumen; they develop into one of three distinct mature secretory cell types which release their secretory product in different ways. Offprint requests to: N.J. Lane     

The fine structure of the nephronic tubule of the mudskipper Periophthalmus koelreuteri (Pallas)

Ultrastructural examination of the head kidney of Periophthalmus koelreuteri (Pallas) (Teleostei, Gobiidae) revealed that the nephronic tubule cells are bound by tight junctions and desmosomes with little intercellular space. The first proximal segment (PI) consists of low columnar cells with well developed brush borders, indented nuclei, and numerous apical endocytic vesicles and lysosomes. A second cell type possessing clusters of apical cilia and lacking brush border and lysosomes is occasionally found between PI cells. The second proximal segment (PII) is formed of high columnar cells with brush border, regular spherical nuclei and numerous mitochondria located between well developed infoldings of the basal membrane. Single ciliary structures protrude into the lumen from PI and PII cells. The distal segment is lined by low columnar epithelium with few microvilli, regular spherical nuclei, numerous scattered mitochondria, and microbodies. The collecting tubule cells are cuboidal with few euchromatic nuclei, some mitochondria, and secondary lysosomes.     

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.     

The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila

Background

Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes.

Methodology/Principal Findings

In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component.

Conclusion/Significance

We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.     

The fine structural organization of sternal glands of pseudergates and workers in termites (Isoptera): a comparative survey

Thirty-nine species belonging to different families of termites are studied to give a comprehensive view of the evolution of the sternal glands. Several modifications occurring at cuticular and cytological levels are described in neuter castes. The outer epicuticle is always pierced by epicuticular pores. In advanced termites the epicuticular filaments greatly increase in number and length creating a thick layer. The pore canals gradually enlarge while the cuticle changes into a lattice structure lining an extracellular space in which the secretion is stored. Two classes of cells are present in basal termites (Mastotermitidae, Hodotermitidae, Termopsidae and Kalotermitidae) but their glandular structures greatly differ between families. A more complex organization with three classes of cells is found in the Serritermitidae and Rhinotermitidae. A regressive evolution occurs in the Termitidae where only two classes of cells are present. A dual nervous control (campaniform sensilla and neurosecretory fibers) is found in lower termites, except for the Hodotermitidae which have mechanosensory bristles. In the other families, neurosecretory fibers are lacking. A comparison with phylogenetic data is given. A more versatile role of sternal glands in neuter castes is hypothesized.     

THE STRUCTURAL ORGANIZATION OF THE SEPTATE AND GAP JUNCTIONS OF HYDRA

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50–60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80–100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15–0.5 µ in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Studies on perineural junctional complexes and the sites of uptake of microperoxidase and lanthanum in the cockroach central nervous system

The perineurial junctional complexes in the nerve cord of Periplaneta americana have been shown to consist of septate desmosomes, extensive gap junctions and relatively limited regions of tight junctions. Microperoxidase (M.W. 1,900) undergoes limited intercellular penetration into the septate desmosomes. Lanthanum penetrates both the septate desmosomes and gap junctions. It is concluded that the restricted access of these substances to the underlying extracellular spaces results from the presence of the perineurial tight junctions. These results contrast with those for small peripheral nerves, which lack equivalent junctional complexes, and in which the extracellular spaces are found to be accessible to externally applied lanthanum. The results are discussed in relation to current concepts of the insect blood-brain barrier.     

Neural projection patterns from homeotic tissue of Drosophila studied in bithorax mutants and mosaics.

The central projection patterns of sensory cells from the wing and haltere of Drosophila, as revealed by filling their axons with cobalt, consist of dorsal components arising from small campaniform sensilla and ventral components arising from large campaniform sensilla and from bristles. All of the bristles of the wing are innervated, some singly and some multiply. All three classes of sensilla are strongly represented on the wing, but the haltere carries primarily small campaniform sensilla and has a correspondingly minute ventral projection. In bithorax mutants in which the haltere is transformed into wing, ventral components are added to the projection pattern, while the dorsal components appear as if haltere tissue were still present. Thus, the three classes of receptors not only produce different projection patterns when they develop in their native mesothoracic segment, but also behave differently in the homeotic situation. Consequently, different developmental programs are inferred for each class. When somatic recombination clones of bithorax tissue are generated in phenotypically wild-type flies, they also produce ventral projections. However, these projections of mutant fibers into wild-type ganglia differ in certain details from the projections of mutant fibers into mutant ganglia. Thus, homeotic changes are inferred to occur in the CNS of mutant flies, but these are not required for the execution of those developmental instructions carried in the genome of large campaniform and bristle sensory cells which specify that their axons should grow ventrad in the CNS.     

Sexual dimorphism of antennal and ovipositor sensilla of Tetrastichus sp. (Hymenoptera: Eulophidae)

Tetrastichus sp. (Hymenoptera: Eulophidae) is a primary parasitoid of the Metisa plana (Lepidoptera: Psychidae), an oil palm bagworm. The sensilla on the surface of the antenna and ovipositor of Tetrastichus sp. were examined using a scanning electron microscope. The antennae of both male and female Tetrastichus sp. are geniculate in shape and hinged at the scape-pedicel joint. The female antenna is about 200 µm longer than the male antenna. However, the male antenna has an additional flagellomere compared to the female antenna. In total, eight different types of antennal sensilla were observed on the antenna of Tetrastichus sp.: trichoid sensilla type 1, 2, 3, 4, placoid sensilla type 1 and 2, basiconic sensilla, and campaniform sensilla. The antenna of the female Tetrastichus sp. lacks placoid sensilla type 2 and campaniform sensilla. The distribution and abundance of the antennal sensilla were compared between the male and female Tetrastichus sp. and discussed. On the ovipositor stylet of Tetrastichus sp., coeloconic sensilla, styloconic sensilla and campaniform sensilla were observed. Trichoid sensilla were observed at the medial part of the distal extremity of the ovipositor.     

Junctional types in the tissues of an onychophoran: the apparent lack of gap and tight junctions in Peripatus

The Onychophora are a rare group of primitive invertebrates, relatively little investigated. Tissues from a range of their digestive, secretory and excretory organs have been examined to establish the features of their intercellular junctions. Glutaraldehyde-fixed cells from the midgut and rectum, as well as the renal organ, mucous gland, salivary gland, epidermis, CNS and testis from specimens of Peripatus acacioi, have been studied by thin section and freeze-fracture electron microscopy. Adjacent cells in the epithelia of all these tissues are joined by apical zonulae adhaerentes, associated with a thick band of cytoskeletal fibrils. These are followed by regular intercellular junctional clefts, which, in thin sections, have the dense, relatively unstriated, appearance of smooth septate junctions (SSJ). However, freeze-fracture reveals that only the midgut has what appear to be characteristic SSJs with parallel alignments of closely-packed rows of intramembranous particles (IMPs); these IMPs are much lower in profile than is common in such junctions elsewhere. The mucous gland, testis, rectal and renal tissues exhibit, after freeze-fracture, the characteristic features of pleated septate junctions (PSJ) with undulating rows of aligned but separated junctional particles. Suggestions of tricellular septate junctions are found in replicas at the interfaces between 3 cells. In addition, renal tissues exhibit scalariform junctions in the basal regions of their cells. Between these basal scalariform and apical septate junctions, other junctions with reduced intercellular clefts are observed in these renal tissues as well as the rectum, but these appear not to be gap junctions. Such have not been unequivocally observed in any of the tissues studied from this primitive organism; the same is true of tight junctions.