Mechanistic Determinants of Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

SUMMARYClathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable ‘transport-packets’ that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.

Ultrastructure of the Salivary Glands of the Stink Bug Predator Podisus distinctus

Podisus distinctus (Hemiptera: Pentatomidae) is a zoophytophagous insect with significant potential for use as a biological control agent in agriculture and forestry because their nymphs and adults actively prey on diverse insect species. The saliva of this insect possesses active substances that cause paralysis and death of the prey. As the first step in identifying compounds of P. distinctus saliva, this study describes the ultrastructure of the salivary glands of this predator. The salivary system of P. distinctus possesses a pair of main salivary glands with a short anterior lobe, a long posterior lobe, and a pair of tubular accessory glands. The main salivary gland of P. distinctus has no associated muscles, suggesting that the saliva-release mechanism occurs with the help of certain thorax muscles. The main salivary gland epithelium has a single layer of cells (varying from cubical to columnar) with cytoplasm rich in rough endoplasmic reticulum, spherical granules of different sizes, a nucleus with a predominance of decondensed chromatin, and nucleolus. The apical cell region has a few short microvilli and the basal region has plasma membrane infoldings. The epithelium of the accessory salivary glands possesses a single-layered epithelium of cubic cells delimiting a narrow lumen. The apical cell region has a high density of microvilli and pleomorphic mitochondria, whereas the central cell region is rich in rough endoplasmic reticulum with a well-developed nucleus and decondensed chromatin. The basal cell region is characterized by the presence of several basal plasma membrane infoldings associated with mitochondria and numerous openings to the hemocoel forming large channels. The ultrastructural characteristics suggest that the main salivary glands and accessory salivary glands play a vital role in protein synthesis for saliva production and that the accessory glands are involved in transport of materials of the hemolymph.

Live imaging of bulk endocytosis in frog motor nerve terminals using FM dyes

We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, high-frequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity.

Planum Semilunatum of the Rat: New Light and Electron Microscopy Observations

Planum semilunatum (PSL) cells of the rat ampullae were studied by light, transmission, and scanning electron microscopy. The observed ultrastructure of rat PSL cells was similar to that described in other species, but is in disagreement with earlier reports of rat PSL cells, most probably because of previous divergent definitions of the PSL. Regions of PSL cells were easily distinguished from other nonsensory epithelia at the lateral ends of the crista ampullaris. The PSL region consisted of irregular-shaped columnar to cuboidal pentagonal or hexagonal cells that interdigitated with one another by lateral membrane infoldings. In the PSL region the subepithelial reticular layer appeared thickened and formed wartlike impressions in the basal surface of the PSL cells. These morphological characteristics of the subepithelial reticular layer were unique to the PSL region in the ampulla and may reflect special adaptations of the PSL region to mechanical stress. Furthermore, the thick subepithelial reticular layer may have implications for transport across the PSL region.

Ultrastructure and microanalyses of the protoscolex hooks ofEchinococcus granulosus

The rostellar distal cytoplasm ofEchinococcus granulosusprotoscoleces is characterized by extensive basal membrane infoldings, prominent hemidesmosomes and is subtended by a lamina reticularis with microfibrils of approximately 10 nm diameter that occasionally show a 55 nm banding periodicity. The rostellar hooks, in 2 rows, each have a blade, guard and handle region and possess a central amorphous pulp, a middle microfibrillar medulla with microfibrils of approximately 4 nm diameter, and a complex outer cortex in all but the proximal region of the guard and the base of the handle. In these regions additional material, of similar electron density to the medulla, but lacking the fibrillar substructure, occurs and gives the areas a lobed appearance. Energy-dispersive X-ray microanalysis of whole hooks demonstrated the presence of sulphur and trace quantities of phosphorus. X-ray near-edge absorption spectra resembled those of cystine, feather and hair and showed the sulphur to be predominantly in the form of disulphide linkages. X-ray diffraction patterns of whole hook preparations revealed 2 diffuse rings with equatorial spacings of 7·99 Å and 15·22 Å, thus differing from vertebrate keratins.

The relationship between talin and acetylcholine receptor clusters in Xenopus muscle cells

Talin is involved in mediating the cytoskeleton-extracellular matrix interaction at focal contacts in cultured fibroblasts. Recently this protein has been localized at both the myotendinous junction (MTJ) and the neuromuscular junction (NMJ) in skeletal muscle. At the MTJ, talin may mediate the insertion of myofibrils into the plasma membrane, thus serving a function similar to that seen at focal contacts. However, the function of talin at the NMJ is unknown. In this study, we examined its distribution at both mature and developing acetylcholine receptor (AChR) clusters in Xenopus muscle cells both in vivo and in vitro with immunofluorescence. At the NMJs of both myotomal and submaxillaris muscles, talin was absent from the AChR clusters. In cultured myotomal muscle cells, it was absent from 40% of both the nerve-associated AChR patches and the spontaneously formed AChR clusters located on the top surface of the cells. We therefore conclude that this protein is not essential for maintenance of AChR clusters at the NMJ. In addition to MTJs, talin was invariably associated with AChR clusters induced by polyornithine-coated beads, and, to a large extent, with spontaneously formed clusters on the ventral side of cultured cells. A common feature of these talin-positive domains is the deep membrane infoldings, where bundles of actin filaments are inserted into the membrane. Thus, talin may be involved in the formation and maintenance of these structures. The deep membrane infoldings, though prominent at most NMJs, are absent from the two muscles under study in vivo. Our work thus suggests that the postjunctional membrane at the NMJ is heterogeneous, consisting of an AChR cluster domain and, often but not always, a domain for proteins involved in cytoskeleton-membrane linkage as exemplified by talin.

Fine structure of the kidneys of osmotically stressed Mytilus, Mercenari, and Anodonta

Osmotically induced ultrastructural changes in the kidneys of the freshwater bivalve Anodonta and the marine bivalves Mytilus and Mercenaria were studied. Osmotic stresses were given to Anodonta by keeping them in distilled water or in 6% seawater, and to Mytilus and Mercenaria by keeping them in 50% seawater for various periods. In all of these bivalves, the convoluted, single cell layered kidney epithelia displayed wide lateral intercellular spaces as well as extracellular spaces in the basal membrane infoldings during hyposmotic stress. These spaces were greatly reduced when the animals were kept in isosmotic media (i.e., isosmotic to their respective hemolymphs). The kidney cells contained abundant cytoskeletal elements and microfilaments were often observed in bundles in the basal membrane infoldings. Actin was observed in the basal membrane infoldings using the specific fluorescent stain nitrobenzoxadiazole-phallacidin. The cell contacts of the kidney epthelia were studied in platinum replicas of freeze-fractured tissues. The lateral cell membrane and basal membrane infoldings contained many gap junctions. Many rows of dense intramembrane particles of septate junctions were observed in the kidneys of animals from isosmotic media. The septate junctions in the kidneys of aminals from hyposmotic media contained either fewer intramembrane particle rows or many sinuous intramembrane particle rows. The site of prourine formation in mollusks are discussed.

Topography and Ultrastructure of the tegument of adultSchistosoma mekongi

SUMMARYThe tegument of adultSchistosoma mekongihas been studied by both scanning and transmission electron microscopy. The gross surface topography of the parasite resembles that ofS. japonicum, in that branched ridges, microvilli and sensory papillae predominate; such characteristics distinguish these two species from the non-oriental schistosomes. A unique feature ofS. mekongi, however, is the numerous pleomorphic protruberances which are concentrated particularly on the middle three-fourths of the body surface. Transmission electron microscopy has revealed that these protruberances enclose bundles of microfilaments which appear to insert into the tegumental outer membrane. The microfilaments are suggested to have a supportive or stabilizing function, and may compensate for the absence of more typical crystalline spines. The tegumental outer membrane is typically heptalaminate in section, while the basal membrane infoldings are surrounded by concentrations of mitochondria. Three types of tegumental inclusion bodies have been recognized. Discoid bodies and membraneous bodies are morphologically identical to those described in all other schistosome species, except that the latter inclusions have been seen connected to each other and to the tegumental outer membrane by unique channels lined with trilaminate membrane. The third inclusion takes the form of spherical, lucent vesicles containing membrane fragments; these may represent the remains of spent membraneous bodies.

The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle.

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.