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Purkinje cells labeled for NF68 at E18 chick embryo, p: Purkinje cells; Arrowhead: axon with NF68 labeling. Fig. 20. Perikaryal labeling of NF68 at E19 chick embryo cerebellum, 320x. p: Purkinje cells with punctate labeling round nucleus. Dashed lines show contour of a Purkinje cell. Fig. 21. Immunolabeling for NF68 in adult chicken cerebellar cortex, 80x. G: granular layer; M: molecular layer; p: Purkinje cells are unlabeled but are surrounded by immunolabeled basket cell axons. Fig. 22. Same as before showing NF 68 in basket cell axons at 128x p-contour of Purkinje cells also shown by arrowheads. Fig. 23. Same as before showing NF68 labeled basket cell axons around a Purkinje cell in adult chicken cerebellum, 320x. Fig. 24. Immunolabeling for GFAP in the molecular layer of E17 chick embryo cerebellum. The Bergmann fibers are exclusively labeled. Fig. 25. Vimentin in Bergmann glial fibers of E16 chick embryo cerebellum, bf: parallel lines are individual fibers of Bergmann glia.
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Evidence is presented here that demonstrates the presence of NP185 (AP3) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associate...
Citations
... AP180 was originally discovered as a prominent component of Clathrin-coated vesicles which were purified from brain (Ahle and Ungewickell, 1986;Keen and Black, 1986;Kohtz and Puszkin, 1988). In fact, it was soon recognized that AP180 is an exclusively brain-expressed protein (Puszkin et al., 1992;Zhou et al., 1992) which localizes specifically to the presynapse (Yao et al., 2002(Yao et al., , 2005Koo et al., 2015) in line with a potential sorting function for Synaptobrevin2. CALM on the other hand is ubiquitously expressed (Dreyling et al., 1996) and was also found at the postsynapse (Yao et al., 2005). ...
Communication between neurons relies on neurotransmitters which are released from synaptic vesicles (SVs) upon Ca2+ stimuli. To efficiently load neurotransmitters, sense the rise in intracellular Ca2+ and fuse with the presynaptic membrane, SVs need to be equipped with a stringently controlled set of transmembrane proteins. In fact, changes in SV protein composition quickly compromise neurotransmission and most prominently give rise to epileptic seizures. During exocytosis SVs fully collapse into the presynaptic membrane and consequently have to be replenished to sustain neurotransmission. Therefore, surface-stranded SV proteins have to be efficiently retrieved post-fusion to be used for the generation of a new set of fully functional SVs, a process in which dedicated endocytic sorting adaptors play a crucial role. The question of how the precise reformation of SVs is achieved is intimately linked to how SV membranes are retrieved. For a long time both processes were believed to be two sides of the same coin since Clathrin-mediated endocytosis (CME), the proposed predominant SV recycling mode, will jointly retrieve SV membranes and proteins. However, with the recent proposal of Clathrin-independent SV recycling pathways SV membrane retrieval and SV reformation turn into separable events. This review highlights the progress made in unraveling the molecular mechanisms mediating the high-fidelity retrieval of SV proteins and discusses how the gathered knowledge about SV protein recycling fits in with the new notions of SV membrane endocytosis.
... Punctae are larger and more localized to processes in mature neurons (14-18 DIV). Significantly, the localization of both these proteins to punctae follows a similar time course to that expected for the development of synapses (Puszkin et al. 1992). Weak but extensive colocalization of NECAP 1 with clathrin pits at early times (6 DIV) would be difficult to detect due to the large diffuse pool. ...
NECAPs (adaptin ear-binding clathrin-associated protein) are a new family of clathrin accessory proteins identified through a proteomic analysis of clathrin-coated vesicles (CCVs) from the brain. One member of this family, NECAP 1, is found primarily in tissues from the central nervous system and has been shown to be complexed tightly with a substantial portion of adaptor protein-2 (AP-2) in brain extracts. However, the function and intracellular location of this protein is unknown. In this study, we find that endogenous and epitope-tagged NECAP 1 co-localizes well with clathrin and AP-2 in punctate structures, many of which also contain the presynaptic markers synaptophysin, synaptotagmin or synaptic vesicle protein 2 (SV2). NECAP 1 was also detected by western blot in synaptic vesicle preparations. Overexpression of a truncation mutant of NECAP 1 (BC-NECAP 1) in neurons inhibited transferrin endocytosis but not epidermal growth factor (EGF) endocytosis, and this inhibition was dependent on an AP-2-binding WVQF motif. Moreover, overexpression of BC-NECAP 1 results in inhibition of synaptotagmin endocytosis both in unstimulated neurons and in neurons stimulated with potassium chloride. This inhibition was abrogated by truncation of the WVQF domain. We conclude from these observations that NECAP 1 plays a role in clathrin-mediated neuronal endocytosis, including a role in presynaptic endocytosis.
... In mammals there are at least two AP180 homologues . The related protein CALM is ubiquitously expressed (Dreyling et al., 1996 ), whereas AP180 is expressed specifically in neurons and localizes to synapses (Puszkin et al., 1992; Morris et al., 1993; Zhou et al., 1993). In spite of the large number of studies that suggest that AP180-like proteins function in endocytosis , yeast mutants lacking two AP180 homologues exhibit no detectable abnormalities in endocytosis (Wendland and Emr, 1998). ...
The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.
... With deletion mutagenesis, we mapped the epitopes of two distinct mAbs directed against NP185 to a 60 amino acid residue region of murine NP185/ AP3; we detected that NP185/AP3 is phosphorylated by casein kinase II in vitro and that NP185/AP3 directly binds to brain tubulin. Bovine NP185 has been discovered and characterized biochemically in our lab (Kohtz and Puszkin, 1988;Kohtz and Puszkin, 1989;Su et al., 1991), and immuno-histochemically in mouse and chicken Perry et al., 1992;Puszkin et al., 1992); while murine F1-20 has been cloned by immunoscreening of cDNA expression library without any evidence showing a relationship with other proteins . Indeed, a survey of previously published reports indicates that NP185, pp155, AP180 and F1-20 have a number of striking similarities: i) expression only in neuronal cells (Kohtz and Puszkin, 1988;Sousa et al., 1992); ii) synapse-specific Sousa et al., 1992) ; iii) all are phosphoproteins (Keen and Black, 1986;Morris et al., 1989;Morris et al., 1990;Zhou et al., 1992); iv) all are acidic and with anomalous migration on SDS-PAGE (Ahle and Ungewickell, 1986;Keen and Black, 1986;Kohtz and Puszkin, 1988;Zhou et al., 1992); v) all are developmentally regulated in their expression Sousa et al., 1992); and vi) all are different isoforms (Kohtz and Puszkin, 1988;this report;Zhou et al., 1992Zhou et al., , 1993Morris, et al., 1993). ...
NP185, a neuronal-specific protein of 185 kDa, was first discovered when we prepared monoclonal anti-bodies (mAbs) against bovine brain clathrin coated vesicles. Two mAbs, 8G8 and 6G7, permitted us to characterize this protein both biochemically and in development (NP185 is expressed in a NGF-dependent manner in PC12 cells). The expression of NP185 coincides with synaptogenesis. In this work, we have further characterized this protein as follows: Microsequence analysis of immuno-purified native NP185 from bovine brain yielded five peptides that corresponded exactly to the known sequences of murine F1-20 and rat AP180 (renamed AP3); ii) Using an established assay, we show that purified recombinant NP185/AP3 can facilitate clathrin cages assembly; iii) Using deletion mutagenesis, we mapped the epitopes of two distinct mAbs directed against bovine NP185 to a 60 amino acid residue region of the murine recombinant NP185/AP3; iv) Recombinant NP185/AP3 can be phosphorylated by purified casein kinase II in vitro; and v) Recombinant NP185/AP3 directly binds to purified brain tubulin. Since NP185/AP3 binds to tubulin and stimulates the clathrin assembly, it may be involved in the regulation of the transport of clathrin-coated vesicles. Casein kinase II, an enzyme known to be present in clathrin-coated vesicles, may play a role in the regulation of NP185/AP3 for the promotion of clathrin assembly.
... With deletion mutagenesis, we mapped the epitopes of two distinct mAbs directed against NP185 to a 60 amino acid residue region of murine NP185/ AP3; we detected that NP185/AP3 is phosphorylated by casein kinase II in vitro and that NP185/AP3 directly binds to brain tubulin. Bovine NP185 has been discovered and characterized biochemically in our lab (Kohtz and Puszkin, 1988;Kohtz and Puszkin, 1989;Su et al., 1991), and immuno-histochemically in mouse and chicken Perry et al., 1992;Puszkin et al., 1992); while murine F1-20 has been cloned by immunoscreening of cDNA expression library without any evidence showing a relationship with other proteins . Indeed, a survey of previously published reports indicates that NP185, pp155, AP180 and F1-20 have a number of striking similarities: i) expression only in neuronal cells (Kohtz and Puszkin, 1988;Sousa et al., 1992); ii) synapse-specific Sousa et al., 1992) ; iii) all are phosphoproteins (Keen and Black, 1986;Morris et al., 1989;Morris et al., 1990;Zhou et al., 1992); iv) all are acidic and with anomalous migration on SDS-PAGE (Ahle and Ungewickell, 1986;Keen and Black, 1986;Kohtz and Puszkin, 1988;Zhou et al., 1992); v) all are developmentally regulated in their expression Sousa et al., 1992); and vi) all are different isoforms (Kohtz and Puszkin, 1988;this report;Zhou et al., 1992Zhou et al., , 1993Morris, et al., 1993). ...
... Furthermore, if NP 185 molecules were present in the neuromuscular junction and only at a presynaptic location, we could postulate a unique role for NP185 at this subcellular location for all neurons. In our attempts to visualize NP185, we used our well-characterized monoclonal antibody 8G8, developed against NP185, which had shown cross-reactivity with chick brain tissue (Su et al., 1990;Perry et al., 1991;Puszkin et al., 1992). We dissected late embryonic chick striated muscle fibers and followed a double-labelling procedure and confocal microscopy. ...
The NP185 polypeptide (AP3) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptosomal plasma membrane (Su et al., 1991). The NP185 molecules are expressed during avian cerebellar synaptogenesis and appear to function in CNS regions rich in synaptic terminals (Perry et al., 1991). In this report we describe double-labelling experiments with avian embryonic striated muscle fibers demonstrating the exclusive presence of the brain-specific protein at the neuromuscular junction. We used indirect rhodamine immunofluorescence labeling with a monoclonal antibody (mAb-8G8) to mark the location of NP185 in muscle combined with fluorescein-alpha-bungarotoxin to mark the postsynaptic location of the acetylcholine receptors (AChRs). We show that the distribution of both NP185 and AChRs has an overall correlation, but the location of NP185 is circumscribed to presynaptic structures adjacent but not overlapping with postsynaptic structures displaying the AchRs. To confirm the identity of NP185, the molecule was extracted from both tissues, partially purified, immunoprecipitated, and identified in Western blots with the mAb 8G8. The mAb reacted with an identical 185 kD protein band purified from both tissues. Based on its properties and specific neuronal location, the NP185 molecule may function in motor nerve terminals by screening membrane proteins, identifying areas of the synaptic plasma membrane, and to anchor these elements with structural proteins for their recycling and transport within the neuronal cellular compartments.
https://searchworks.stanford.edu/view/8166459
Bioquímica y patología clínica : ByPC : revista de la Asociación Bioquímica Argentina
LANGUAGE
Spanish, English. In Spanish, with occasional articles and summaries in English.
IMPRINT
Buenos Aires, Argentina : Asociación Bioquímica Argentina, 1997-
PHYSICAL DESCRIPTION
volumes : illustrations ; 28 cm
Creators/Contributors
CONTRIBUTOR
Asociación Bioquímica Argentina.
Subjects
SUBJECT
Biochemistry > Periodicals.
Clinical biochemistry > Periodicals.
Diagnosis, Laboratory > Periodicals.
Biochemistry.
Chemistry, Clinical.
Laboratory Techniques and Procedures.
Pathology, Clinical.
Biochemistry.
Clinical biochemistry.
Diagnosis, Laboratory.
GENRE
Periodicals.
Periodicals.
Bibliographic information
BEGINNING DATE
1997
TITLE VARIATION
ByPC
FREQUENCY
Three issues yearly
VOL/DATE RANGE
Vol. 61, no. 1 (agosto 1997)-
NOTE
Title from cover.
AVAILABLE IN ANOTHER FORM
Online version: Bioquímica y patología clínica (Online)
AVAILABLE IN ANOTHER FORM
CD-ROM version: Bioquímica y patología clínica ( 2250-5903 )
CONTINUES
Asociación Bioquímica Argentina. Revista de la Asociación Bioquímica Argentina ( 0004-4768 )
ISSN
1515-6761
KEY TITLE
Bioquímica y patología clínica
Purification and Molecular Characterization of NP185, a neuron and synapse-specific protein: Purification of bovine brain NP185 was performed by differential centrifugation, multiple column (size column, ion column, immuno-affinity column). Peptide sequencing was obtained from purified NP185 protein. Molecular cloning of NP185, a synapse-specific protein, was performed and epitopes of 2 mAbs were mapped.
Purification and Molecular Characterization of NP185, a neuron and synapse-specific protein: Purification of bovine brain NP185 was performed by differential centrifugation, multiple column (size column, ion column, immuno-affinity column). Peptide sequencing was obtained from purified NP185 protein. Molecular cloning of NP185, a synapse-specific protein, was performed and epitopes of 2 mAbs were mapped.
This chapter introduces the classes of synaptic vesicle and discusses the structure and components of small synaptic vesicles (SSVs) in detail. It highlights the relationship of SSV structure to their dynamics in the nerve terminal, especially the mechanisms that organize SSV within the synaptic cytoplasm, and at the active zone where exocytosis takes place. It also discusses the recycling of SSV from components retrieved from synaptic plasma membranes by endocytosis. SSVs store and release the classical neurotransmitters such as acetylcholine, γ-aminobutyric acid (GABA), glutamate, and glycine. These vesicles are remarkably homogenous in both size and density and they account for approximately 6% of total brain protein. Electron microscopic studies reveal that they have a clear core, except for catecholamine containing SSVs, which appear to have an electron-opaque core under certain fixation conditions; these particular vesicles are sometimes referred to as small dense core vesicles. SSV exocytosis is initiated by the Ca2+ influx that is mediated by voltage-sensitive Ca2+ channels upon depolarization of the presynaptic membrane. Within 200 μsec of calcium influx, SSV exocytosis has occurred. A second essential requirement for exocytosis is guanosine triphosphate GTP.
