A novel AP180-related protein in vesicles that concentrate at acetylcholine receptor clusters

Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305–598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with γ adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters. J. Cell. Biochem. 68:457–471, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural evidence for dendritic release of acetylcholinesterase in the rat substantia nigra

Morphological evidence for dendritic secretion of acetylcholinesterase (AChE) in rat substantia nigra--a physiologically known phenomenon--was searched by means of a modified cytochemical method devised for fine localization of AChE activity at the electron microscopic level. DAB precipitate was observed in cluster of small vesicles in contact with the plasma membrane and in the extracellular space in the vicinity of the vesicles. Single coated or uncoated large vesicles filled with stained material were found in the cytoplasm of the dendrites at distance from or in contact with the plasma membrane. Immunoperoxidase staining with specific anti-serum against rat AChE gave similar localization of AChE. These results suggest that AChE is released from the dendrites of the nigral neurons by a process of vesicular exocytosis and captured by endocytosis. The relation of this process to a putative release from the smooth endoplasmic reticulum remains to be elucidated.     

Legumin antibodies recognize polypeptides in coated vesicles isolated from developing pea cotyledons

Summary A highly enriched coated vesicle fraction has been isolated from cotyledons of developing pea seeds. This, and coated vesicles isolated from bovine brain as well as from bean leaves were subjected to SDS-PAGE followed by Western blotting with legumin antibodies. A distinct cross reaction with two polypeptides at around 60 kDa was seen, but only with the coated vesicles isolated from peas. Since legumin is synthesized as a 60 kDa precursor, but occurs as 40 and 20 kDa polypeptides in the protein body, we interpret our results as giving support to the idea that reserve proteins, like lysosomal proteins, are transported via coated vesicles.Abbreviations CV coated vesicle - DTT dithiothreitol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.     

Ultrastructural Observations on CTC-Induced Callose Formation in Riella helicophylla

The Ca²⁺-chelator CTC binds to a specific site on both outer surfaces of all non-meristematic cells of the unistratose thallus of Riella, known to be rich in anionic wall components and calcium, and induces there the deposition of callose. Structural changes in this region during prolonged CTC treatment have been followed by light and transmission electron microscopy. With fluorescence microscopy punctate structures can be detected after 10 min, which upon longer incubation in CTC develop into large vesicular bodies, surrounded by a circular structure. The aniline blue-derived fluorescence intensity of these structures is highest in cells of the extension growth zone. At the ultrastructural level a mosaic of numerous smooth-surfaced vesicles, presumably containing callose, initially appears subjacent to the plasma membrane. These vesicles swell and fuse with each other, forming ultimately a circular fusion profile with the plasma membrane. This complex of callose-forming vesicles is thought to develop from elements of the partially coated reticulum (PCR), based on the presence of coated vesiculation profiles on the callose vesicles and numerous aggregates of coated vesicles in their immediate vicinity. After 30 min in CTC osmiophilic particles appear around these callose vesicles and at the cytoplasmic face of mitochondria. They are later (after 60 min) deposited in the periplasmic space between wall and plasma membrane and are also released into the surrounding medium. As judged by their reaction with FeCl₃, the osmiophilic particles appear to be phenolic in nature. We propose that upon binding of CTC a local increase of cytoplasmic calcium triggers callose synthesis in PCR-like compartments beneath the plasma membrane. However it remains to be shown as to why callose is synthesized exclusively in these intracellular compartments and not at the plasma membrane.     

A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle

An mAb library was produced against proteins from the germinal vesicle (GV) of the frog Xenopus laevis; mAb 104 was selected from this library on the basis of its immunofluorescent staining of lampbrush chromosome loops. Chromosomes from several species of frogs and salamanders stained equally well. The antibody also stained the surface of numerous small granules in the GV nucleoplasm. The interior of the same granules was stained by antibodies against small nuclear ribonucleoproteins (snRNPs). mAb 104 also stained somatic nuclei from many vertebrate and invertebrate species, usually in a finely punctate pattern similar to that described for anti-snRNP and other antinuclear antibodies. The staining of somatic nuclei was much stronger during the mitotic stages than during interphase. Immunoblot analysis showed that mAb 104 recognizes a phosphorylated epitope.     

Molecular forms of cathepsin D in coated vesicle preparations

We have studied the polypeptide pattern of cathepsin D associated with coated vesicle fractions prepared from human placenta. In these fractions cathepsin D was about 35-fold enriched in the precursor polypeptides as compared to the unfractionated tissue extract. The enrichment was more prominent if the vesicles were fractionated in the presence of Triton X-100. Adsorption of exogenously added metabolically labelled cathepsin D precursor to the fractionated material was negligible. It is likely that the precursor and may be also the mature cathepsin D polypeptides are present in the matrix of the coated vesicles. This finding substantiates the idea that coated vesicles participate in the transport of newly synthesized cathepsin D into the lysosomes.     

Location of the 100 kd-50 kd accessory proteins in clathrin coats.

We present a three-dimensional map of the clathrin coat of coated vesicles, generated from tilt series of electron micrographs of unstained specimens embedded in vitreous ice. We have examined native placental coated vesicles and coats reassembled from their purified constituents, namely clathrin triskelions and accessory proteins of approximate mol. wts 100 kd and 50 kd. Our results show that the accessory proteins contribute a further shell of density within the double shell of the clathrin cage, extending from the terminal domains of the clathrin to the membrane of the vesicle. The thickness of the complete coat is approximately 22 nm.     

Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.     

100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies

Proteins with molecular weights of around 100,000 (designated 100K) are found in all coated vesicles. Five monoclonal antibodies have been raised against the major 100K proteins of bovine brain coated vesicles, which migrate on SDS gels as three closely spaced bands. One antibody stains the middle band (band B), two stain both upper and lower bands (bands A and C), and two stain the lower band (band C) only. Thus, the polypeptides in bands A and C are related (but not identical), a result confirmed by NH2-terminal sequencing. Other tissues were found to express proteins corresponding to, and co-migrating with, bands B and C but not band A. Only the two antibodies that recognize both A and C stained fixed and permeabilized tissue culture cells; they both showed a punctate pattern in the plane of the plasma membrane. Double labeling with anti-clathrin antibodies confirmed that the dots correspond to coated pits and vesicles. However, perinuclear staining seen with anti-clathrin, corresponding to Golgi-derived coated vesicles, was conspicuously absent with the two monoclonal antibodies. Affinity-purified polyclonal antisera against the 100K proteins, reported earlier, gave perinuclear as well as punctate staining; these included one antiserum which gave mainly perinuclear staining (Robinson, M. S., and B. M. F. Pearse, 1986, J. Cell Biol., 102:48-54). Thus, different 100K proteins appear to be found in different membrane compartments. Since the 100K proteins are thought to lie between clathrin and the membrane proteins of the vesicle, these results may help to explain how different membrane proteins can be sorted into coated vesicles in different parts of the cell.     

Cotton fibre differentiation: Occurrence and distribution of coated and smooth vesicles during primary and secondary wall formation

Summary Coated vesicles occur in differentiating cotton fibres during primary and secondary wall formation. The coated vesicles are often associated with the plasmalemma, or with membranes at the secreting face of dictyosomes, corresponding positionally to GERL. During secondary wall formation the number of dictyosome-associated coated vesicles seems to be smaller than during primary wall formation. When sections are stained for periodateoxidizable polysaccharides (Thiéry reaction) the membrane of plasmalemma-associated coated vesicles is intensely stained. The membrane of dictyosome-associated coated vesicles is only weakly stained. On the basis of the present evidence it is not possible to clearly decide, whether the staining in plasmalemma-associated coated vesicles is due to obliquely cut membrane or to vesicle contents. The vesicle coat material is not stained. Possible functions of coated vesicles in differentiating cotton fibres are discussed.Vesicles with contents positively stained with the Thiéry reaction are observed only during primary wall formation. The membrane of these vesicles is smooth and seems to bud from the same cisternae, probably GERL, as do the coated vesicles. During secondary wall formation no vesicles containing periodate-oxidizable polysaccharides could be detected, even under conditions that result in a strong, specific reaction in the cellulosic secondary wall. In some instances polysaccharidic material, resembling secondary wall material, has been seen to adhere to the outside of the plasmalemma. These results are consistent with the hypothesis that, in higher plants, at least part of primary wall material may already be synthesized in dictyosome vesicles, whereas cellulose biosynthesis occurs at the cell surface.     

Immunocytochemical localization of coated vesicle protein in rodent nervous system

Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.     

Enrichment of Presenilin 1 Peptides in Neuronal Large Dense-Core and Somatodendritic Clathrin-Coated Vesicles

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.     

Acetylcholine receptor clusters are associated with nuclei in rat myotubes

Clustered and diffuse acetylcholine receptors are present in cultured myotubes. These clustered AChRs represent regions of myotube membrane containing high receptor density. We have studied the distribution of the AChR clusters and nuclei to determine whether there is an association in the distribution of nuclei beneath AChR clusters. AChR clusters were visualized with alpha-bungarotoxin conjugated to tetramethylrhodamine (alpha BTX-TMR) and the nuclei were stained with bisbenzimide which binds specifically to DNA. This double label procedure, and the computerized analysis of the data allowed us to determine the distribution of nuclei and AChR clusters in the same myotube. During early stages of myotube development the nuclei formed aggregates which were comprised of 4 to 10 nuclei in close apposition to one another. This association of AChR clusters with nuclear aggregates was greatest at Day 4 after plating. As the number of nuclear aggregates associated with clusters decreased the number of nuclei in the aggregates also decreased and the AChR clusters decreased in size as well as number. At all time points examined, the concentration of myotube nuclei in the cells was 3 to 12 times higher beneath areas of AChR clusters than away from clusters. Our computerized analysis shows that there is an association of the AChR clusters with the nuclear region during myotube development.     

Several ADP-ribosylation factor (Arf) isoforms support COPI vesicle formation

Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and Arf6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.     

The isolation and enrichment of coated vesicles from suspension-cultured carrot cells

Summary A method has been developed to isolate and purify coated vesicles from suspension cultured carrot (Daucus carota L.) cells. It incorporates features of centrifugation methods (sucrose step gradient; Ficoll/D₂O gradient) previously employed in the isolation of coated vesicles from mammalian brain tissue. Most important is the treatment of the crude coated vesicle fraction (postmicrosomal supernatant) with ribonuclease to remove ribosomes which are a serious source of contamination in such fractions. The fraction finally obtained is contaminated to the extent of 30% of total observed particles in negatively stained preparations with naked vesicles whose diameter are smaller than those of the coated vesicles. These vesicles are interpreted as being coated vesicles which have been stripped of their coats. SDS-PAGE of coated vesicle fractions purified by this method reveal significant differences in the polypeptide patterns obtained from plant and animal systems.     

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.     

Concentration-dependent staining of lactotroph vesicles by FM 4-64

Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution dissolve first in the plasma membrane and are then transported into the vesicle by lateral diffusion in the plane of the membrane, and finally the vesicle matrix is stained from the vesicle membrane. On the other hand, these molecules may enter the vesicle lumen and reach the vesicle matrix by permeation through an open aqueous fusion pore. To address these questions, we exposed pituitary lactotrophs to different concentrations of FM 4-64 to monitor the fluorescence increase of single vesicles by confocal microscopy after the stimulation of cells by high K(+). The results show that the membrane and the vesicle matrix exhibit different concentration-dependent properties: the plasma membrane staining by FM 4-64 has a higher affinity in comparison to the vesicle matrix. Moreover, the kinetics of vesicle loading by FM 4-64 exhibited a concentration-dependent process, which indicates that FM 4-64 molecules stain the vesicle matrix by aqueous permeation through an open fusion pore.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

Agonists that Increase [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Halt the Movement of Acidic Cytoplasmic Vesicles in MDCK Cells

Translocation of vesicles within the cytoplasm is essential to normal cell function. The vesicles are typically transported along the microtubules to their destination. The aim of this study was to characterize the vesicular movement in resting and stimulated renal epithelial cells. MDCK cells loaded with either quinacrine or acridine orange, dyes taken up by acidic vesicles, were observed at 37°C in semiopen perfusion chambers. Time-lapse series were analyzed by Imaris software. Our data revealed vigorous movement of stained vesicles in resting MDCK cells. These movements seem to require intact microtubules because nocodazole leads to a considerable reduction of the vesicular movements. Interestingly, we found that extracellular ATP caused the vesicular movement to cease. This observation was obvious in time lapse. Similarly, other stimuli known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in MDCK cells (increment in the fluid flow rate or arginine vasopressin) also reduced the vesicular movement. These findings were quantified by analysis of single vesicular movement patterns. In this way, ATP was found to reduce the lateral displacement of the total population of vesicles by 40%. Because all these perturbations increase [Ca²⁺]_i, we speculated that this increase in [Ca²⁺]_i was responsible for the vesicle arrest. Therefore, we tested the effect of the Ca²⁺ ionophore, ionomycin (1 μM), which in the presence of extracellular Ca²⁺ resulted in a considerable and sustained reduction of vesicular movement amounting to a 58% decrease in average lateral vesicular displacement. Our data suggest that vesicles transported on microtubules are paused when subjected to high intracellular Ca²⁺ concentrations. This may provide an additional explanation for the cytotoxic effect of high [Ca²⁺]_i.