Neurosecretion of the mediodorsal cells of the central nervous system of the snail Helisoma duryi

Summary The neurosecretory mediodorsal cells that produce a putative growth hormone of the snail Helisoma duryi were studied in fast-growing virgin snails and in slow-growing reproducing snails. There are about 60 mediodorsal cells in clusters on each side of the cerebral commissure of the central nervous system, and they contain dense-cored granules which are 100–200 nm in diameter. The cells of virgin snails have dense Golgi bodies, scattered ER cisternae, and few granules, while those of reproducing snails have pale Golgi bodies, stacked ER cisternae, and numerous granules. Thus the mediodorsal cells of the virgin snails appear to be more active synthetically than those of the reproducing snails. The cells near the endocrine dorsal bodies contain many dorsal body precesses in their membrane interdigitations. There are junction-like interactions between some of the interdigitations. Gap junction-like contacts are seen between mediodorsal cells and glial cells. The axon endings of the mediodorsal cells at the neurohemal area in the labial nerve show more release profiles in virgin snails than in reproducing snails. A daily pattern of release has been observed in reproducing snails, and rates of release are higher in the evening than in the morning.     

Cell junctions and other membrane specializations in the ocellus of the wasp,Paravespula Germanica L. (Hymenoptera : Vespidae)

Five types of cell contacts and other membrane specializations were found in the ocellus of the adult wasp, Paravespula germanica L. (Hymenoptera : Vespidae), based on freeze-fracture replicas and thin sections.Septate junctions alongside small gap junctions are present between iris cells and between corneagenous cells. Gap junctions are sometimes observed between glial cell processes. Photoreceptor cells and glial cells are frequently connected by scalariform junctions. Tight junction-like structures are found on receptor-cell membranes near rhabdomeric microvilli. Desmosomes are widespread in the ocellus, connecting iris cells, corneagenous cells, receptor cells, and glial cell processes. Desmosomes are found next to septate junctions.Glial membranes connected to receptor cells have a non-junctional type of membrane specializations, consisting of intramembraneous particles arranged in a rhombic pattern. Interestingly, both particle arrays and scalariform junctions are often adjacent to each other. Furthermore, a conspicuous modification of the cell surface in freeze cleaved cells is seen between adjacent glial cells intermediating two receptor cells.     

Molecular basis for pacemaker cells in epithelia

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.     

Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells

In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, beta-GA (10 muM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca(2+) current in a dose-dependent manner without affecting the voltage dependence of this current. The IC(50) for Ca(2+) currents was 1.9 muM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca(2+)-activated K(+) currents but decreased net delayed rectifier K(+) currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca(2+) currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K(+) conductances in smooth muscle cells.     

Cadherins mediate intercellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium-permeable channels

Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calcium-permeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca(2+) transients (65 +/- 9.4 nm above base line). Similar Ca(2+) transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca(2+) responses depended on influx of extracellular Ca(2+) through mechanosensitive channels because both Ca(2+) chelation and gadolinium chloride abolished the response and MnCl(2) quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.     

Actin stress fibres and cell-cell adhesion molecules in tendons: organisation in vivo and response to mechanical loading of tendon cells in vitro.

Tendons consist of parallel longitudinal rows of cells separated by collagen fibres. The cells are in intimate contact longitudinally within rows, and laterally via sheet-like lateral cell processes between rows. At points of contact, they are linked by gap junctions. Since tendons stretch under load, such cell contacts require protection. Here we describe the organisation of the actin cytoskeleton and actin-based cell-cell interactions in vivo and examine the effect of cyclic tensile loading on tendon cells in vitro. Cells within longitudinal rows contained short longitudinally running actin stress fibres. Each fibre was aligned with similar fibres in the cells longitudinally on either side, and fibres appeared to be linked via adherens junctions. Overall, these formed long oriented rows of stress fibres running along the rows of tendon cells. In culture, junctional components n-cadherin and vinculin and the stress fibre component tropomyosin increased in strained cultures, whereas actin levels remained constant. These results suggest that: (1) cells are linked via actin-associated adherens junctions along the line of principal strain; and (2) under load, cells appear to attach themselves more strongly together, and assemble more of their cytoplasmic actin into stress fibres with tropomyosin. Taken together, this suggests that cell-cell contacts are protected during stretch, and also that the stress fibres, which are contractile, may provide an active mechanism for recovery from stretch. In addition, stress fibres are ideally oriented to monitor tensile load and thus may be important in mechanotransduction and the generation of signals passed via the gap junction network.     

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     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Possible involvement of ATP-purinoceptor signalling in the intercellular synchronization of intracellular Ca2+ oscillation in cultured cardiac myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The contraction rhythms of two isolated cardiac myocytes, each of which beats at different frequencies at first, become synchronized after the establishment of mutual contacts, suggesting that mutual entrainment occurs due to electrical and/or mechanical interactions between two myocytes. The intracellular concentration of free Ca(2+) also changes rhythmically in association with the rhythmic contraction of myocytes (Ca(2+) oscillation), and such a Ca(2+) oscillation was also synchronized among cultured cardiac myocytes. In this study, we investigated whether intercellular communication other than via gap junctions was involved in the intercellular synchronization of intracellular Ca(2+) oscillation in spontaneously beating cultured cardiac myocytes. Treatment with either blockers of gap junction channels or an un-coupler of E-C coupling did not affect the intercellular synchronization of Ca(2+) oscillation. In contrast, treatment with a blocker of P2 purinoceptors resulted in the asynchronization of Ca(2+) oscillatory rhythms among cardiac myocytes. The present study suggested that the extracellular ATP-purinoceptor system was responsible for the intercellular synchronization of Ca(2+) oscillation among cardiac myocytes.     

Structural reorganization of proteins revealed by radiolysis and mass spectrometry: G-actin solution structure is divalent cation dependent

The solution structures of isolated monomeric actins in their Mg(2+)-ATP and Ca(2+)-ATP bound forms and in complexes with gelsolin segment-1 have been probed using hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry analysis of 28 peptides containing 58 distinct reactive probe sites within actin were used to monitor conformational variations linked to divalent cation and gelsolin segment-1 binding. The solvent accessibilities of the probe sites, as measured by footprinting in solution for the Ca(2+)-G-actin and Mg(2+)-G-actin complexes with gelsolin segment-1, were consistent with available crystallographic data. This included a specific protection at the contact interface between the partners, as revealed by reduced reactivity of peptide 337-359 in the complex. Aside from the specific protection indicated previously, the oxidation rates for the reactive residues of the isolated Ca(2+)-G-actin were similar to those of the actin gelsolin segment-1 complexes; however, the reactivity of numerous residues in the isolated Mg(2+)-G-actin form was significantly reduced. Specifically, Mg(2+)-G-actin has a set of protected sites relative to Ca(2+)-G-actin that suggest a structural reorganization in subdomains 4 and 2 and a C-terminus more closely packed onto subdomain 1. These conformational variations for isolated Mg(2+)-G-actin provide a structural basis for its greater tendency to polymerize into filaments as compared to Ca(2+)-G-actin.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

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.     

Morphological changes and spatial regulation of diacylglycerol kinase-zeta, syntrophins, and Rac1 during myoblast fusion

The fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well-defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase-zeta (DGK-zeta), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK-zeta has a role in the fusion of cultured C2C12 myoblasts. We show that DGK-zeta and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK-zeta, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK-zeta codistributed with the Ca(2+)-dependent cell adhesion molecule N-cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell-cell junctions by N-cadherin-containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell-cell junctions. Reconstruction of confocal optical sections showed these form dome-like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK-zeta and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion.     

Intercellular junctions and rhombic particle arrays in the developing and adult dorsal ocelli of the honeybee

Intercellular junctions and particle arrays in the developing and mature dorsal ocelli of the honeybee Apis mellifera have been studied with conventional and freeze-fracture electron microscopy. Four types of junctions are found in the lentigenic and retinogenic part during development. These are desmosomes, septate junctions, tight junctions, and gap junctions. Gap junctions and septate junctions are found between differentiating photoreceptor cells only as long as the rhabdoms are beginning to form. Their disappearance after differentiation indicates that they could play a part in cell determination. Desmosomes connect photoreceptor cells into the early imaginai stage and then disappear. Other junctions, once they have formed, remain for the life of the animal, but can change considerably in structure, distribution and frequency. The cells of the perineurium surrounding the ocellus are connected by septate and gap junctions, which may be the basis of the blood-eye barrier. Rhombic particle arrays on the E-face of the glial membrane attached to the photoreceptor cell membrane first appear in small groups one day before emergence. In the further course of life these arrays become more extensive and apparent. Their significance may be to play some role in receptor function.     

Gap junction dynamics: reversible effects of divalent cations

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode''s solutions containing a Ca++ ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.     

The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures

The blood brain barrier (BBB) is essential for insulation of the nervous system from the surrounding environment. In Drosophila melanogaster, the BBB is maintained by septate junctions formed between subperineurial glia (SPG) and requires the Moody/G protein-coupled receptor (GPCR) signaling pathway. In this study, we describe novel specialized actin-rich structures (ARSs) that dynamically form along the lateral borders of the SPG cells. ARS formation and association with nonmuscle myosin is regulated by Moody/GPCR signaling and requires myosin activation. Consistently, an overlap between ARS localization, elevated Ca(2+) levels, and myosin light chain phosphorylation is detected. Disruption of the ARS by inhibition of the actin regulator Arp2/3 complex leads to abrogation of the BBB. Our results suggest a mechanism by which the Drosophila BBB is maintained by Moody/GPCR-dependent formation of ARSs, which is supported by myosin activation. The localization of the ARSs close to the septate junctions enables efficient sealing of membrane gaps formed during nerve cord growth.     

Low resistance junctions in crayfish. Structural changes with functional uncoupling

Electrical uncoupling of crayfish septate lateral giant axons is paralleled by structural changes in the gap junctions. The changes are characterized by a tighter aggregation of the intramembrane particles and a decrease in the overall width of the junction and the thickness of the gap. Preliminary measurements indicate also a decrease in particle diameter. The uncoupling is produced by in vitro treatment of crayfish abdominal cords either with a Ca++, Mg++-free solution containing EDTA, followed by return to normal saline (Van Harreveld's solution), or with VAn Harreveld's solution containing dinitrophenol (DNP). The uncoupling is monitored by the intracellular recording of the electrical resistance at a septum between lateral giant axons. The junctions of the same septum are examined in thin sections; those of other ganglia of the same chain used for the electrical measurements are studied by freeze-fracture. In controls, most junctions contain a more or less regular array of particles repeating at a center to center distance of approximately 200 A. The overall width of the junctions is approximately 200 A and the gap thickness is 40-50 A. Vesicles (400-700 A in diameter) are closely apposed to the junctional membranes. In uncoupled axons, most junctions contain a hexagonal array of particles repeating at a center to center distance of 150-155 A. The overall width of the junctions is approximately 180 A and the gap thickness is 20-30 A. These junctions are usually curved and are rarely associated with vesicles. Isolated, PTA-stained junctions, also believed to be uncoupled, display similar structural features. There are reasons to believe that the changes in structure and permeability are triggered by an increase in the intracellular free Ca++ concentration. Most likely, the changes in permeability are caused by conformational changes in some components of the intramembrane particles at the gap junctions.     

Dynamics of gap junctions observed in living cells with connexin43-GFP chimeric protein

To study the aggregation of cell-to-cell channels into gap junctions at individual cell-cell contacts, we transfected cells with an expression vector for a chimeric protein composed of the cell-to-cell channel protein connexin43 and a green fluorescent protein. The chimeric channel protein was visualized in the fluorescence microscope and was found to form gap junctions at the cell-cell contacts just like wild-type connexin43. Cells expressing the chimeric protein had functional cell-to-cell channels. Using timelapse videomicroscopy on live cells we observed individual gap junctions over long periods and recorded the time course of aggregation of the chimeric channel protein into gap junctions at newly formed cell-cell contacts. We found that individual small gap junctions were very dynamic, moving about or becoming assembled and disassembled in the course of minutes. Larger gap junctions were more stable than small punctate ones. In control condition, stable new gap junctions were not formed during observation times of 30 min or longer. But at elevated levels of cyclic adenosine monophosphate, the chimeric channel protein began aggregating at new junctions 5-10 minutes after cell-cell contact and continued to concentrate there for at least one hour. Also already established junctions grew in size. The fluorescent chimeric channel protein will be an excellent tool to investigate the regulation of trafficking of connexin from and to the membrane and the mechanism of connexin channel aggregation into gap junctions.