Regions Of Apposition Research Articles

The Structural Context of Disease-causing Mutations in Gap Junctions

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.

Synaptic relationships between serotonin-immunoreactive axons and dorsal horn spinocerebellar tract cells in the cat spinal cord.

Dorsal horn spinocerebellar tract cells were identified according to electrophysiological criteria in adult cats and labelled intracellularly with horseradish peroxidase. Sections containing labelled neurons were processed to reveal serotonin immunoreactivity and examined with light and electron microscopy. Numerous contacts were observed on cell bodies, and on proximal and intermediate parts of dendrites. Electron microscopic examination of contacts revealed that synaptic junctions were usually present at the region of apposition. It is concluded that serotonin has a postsynaptic action on dorsal horn spinocerebellar tract cells and that this action is mediated through conventional synapses.

Dendritic reduction in Passover, a Drosophila mutant with a defective giant fiber neuronal pathway.

The jump response to a light-off startle stimulus in Drosophila melanogaster occurs when the Giant Fiber (GF), a neuron descending from the brain to the thorax, drives the jump (tergotrochanteral) muscle motorneuron (TTMn). Nonjumping mutants have been isolated in which this response is disrupted. Flies bearing the X-chromosome mutation Passover (Pas) fail to jump in response to a light-off stimulus, and electrical stimulation of the GF in the brain no longer elicits the normal response in the TTM. We have used retrograde HRP labelling to examine the TTMn motorneuron in wild-type flies and in a variety of newly identified Pas alleles. In wild type the medial branch (MB) of the TTMn has an extensive region of apposition with the GF. In Pas alleles, there is a general reduction in anterior-posterior (A-P) extent of the medial branch but not of the posterior branch. Nevertheless, Pas alleles usually leave the TTMn close enough to the GF so that contact would not be precluded. In flies carrying a particular deficiency of Pas, Df(1) 16-3-22, including Pas/Df(1) 16-3-22 heterozygotes, there can be extensive growth of the medial-branch including a contralateral projection; these heterozygotes have more than the normal amount of overlap between the GF and the TTMn. This phenotype, originally ascribed to Pas mutants, is associated with Df(1) 16-3-22, but not with other deletions of the Pas gene. The driving of the TTMn by the GF is defective in mutant genotypes with extensive medial branches as well as in mutants where GF-TTMn contact is reduced. The fact that the TTMn grows into its normal synaptic region in mutant genotypes, but the GF pathway functions abnormally suggests that pathfinding by the TTMn is not impaired. It is more likely that the Pas mutation disrupts cell recognition, synaptogenesis, or synaptic function in the TTMn or its presynaptic partners.

Reversible Epithelial Adhesion Closes the Mouth of Beroe, a Carnivorous Marine Jelly.

We investigated how the ctenophore Beroe, a carnivore of the marine zooplankton, keeps its mouth shut to maintain a streamlined body shape during forward swimming in search of prey. In big-mouthed, thin-walled beroids we found that mouth closure requires neither muscular nor nervous activity. Instead, mouth adhesion is due to paired strips of adhesive epithelial cells on opposing stomodaeal walls. The two joined epithelial layers make numerous close appositions interrupted by vacuolar intercellular spaces. At regions of apposition, the plasma membranes are highly folded and interdigitated with each other, and are separated by a uniform distance of about 15 nm. A dense cytoplasmic coat underlies the membranes at such appositions. Synapses of neurites are found on the basal ends of the adhesive cells. We found two orthogonally different orientations of the stomodaeal adhesive strips in B. sp. vs. B. forskali, correlated with different distributions of feeding macrocilia inside the stomodaeum. Mouth opening in response to food requires muscular contractions of the lips. However, the stomodaeal adhesive strips are not pulled apart all at once, but are peeled apart starting from a site of vigorous muscular tension. The mouth re-seals after feeding, or after being experimentally pulled open, showing that tissue adhesion is functionally reversible. Epithelial adhesion in Beroe appears to be a useful method for closing the mouth and streamlining the body of a gelatinous predator that spends most of its time swimming mouth-forward in search of prey. Opening of the mouth appears to be an efficient process as well, because peeling apart of the adhesive strips requires a smaller applied force than does separating them all at once. Tissue adhesion in Beroe shares many structural and functional properties with transient adhesions formed between moving cells in embryos and in culture, and may be a useful experimental system for studying the mechanisms and regulation of dynamic cell adhesions. "Loose lips Sink ships." --U. S. Navy slogan, WW II.

The ultrastructure of cones in the walleye retina

The ultrastructure of single and twin cone photoreceptors in the retina of the walleye was analyzed by scanning and transmission electronmicroscopy. The outer segment disks resemble those of other vertebrates. An accessory outer segment arises from the inner segment, makes frequent contacts with the outer segment proper, and may thus provide a bridge for signal transmission from outer to inner segment. A palisade of some 30 calycal processes surrounds and may provide structural support for the outer segment. The region of apposition between the inner segments of twin cones consists of a space of some 28 nm with no indication of gap junctions. The proximal quarter of the inner segment displays a profusion of some 50 lateral fins which increase the inner segment surface by 3–4 x and do not contact fins of neighboring cones. The fins surround a profusion of Müller cell microvilli and probably promote metabolic exchange between cones and Müller cells. Apart from differences in size and the presence of apposed inner segments, twin and single cones appear to be morphologically similar.

Membrane structures and physiology of an immature synapse

Immature synapses, developing moth neuromuscular junctions, were studied using electro-physiological and ultrastructural techniques, and were compared with synapses from the flight muscles of adult moths. Neuromuscular junctions, formed by short side branches of the single fast motor axon, were assessed for functional state by stimulating the nerve and recording the endplate potential intracellularly from the muscle fibre. The muscle was then fixed and prepared for scanning, thin-section, and freeze-fracture microscopy. The immature stage differs from the adult by having very small (average 7.8 mV, compared with 20-30 mV), long duration ejp's that fatigue rapidly. The immature junctions are, however, only 13% shorter than those of the adult. Within the junction, the nerve terminal comes into direct contact with the muscle membrane in a series of oval patches separated by glial processes. These regions of apposition or 'plaques' in the immature synapse are about half the diameter of the adult plaques. In freeze-fractured material, the nerve terminal membrane in the plaque region bears an irregular band of particles on the cytoplasmic leaflet; the length of the band is essentially the same in the immature synapse as in the adult. This band marks the location of the active zone, an electron dense bar of the same length in thin section. The apposing external leaflet of the muscle membrane bears a patch of postsynaptic particles; the patch is much smaller than in the adult plaque. These immature patches, presumably representing clusters of receptors, range in size from a dozen particles to a hundred or more. We consider it likely that a lack of postsynaptic receptors may partially explain the very small ejp in the developing synapse, but that other factors may also be limiting. Desmosome-like contacts between glial cells and the muscle fibre were observed. Small wisps of electron dense material appear to bridge the extracellular space between the nerve terminal and the muscle fibre or between the glial processes and the muscle fibre in some locations. They are found in the same regions of the neuromuscular junction as small groups of large particles, suggesting that these two features are different aspects of the same structure. From their location one could hypothesize that they have either a mechanical function of stabilizing the glial invaginations, or a role in communication between the three types of developing cells.

Ultrastructural changes at gap junctions between lesioned crayfish axons.

In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segme;t retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal usually contains gap junctions and 50-60 nm vesicles near the axolemma of both SLGAs. From 1-14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find. These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic - pinocytotic action of 50-60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.

Junctions between the cells of the trabecular meshwork

In an electron-microscopic study of cell-to-cell attachments in the trabecular meshwork of three primate species and the rabbit, two types of junctions were identified. These junctions were common to all four species. One was classified as a macula adhaerens while the other, after uranyl acetate “en bloc” treatment of the tissue, was thought to be a macular gap junction. These junctions were common to all regions of apposition in the meshwork although their incidence in isolated sections was low; their possible role as an adhesive mechanism was discussed.

A study of the T system in rat heart.

The technique of extracellular space tracing with horseradish peroxidase is adapted for labeling the transverse tubular system (T system) in rat heart. In rat ventricular muscle the T system shows extensive branching and remarkable tortuosity. The T system can only be defined operationally, since it does not display specific morphological features throughout its entire structure. Owing to branching of the T system, a sizable proportion of the apposition between the T system and L system (or closed system) occurs at the level of longitudinal branches of the T system and is not restricted to the Z line region. The regions of apposition between the T system and L system are analyzed in rat ventricular muscle and skeletal muscle (diaphragm) and compared with the intercellular tight junctions (nexuses) of heart muscle by the use of a photometric method. The over-all thickness of the nexus is significantly smaller than that of T-L junctions in both cardiac and skeletal muscles. The thickness of the membranes of the T and L systems are not significantly different in the two muscles, but the gap between both membranes is larger in the heart. In atrial muscle the following two types of cells are found: (a) those cells with a well-developed T system in which the tubular diameter is quite uniform and the orientation predominantly longitudinal and, (b) cells with no T system, but with a well-developed L system. Atrial cells possessing a T system are richly provided with specific granules and show little micropinocytotic activity, whereas cells devoid of T system show intense micropinocytotic activity and few specific granules. The possible functional implications of these findings are discussed.