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Biomedical subjects

R B Kelly

Publications and source records attributed to R B Kelly.

At least 55 records · Page 3Linked to original sources
PubMed1995

A targeting signal in VAMP regulating transport to synaptic vesicles.

VAMP is a synaptic vesicle membrane protein required for fusion. Synaptic vesicle targeting was measured for mutants of an epitope-tagged form of VAMP in transfected PC12 cells. A signal within a predicted amphipathic alpha helix is essential for targeting to synaptic vesicles. Cellubrevin, a nonneural VAMP homolog, contains this signal and is also targeted to synaptic vesicles. Amino acid substitutions within the synaptic vesicle targeting signal either enhance or inhibit sorting of VAMP to synaptic vesicles, but do not affect the ability of VAMP to form complexes with syntaxin and SNAP-25.

Amino Acid Sequence↗
PubMed1995

Biogenesis of synaptic vesicles in vitro.

Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.

Animals↗
PubMed1995

Maintained PC1 and PC2 expression in the AtT-20 variant cell line 6T3 lacking regulated secretion and POMC: restored POMC expression and regulated secretion after cAMP treatment.

Two variant cell lines were recently established from parent AtT-20 cells. Whereas HYA.15.10.T.2 have a reduced level of secretory granules, HYA.15.6.T.3 are completely devoid of both the regulated pathway of secretion and of dense-core secretory granules. AtT-20 cells normally express the processing enzymes PC1, PC2, furin, carboxypeptidase E, and peptidylglycine alpha-amidating monooxygenase, as well as proopiomelanocortin, chromogranin B, and 7B2. We measured the expression of these mRNAs in both variant cell lines. Although some differences in mRNA level were noted, HYA.15.10.T.2 and HYA.15.6.T.3 cell lines maintained their expression of the processing enzymes and of 7B2. Furthermore, PC1 and PC2 were shown to be functionally active in the HYA.15.6.T.3 cells. In contrast, proopiomelanocortin and chromogranin B mRNA levels were no longer detectable in HYA.15.6.T.3 cells. Interestingly, stimulation of the HYA.15.6.T.3 cells with cAMP restored proopiomelanocortin mRNA, beta-endorphin immunoreactivity, and dense-core granules. Furthermore, at the ultrastructural level, beta-lipotropin immunoreactivity was detected in granules of cAMP-induced HYA.15.6.T.3 cells. Finally, depolarization of cAMP-induced HYA.15.6.T.3 cells with 56 mM potassium chloride resulted in a marked increase in the release of beta-endorphin immunoreactivity. These observations demonstrate that cAMP restores the regulated pathway of secretion in HYA.15.6.T.3 cells, which under untreated conditions do not demonstrate regulated release. These variant cell lines are unique models to understand better the relationship of the regulated pathway and the expression of the processing enzymes.

Animals↗
PubMed1994

A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4.

Some cell types have cytoplasmic storage vesicles whose fusion with the cell surface is triggered by an extracellular signal. To explore the relationship between different classes of storage vesicles, we expressed, in the neuro-endocrine cell line PC12, the facilitative glucose transporter GLUT4, which is stored in small cytoplasmic vesicles in fat and muscle cells and mobilized to the cell surface when insulin is present. PC12 cells have two known types of storage vesicles, secretory granules and synaptic vesicles, but GLUT4 is targeted to neither. It is recovered, however, in a class of small vesicles that sediment approximately twice as fast as synaptic vesicles. Immunoelectron microscopy confirmed the presence of such small vesicles in transfected PC12 cells. By velocity sedimentation analysis, GLUT4 vesicles efficiently exclude the synaptic vesicle markers synaptophysin, SV2, and synaptobrevin; the transferrin receptor, a marker of conventional endocytosis; and the polymeric immunoglobulin receptor, a marker of transcytosis. The exclusion of synaptophysin and the transferrin receptor from most of the GLUT4-containing structures was confirmed by confocal immunofluorescence microscopy. Like synaptic vesicles, therefore, GLUT4 vesicles of PC12 cells appear to be a unique type of organelle. A GLUT4-containing organelle of identical sedimentation properties was found in transfected fibroblast cell lines and in rat adipocytes. On stimulation of the adipocytes with insulin, GLUT4 was translocated from the peak of small vesicles to faster sedimenting membranes. We propose that the class of vesicles described here is present in a wide range of cell types and is involved in transient modification of the cell surface.

3T3 Cells↗
PubMed1994

Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions.

We show that uptake and release of the styryl dye FM1-43 may be used to monitor synaptic vesicle exocytosis and recycling at Drosophila larval neuromuscular junctions. At Drosophila nerve terminals, FM1-43 specifically labels subsynaptic domains enriched in synaptotagmin, in a manner that requires Ca2+, membrane depolarization, and shibire (shi) function. Endocytosis rates, very low in unstimulated synapses, are induced severalfold by the exocytosis of synaptic vesicles. Using shi(ts)1 mutant synapses to separate synaptic vesicle fusion and recycling temporally, we show that recycling events subsequent to the shi block do not require extracellular Ca2+. We suggest that two distinct intermediate stages in vesicle recycling may be trapped and analyzed at Drosophila neuromuscular junctions.

Animals↗
PubMed1994

The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes.

P-selectin is a cell adhesion molecule required transiently on the surface of activated platelets and endothelial cells as a receptor for leukocytes. It is stored in secretory granules in platelets, endothelial cells, and transfected neuroendocrine cells and is rapidly delivered to the plasma membrane upon exocytosis of the secretory granules. It is then rapidly internalized in endothelial cells and transfected cells. We find that in transfected neuroendocrine PC12 cells, the fraction of P-selectin that is not targeted to secretory granules is rapidly degraded. In transfected CHO fibroblasts, which lack secretory granules, P-selectin was degraded with a half time of 2.3 h in plated cells, while low density lipoprotein receptor (LDL-R) had a half life of 9 h. In cells cultured in ammonium chloride to inhibit lysosomal proteinases, P-selectin was protected from degradation and rapidly accumulated in vesicles enriched in lgp-B, a resident lysosomal membrane protein. The cytoplasmic domain of P-selectin was sufficient to confer rapid turnover on LDL-R. Deletion of 10 amino acids from the cytoplasmic domain of P-selectin extended the half life to 9.5 h and abrogated rapid lysosomal targeting in the presence of ammonium chloride, implicating this sequence as a necessary element of a novel lysosomal targeting signal. The rate limiting step in degradation occurred after internalization from the cell surface, indicating that sorting of P-selectin away from efficiently recycled proteins occurs in endosomes. We propose that this sorting event represents a constitutive equivalent of receptor down regulation, and may function to regulate the expression of P-selectin at the surface of activated endothelial cells.

Amino Acid Sequence↗
PubMed1994

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells.

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.

Animals↗
PubMed1994

Identifying oral lesions associated with crack cocaine use.

Dental patients who smoke crack cocaine are at higher risk for HIV infection and other medical concerns including stroke, heart failure and pulmonary hemorrhage. Four cases are reported which illustrate oral ulcers caused by crack cocaine usage.

Adult↗
PubMed1993

Storage and release of neurotransmitters.

Because synaptic vesicles and secretory granules are simple in composition and easy to purify, many of their protein components have been identified and often sequenced. Attempts are underway to link the small number of membrane proteins to the small number of functions the vesicles perform. The discovery of sequence homologies has helped greatly with this. In addition, techniques that have begun to prove successful involve microinjection, identification of proteins that bind synaptic vesicle proteins, DNA transfection into cells and oocytes, and more recently, in vitro reconstitution of exocytosis, endocytosis, and vesicle biogenesis. Advances in the latter areas have been strongly influenced by the breakthroughs in our knowledge of membrane traffic in nonneuronal cells. The budding reactions involved in making synaptic vesicles and secretory granules resemble in many ways the generation of carrier vesicles from the ER and the Golgi complex. Finally, exocytosis in neurons may closely resemble fusion of carrier vesicles with target organelles in nonneuronal cells, using complexes of peripheral membrane proteins, GTP hydrolysis, and integral membrane proteins with fusogenic domains. The usefulness of in vitro reconstitution, reverse genetics, and the parallels with better understood systems compensates in part for a major weakness in the field, namely the difficulty in obtaining viable mutants that are defective in the storage and release of secretory vesicle content.

Animals↗