A function for plasma membrane reticular systems

The basal surface in transporting epithelia is infolded in a way that encourages the formation of standing gradients. Many insect cells have a similar infolded reticular system (RS) although they are clearly not transporting epithelia. These cells are like one another metabolically in that they sequester lipid from hemolymph lipophorins (lipid transporting proteins). Dietary lipids enter the hemolymph from the midgut RS which may be an adaptation for lipophorin loading. The plasma membrane reticular system of tissues metabolizing lipids (fat body, wax glands, oenocytes, lenticles) may be an adaptation for lipophorin reception and unloading. Cationic ferritin (pI 8.5) shows all RSs are covered by a lamina functioning as a negatively charged sieve. The basal plasma membrane leading to the RS is also negatively charged. The RS is a container with charged entrances that would be expected to affect the composition of the contents. Midgut cells release lipid particles into their RS. The particles are positively charged since in tracer studies they associate with anionic but not cationic ferritin. Lipophorins are anionic. The electrostatic binding of lipid to lipophorin would make it less anionic and more likely to leave the RS when loaded, thus carrying lipid to the hemolymph. Conversely, at the destination RS, loaded lipophorin would penetrate more easily than unloaded. A change in charge with unloading would be expected to alter the equilibrium between entering and leaving lipophorin, causing protein concentration in the RS of lipid receiving tissues as has been observed in the fat body. Reticular systems may thus be reaction vessels for interactions between carrier proteins and their load.     

The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect

The structure and development of the permanent oenocytes of Calpodes ethlius (Lepidoptera, Hesperiidae) are described. There are three sorts of oenocyte. The permanent oenocytes are arranged ventral to the last two pairs of spiracles on abdominal segments 7 and 8 in four clusters of about 45 cells each. The molt cycle oenocytes are ventral to the other spiracles and only enlarge at molting. The subdermal oenocytes differentiate from the epidermis in large numbers shortly before pupation. The permanent oenocytes are large polyploid cells characterized by a cytoplasm of densely packed smooth tubular endoplasmic reticulum, and a plasma membrane invaginated in a meshwork of tubes ending in a reticular layer about 12 micro below the surface. There are two sorts of Golgi complex, one small and of conventional form, the other composed of clouds of microvesicles. 'Dense bodies', believed to belong to the microbody class of organelles, arise directly from the STER. There is a variety of membranous and 'crystalline' inclusions. The formation of isolation membranes from the tubular endoplasmic reticulum, and the origin of isolation bodies and autophagic vacuoles are described. Some autophagy takes place at all times in the molt/intermolt cycle, but there are phases of massive autophagy before the 4th-5th molt and the 5th-pupal molt. These phases coincide with pinocytosis of blood proteins and overlap with or are followed by phases of nuclear replication, RNA synthesis (ribosomes) and ER regeneration. Nuclear blebbing occurs before pupation. The morphology of the oenocytes is most like that of vertebrate cells engaged in steroid hormone synthesis. It is pointed out that the oenocytes rather than the prothoracic glands could be the source of ecdysone and the stimulus for molting.     

Apolysis and the turnover of plasma membrane plaques during cuticle formation in an insect.

The apical plasma membranes of Calpodes epidermal cells have small fattened areas or plaques with an extra density upon their cytoplasmic face. The plaques are typically at the tips of microvilli. The are present during the deposition of fibrous cuticle and the cuticulin layer. Since the plaques are close (less than 15nm) to the sites where these kinds of cuticle first appear, they are presumed to have a role in their synthesis and/or deposition and orientation. When fifth stage larval cuticle deposition ceases prior to pupation, the plaques are lost as the area of the apical plasma membrane is reduced. The plaques pass from the surface into pinocytosis vesicles and multivesicular bodies where they are presumably digested. The loss of plaques occurs as the blood level of moulting hormone reaches a peak at the critical period after which the prothoracic glands are no longer needed for pupation. Apolysis or separation of the epidermis from the old cuticle is the stage when plaques are absent, the old ones have been lost but the new ones have yet to form. After the critical period, the epidermis prepared for pupation with a phase of elevated RNA synthesis at the end of which plaques and microvilli reform in time to secrete the new cuticulin layer and later the fibrous cuticle of the pharate pupa. There is a new generation of plaques for each moult and succeeding intermoult and each generation is involved in two kinds of cuticle deposition before involution and redifferentiation.     

The formation of the ecdysial droplets and the ecdysial membrane in an insect

Insect cuticle forms as a result of overlapping sequences of two kinds of process, those involving vesicles of the Golgi complex, and those related to transport through and/or assembly at the apical plasma membrane. The ecdysial droplets are the last layer of old cuticle to be deposited before ecdysis and form from the contents of secretory vesicles from Golgi complexes. Ecdysial droplets and secretory vesicles both stain with PTA and react with silver hexamine after oxidation with periodic acid. The vesicles discharge in localized apical areas devoid of microvilli where they accumulate as droplets measuring about 3 [ x 1 [. The. droplets span the last few lamellae of the endocuticle which becomes the ecdysial membrane. They dissolve to leave the ecdysial membrane full of holes at the time that the rest of the old cuticle is digested.     

Structural lipids in the insect cuticle and the function of the oenocytes

Stabilized lipid (cuticulin), combined with protein, serves to stiffen the cuticle before sclerotization occurs. It is always present in large amounts in exocuticle that will later be tanned to form sclerotin. It is plentiful also in the untanned mesocuticle, including the tracheal taenidia, as well as pore canals, egg shell and spermatophore sheath. Stabilized lipid present in small amounts between the laminae of the endocuticle may perhaps be concerned in reversible stiffening and plasticisation. The oenocytes appear to be the source of the precursors for cuticulin formation in both larva and reproducing adult.     

The vacuolar-type ATPase from insect plasma membrane: immunocytochemical localization in insect sensilla

Summary This comparative immunocytochemical investigation provides evidence that the electrogenic potassium pump of insect sensilla is a vacuolar-type proton ATPase energizing potassium-proton antiport, as was shown recently for the electrogenic potassium pump in the larval midgut of the sphinx moth Manduca sexta. Antennal sensilla of the saturniid moth Antheraea pernyi were probed with antibodies to the midgut vacuolar-type ATPase. The monoclonal antibodies recognized their epitopes in the native and SDS-denatured state, and bound specifically to the subunit with the relative molecular mass (M_r) of 67000 (antibody 86-3) or to the subunits of M_r 28000 and 16000 (antibody 47-5). Both antibodies labelled the apical region of the auxiliary cells, as was demonstrated by immunofluorescence microscopy. Immunogold-electron microscopy localized the binding sites of the 47-5 antibody in the highly folded apical plasma membranes of the auxiliary cells. Labelling was selective and was detected in all types of examined sensilla (S. trichodea, S. styloconica, S. coeloconica). These findings are in agreement with the current view that an electrogenic potassium pump is situated in the apical plasma membrane of the auxiliary cells and that the pump is involved in driving the receptor current. They support the hypothesis that a proton-motive force generated by a vacuolar-type ATPase provides an alternative to the classical Na⁺/K⁺-ATPase to energize animal plasma membranes.     

Evolution of the reticular formation

The reticular formation of mammals contains numerous nuclei which can be recognized by their projection patterns, cytoarchitectonics, and neuropeptide/neurotransmitter content. We have identified reticular nuclei in representatives from numerous reptilian groups and ascertained presence or absence of these reticular nuclei in an attempt to use neuronal occurrence as a tool to determine phylogenetic relationships. Recently these studies have been extended to two elasmobranchs, a galeomorph shark and a ray. In this report, we concentrate on three medullary spinal projecting reticular nuclei, reticularis gigantocellularis, reticularis magnocellularis, and reticularis paragigantocellularis. We found that all three nuclei were present in rats, lizards, and elasmobranchs, but one nucleus was absent in crocodilians, and two nuclei were absent in turtles. Thus brain organization may give us clues to phylogenetic relationships. Moreover, these three reticular nuclei exhibited remarkably similar cellular morphology in mammals, reptiles, and elasmobranchs.     

The reticular formation differentiates heterotopic stimuli

The role of the reticular formation (RF) in discriminative functions was studied in experiments on anaesthetized cats. In this association, a) the variability of responses in the RF (bipolarly recorded potential gradients - PG) to the same stimuli, and b) the responses of the RF to different (heterotopic) stimuli were recorded. The PG pattern in electrical stimulation of the same source (a nerve, skin) was constant during the time periods studied (several tens of minutes); even when the functional state of the RF was modified with strychnine, the basic characteristics of the responses usually remained intact. The pattern of PG generated by natural stimulation (tapping) was likewise reproduced with exactness at the level of statistical significance. Responses evoked from reciprocally remote receptor areas were manifestly different, at least in one of the depths investigated. The differences in stimulation of topically close areas were small. The results show that the RF, through its reactions, can differentiate heterotopic stimuli.     

Multifunctional plasma membrane redox systems

All the biological membranes contain oxidoreduction systems actively involved in their bioenergetics. Plasma membrane redox systems seem to be ubiquitous and they have been related to several important functions, including not only their role in cell bioenergetics, but also in cell defense through the generation of reactive oxygen species, in iron uptake, in the control of cell growth and proliferation and in signal transduction. In the last few years, an increasing number of mechanistic and molecular studies have deeply widened our knowledge on the function of these plasma membrane redox systems. The aim of this review is to summarize what is currently known about the components and physiological roles of these systems.     

The source of lipids and polyphenols for the insect cuticle: The role of fat body, oenocytes and oenocytoids

The newly fed fourth instar larva of Rhodnius lays down the outer epicuticle at 8-9 days, the inner epicuticle at 9 days, and it moults at 12 days. The oenocytes, which supply the lipid precursors, reach their maximum size at 7 days when lipid spheres and lipid-coated vesicles appear in their cytoplasm. The epidermal cells extend cytoplasmic strands to the contracting oenocytes and receive abundant lipid, which they transfer to the plasma membrane for construction of the outer and inner epicuticle. The oenocytes also transfer lipid to the epidermis attached to the basal lamina. This lipid is discharged through the lamina and taken up by ocnocytoids. which apply themselves to the basal lamina and liberate this copious absorbed material into the haemolymph before disintegrating. The synthesis of polyphenols for sclerotization takes place in the fat body, reaching a peak at day 10. After discharge into the haemolymph it is taken (presumably by a carrier protein) to the epidermis, where its uptake and transfer can be monitored by argentaffin staining. The tubular system of pore canals and tubular filaments is formed by invagination of the plasma membrane immediately after the inner epicuticle is complete, and is filled with lipid precursors and polyphenols. There is evidence that these metabolites are carried separately: the lipid in multiple tubular filaments; the polyphenol through the substance of the axial filament. Lipid and polyphenols are still supplied to the epidermis during days 10-12. Both are most richly supplied to the sites forming exocuticle-which illustrates the importance of lipid as well as polyphenol in cuticle hardening.     

The plasma membrane transport systems and adaptation to salinity

Salt stress represents one of the environmental challenges that drastically affect plant growth and yield. Evidence suggests that glycophytes and halophytes have a salt tolerance mechanisms working at the cellular level, and the plasma membrane (PM) is believed to be one facet of the cellular mechanisms. The responses of the PM transport proteins to salinity in contrasting species/cultivars were discussed. The review provides a comprehensive overview of the recent advances describing the crucial roles that the PM transport systems have in plant adaptation to salt. Several lines of evidence were presented to demonstrate the correlation between the PM transport proteins and adaptation of plants to high salinity. How alterations in these transport systems of the PM allow plants to cope with the salt stress was also addressed. Although inconsistencies exist in some of the information related to the responses of the PM transport proteins to salinity in different species/cultivars, their key roles in adaptation of plants to high salinity is obvious and evident, and cannot be precluded. Despite the promising results, detailed investigations at the cellular/molecular level are needed in some issues of the PM transport systems in response to salinity to further evaluate their implication in salt tolerance.     

Depolymerization of actin facilitates memory formation in an insect

In mammals, memory formation and stabilization requires polymerization of actin. Here, we show that, in the honeybee, inhibition of actin polymerization within the brain centres involved in memory formation, the mushroom bodies (MBs), enhances associative olfactory memory. Local application of inhibitors of actin polymerization (Cytochalasin D or Latrunculin A) to the MBs 1 h before induction of long-term memory increased memory retention 2 and 24 h after the onset of training. Post-training application of Cytochalasin D also enhanced retention, indicating that memory consolidation is facilitated by actin depolymerization. We conclude that certain aspects of memory mechanisms could have been established independently in mammals and insects.     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane

In the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required. Apparently no specific cofactors are needed because i), clusters form equally well in nonneuronal cells, and ii), as revealed by nanoscale subdiffraction resolution provided by STED microscopy, the number of clusters directly depends on the syntaxin 1 concentration. For syntaxin 4 clustering the N-terminal domain and the linker region are also dispensable. Moreover, clustering is specific because in both cluster types syntaxins mutually exclude one another at endogenous levels. We suggest that the SNARE motifs of syntaxin 1 and 4 mediate specific syntaxin clustering by homooligomerization, thereby spatially separating sites for different biological activities. Thus, syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions.     

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron-glia co-culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two-photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal-dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin-forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.     

Brush-border formation in the midgut of an insect,Calliphora erythrocephala meigen

Summary The embryonic development of the brush-border of anterior midgut cells of Calliphora was studied by electron microscopy. Dense surface-forming vesicles, as described by Bonneville (1970), are found prior to microvillus formation. These dense vesicles provide membranous and coating material for the moulding of the microvilli. The number of dense vesicles increases rapidly to a maximum just before brush-border formation, after which it decreases very rapidly, accompanied by an increase in the number of microvilli. Formation of microvilli proceeds in essentially the same way as in Xenopus. First, some of the vesicles fuse with the apical cell membrane, resulting in an increase of the cell surface, part of which is coated with filamentous material deriving from the dense vesicles. This in turn leads to bulging, and short irregular microvilli appear. These are erected and elongated.Prefabricated tubular elements are believed to play a part in this erection and elongation, probably due to the unwinding of spirally coiled strands.Microvillus formation proper lasts 2 to 3 hours in Calliphora. Almost the entire amount of membranous and coating material is prefabricated prior to the formation of microvilli.