Ultrastructure of the Olfactory Epithelium in the Australian Lungfish Neoceratodus forsteri

Four cell types are present in the olfactory epithelium of Neoceratodus forsteri, i.e., olfactory receptor cells, supporting cells, non-sensory ciliated cells, and basal cells. Only microvilli and no cilia were observed on the receptor cells. The neurotubules pass out into these microvilli. Conspicuous arrays of agranular endoplasmic reticulum are present in the nuclear region of the receptor cells. The supporting cells are provided with microvilli. These cells may be secretory. The non-sensory ciliated cells produce secretory granules containing acid mucopolysaccharides. A discontinuous zonula occludens appears to be present.     

Toxicity of botulinum toxin: a stoichiometric model for the locus of its extraordinary potency and persistence at the neuromuscular junction.

The number of botulinum toxin molecules calculated to reach either a diaphragmatic endplate after a minimum lethal dose or a gastrocnemius endplate after a minimal paralyzing dose is in close agreement with both the number of vesicles released per impulse and the finite number of active sites on the endplate for efflux of acetylcholine-containing vesicles. The close agreement in size between botulinum toxin molecules, synaptic vesicles, and the width of neurotubules supports a model in which botulinum toxin physically blocks efflux of acetylcholine. The stoichiometric attachment of botulinum toxin may occur at the critical moment that vesicles fuse with the presynaptic membrane, thereby exposing a receptor. While attachment may be to a ganglioside or neuraminidase-sensitive site, the chemical nature of the receptor is neither defined nor critical for the validity of the model.     

Effects of colchicine and vinblastine on neurotubules of the sciatic nerve and cholinesterases in the developing myoneural junction of the rat

Summary Colchicine (0.1 M) or vinblastine (0.01 M) was locally applied on the sciatic nerves of newborn rats. Both colchicine and vinblastine caused reversible disappearance of axonal neurotubules and appearance of increased amounts of neurofilaments at the site of application. Subsequent morphogenesis of myoneural junctions in the tibialis anterior muscle was studied after histochemical demonstration of acetylcholinesterase (AChE; E.C. 3.1.1.7) and non-specific cholinesterase (Ns. ChE; E.C. 3.1.1.8) activity in the myoneural area.Development of the postsynaptic muscle plasma membrane of the myoneural junction was arrested in the ipsilateral, but not in the contralateral control side, for a period of about three weeks following treatment with the test substances. After this delay the myoneural morphogenesis continued normally and neurotubules were seen in the axoplasm.Since disruption of neurotubules is likely to cause blockage of the intratubular axoplasmic transport system, it seems possible that the neurotrophic influence responsible for the development of the postsynaptic muscle membrane is mediated through a secretory product transported along axons intratubularly to the nerve endings.     

Fine structure of the receptors at the myotendinous junction of human extraocular muscles

The myotendinous junction of the human extraocular muscles was studied by electron microscopy. Some peculiar receptorial structures have been found in the majority of the samples examined. These structures are very small and consist of 1) the terminal portion of one muscle fibre, 2) the tendon into which it inserts and 3), within the tendon, a rich nerve arborization, whose branches are always very close to the muscle component. Only one discontinuous layer, made up of flat cells, which lack a basal lamina and often show pinocytotic vesicles, encapsules every musculo-tendinous complex. The tendinous component consists of amorphous ground substance of different electron density, of collagen and elastic fibres and is divided in compartments by ramified cells, which make an inner capsular-like covering to the nerve fibres. Three types of afferent nerve endings can be identified. One type is usually more frequent than the others, possesses a large number of neurotubules and neurofilaments and few mitochondria and is always surrounded by a Schwann cell which forms finger-like processes penetrating into the axoplasm. The second type is only partially enveloped by the Schwann cell. The axoplasm is devoid of neurotubules and contains few neurofilaments, several mitochondria and groups of small clear vesicles placed in the areas uncovered by the glial sheath. The third one is completely surrounded by the Schwann cell, but is devoid of neurotubules and neurofilaments and full of mitochondria. These morphological features correspond well with the probable role of these receptorial structures, which is to ensure very exact and precise ocular movements.     

Two ciliate sense receptors in the larva of Austramphilina elongata Johnston, 1931 (Amphilinidea)

Summary The ultrastructure of a uniciliate and a quadruciliate receptor in the anterior end of the larva of Austramphilina elongata is described on the basis of serial sections. The uniciliate receptor has numerous branched and interconnected microvilli at its surface, several rings forming the electron dense collar, and cross-striated rootlets diverging from the basal body of the cilium. The quadruciliate receptor has four short club-shaped sensory cilia and a single electron-dense collar.Abbreviations used in figures ec electron-dense collar - ep epidermis - m microvilli - nt neurotubules - pe process of electron-dense collar - r rootlet of cilium - sc sensory cilium - sd septate desmosome     

Second Messengers in the Locomotor-Sensory System of First Multicellulars. The IP3-Containing Compartments in Chemoreceptor Cells of the Comb Jelly Beroe cucumis from Data of Cytochemical Study

Result of the performed electron microscopy cytochemical study was an indirect detection of intracellular localization of inositol 1,4,5-triphosphate according to sites of its binding with specific phosphatase in receptor and secretory cells of epithelium surrounding actinostome of the comb jelly Beroe cucumis. The highest level of the deposit of the enzymatic cytochemical reaction product was revealed in chemoreceptor cells of I type. In all cells of the epithelium as well as in nerve terminals and neuroplexus elements, the following compartments had the highest deposit content: cellular membrane and glycocalyx areas, microtubules, microfilaments, mitochondria, neurotubules, and structures of the synaptic apparatus. The obtained data expand the picture of cytochemical distribution and of the contents of the deposit compatible with localization of components of inositol pathway in receptor cells of the locomotor-sensory system of Beroe cucumis [1, 2] and indicate its essential role in the mechanism of chemosensory transduction.     

Effect of colchicine and lumicolchicine on the ultrastructure of non-myelinated axons in autonomic nerves in cat in vitro]

The effects of colchicine and lumicolchicine on the ultrastructure of non-myelinated axons in cat autonomic nerves were studied using in vitro preparations of inferior mesenteric ganglion/hypogastric nerves. After 24 hrs of in vitro incubation with colchicine added to the medium (10 microng/ml) a significant decrease in number of neurotubules per 1 axon was observed. In the presence of a solution of alpha-beta and gamma-lumicolchicine (10 microng/ml) severe degenerative changes occured in axons and Schwann cells. At a lower dose of lumicolchicine (3 microng/ml) these changes were less frequent and the number of neurotubules per 1 axon did not differ from that in control nerves.     

An ultrastructural study of the synaptic densities,nematosomes, neurotubules,neurofilaments and of a further three-dimensional filamentous network as disclosed by the E-PTA staining procedure

Summary After the staining of nervous tissue with phosphotungstic acid in absolute ethanol (E-PTA), a selective opacification occurs at certain specific sites, while other structures, especially the plasma and intra-cellular membranes, remain electron-lucent. Among those selectively stained sites, our studies have been focussed on: (1) The dense synaptic material consisting of several presynaptic clumps, termed projections, an intrasynaptic dense line and a subsynaptic web from which fine fibrillar wisps extend into the surrounding ground substance; (2) Neurofilaments and neurotubules, the surface of which is bristled by numerous side-arms; (3) A microfilamentous network intertwines the neurotubules, the neurofilaments and the mitochondria in the dendrites and axon, and is also connected to the E-PTA dense undercoating delineating the inner aspect of the plasma membrane and to the fine wisps emanating from the subsynaptic web. A three-dimensional microfilamentous latticework is thus formed in the nerve cell processes; (4) Dense cytoplasmic inclusions, termed nematosomes, which are usually located in the ground substance of the perikaryon among or in the vicinity of clusters of ribosomes. Tiny microfilaments emanate from the peripheral strands of these bodies. The presence of basic residues in the chains of structural proteins of which consist the subsynaptic web and the nematosome is plausible, since the specificity of the E-PTA staining procedure for the detection of basic residues has previously been put forth. The occurrence of a three-dimensional microfilamentous network in the nerve cell processes led us to hypothesize that it plays a role in translocation of materials. It may provide the motive force for the axoplasmic transport, for instance, with the neurotubules, as well as, plausibly, with the neurofilaments, serving as attachment sites and guideways.Supported by grant MA-3448 from The Medical Research Council of Canada.     

The homology of spindle tubules and neuro-tubules in the chick embryo retina

Summary An ultrastructural study of the 8 day chick embryo retina has been performed using gluteraldehyde and osmium tetroxide fixation. It has been noted that the centrioles of ganglion cells show clavate extensions similar to those seen in the basal bodies of photoreceptors and that these appear to give rise to the neurotubules. The neurotubules thus are shown to originate from the same organelle and have the same morphology as the spindle tubules in the mitotic cell, although the mode of origin is quite different. Some implications of these observations have been discussed.Supported by Grants NB-02255, 2M-6418, and GM 11558-01 from the National Institutes of Health.     

Functional characterization of the 180-kD ribosome receptor in vivo

A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned and sequenced. The deduced primary structure indicates three distinct domains: an NH2-terminal stretch of 28 uncharged amino acids representing the membrane anchor, a basic region (pI = 10.74) comprising the remainder of the NH2-terminal half and an acidic COOH- terminal half (pI = 4.99). The most striking feature of the amino acid sequence is a 10-amino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem without interruption in the NH2-terminal positively charged region. We postulate that this repeated sequence represents a ribosome binding domain which mediates the interaction between the ribosome and the ER membrane. To substantiate this hypothesis, recombinant full-length ribosome receptor and two truncated versions of this protein, one lacking the potential ribosome binding domain, and one lacking the COOH terminus, were expressed in Saccharomyces cerevisiae. Morphological and biochemical analyses showed all proteins were targeted to, and oriented correctly in the ER membrane. In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or one lacking the COOH-terminal domain were able to bind two to four times as many human ribosomes as control membranes lacking a recombinant protein or microsomes containing a receptor lacking the NH2-terminal basic domain. Electron micrographs of these cells revealed that the expression of all receptor constructs led to a proliferation of perinuclear ER membranes known as "karmellae." Strikingly, in those strains which expressed cDNAs encoding a receptor containing the putative ribosome binding domain, the induced ER membranes (examined in situ) were richly studded with ribosomes. In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding domain were uniformly smooth and free of ribosomes. Cell fractionation and biochemical analyses corroborated the morphological characterization. Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding.     

Subsurface sense receptors in the larva of Austramphilina elongata Johnston, 1931 (Amphilinidea)

Summary The ultrastructure of tegumental and subtegumental receptors in the larva of Austramphilina elongata is described. The receptors are terminal swellings of dendrites and contain numerous small vesicles and neurofilaments which are predominantly peripheral. Tegumental receptors, together with a sheath consisting of basal lamina and tegument, project into the epidermis, and cross-striated rootlets were sometimes found in them. Subtegumental receptors lie below the tegument and ciliary rootlets were never observed in them. Anterior dendrites contain single centrioles and clusters of centrioles. The possible function of receptors and centrioles is discussed.Abbreviations in figures bl basal lamina - c centriole - d dendrite - ep epidermis - m microvillus - nt neurotubules - r rootlet of cilium - re receptor - st subtegumental receptor - t tegument     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

Observations on the ultrastructure of a cephalic sense organ of the nematode Mermis nigrescens

This cephalic sense organ of Mermis nigrescens is unusual in that it is innervated by a dendrite which ends in two dendritic processes. These dendritic processes contain numerous neurotubules. There is a basal plate at the base of the dendritic process, and this is associated with a well-developed system of striated fibres which extend into the cytoplasm of the dendrite.     

Colchicine-binding activity and neurotubule integrity in a rat sympathetic ganglion

The colchicine-binding activity of rat superior cervical ganglia was examined. Ganglia were cooled and re-warmed in the presence of either Cu²⁺ or of metabolic inhibitors. Electronmicroscopy showed that these treatments depolymerized the neurotubules. This depolymerization of neurotubules did not affect the colchicine-binding activity of ganglion homogenates but caused a two-fold increase in colchicine-binding by whole ganglia. This suggests that colchicine binds only to depolymerized neurotubule subunits and that colchicine-binding by whole ganglia can be used as a measure of polymerization of the neurotubule protein.The major part of the colchicine-binding activity of ganglion homogenates was found in the soluble fraction and was unstable. In the absence of divalent cations, 10⁻⁴ M vinblastine stabilised the soluble colchicine-binding activity.     

Involvement of ryanodine receptor type 3 in dopamine release from the striatum: evidence from mutant mice lacking this receptor

Although it is known that ryanodine receptor type 3 is expressed in the striatum, the function of this receptor has not been elucidated. Therefore, we examined whether caffeine- and ryanodine-induced dopamine release in striatal slices is affected in mice lacking ryanodine receptor type 3. Pretreatment with thapsigargin, an inhibitor of the Ca(2+) ATPase pump of the endoplasmic reticulum, abolished caffeine- or ryanodine-induced dopamine release in slices from normal mice. Dopamine concentration in the striatum and KCl-induced dopamine release were unaffected by a ryanodine receptor type 3 deficiency. Ryanodine-induced dopamine release was significantly attenuated in mice lacking ryanodine receptor type 3, whereas caffeine-induced dopamine release was partially attenuated. Caffeine produced a similar hyper-motor activity in both wild and homozygous mice. The present results suggest the involvement of ryanodine receptor type 3 in dopamine release from the striatum.     

Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.     

The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell.

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.     

The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.     

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.