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L Clift-O'Grady

Publications and source records attributed to L Clift-O'Grady.

8 recordsLinked to original sources
PubMed1998

Vesiculation and sorting from PC12-derived endosomes in vitro.

Formation of small vesicles resembling synaptic vesicles can be reconstituted in vitro by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faúndez, V., Horng, J.-T. & Kelly, R. B. (1998) Cell 93, 423-432]. To determine whether AP3 was mediating budding from plasma membranes or endosomes the organelle that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15 degrees C, but not at 4 degrees C, that excluded a marker of plasma membranes and that contained internalized transferrin, indicating that the precursor was an endosome. Vesicles formed from the endosomal precursor in vitro excluded transferrin. We conclude that ARF-mediated vesiculation into synaptic vesicle-sized organelles uses an endosomal precursor and occurs simultaneously in vitro with sorting of synaptic vesicle proteins from other membrane protein constituents of the endosome.

ADP-Ribosylation Factor 1↗
PubMed1998

Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes.

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.

Adenosine Triphosphate↗
PubMed1997

ATP-dependent formation of free synaptic vesicles from PC12 membranes in vitro.

Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor membranes are a rapidly sedimenting fraction derived from the neuroendocrine cell line PC12 (pheochromocytoma 12). These starting membranes contain the synaptic vesicle proteins, synaptophysin and SV2, and the endosomal markers transferrin receptor and cation-independent MPR (mannose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocity sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synaptic vesicle increase in vitro is time-, cytosol-, ATP- and temperature-dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A) and aluminum fluoride, but not GTP gamma S (guanosine-5'O-C3-thiotriphosphate). The production of synaptic vesicles under these conditions is unlike the de novo generation of vesicles from endosomes (1). Incubation in vitro under the conditions described here may allow the final stages of synaptic vesicle formation, uncoating or undocking, to occur but not the initiation of formation de novo.

Adenosine Triphosphate↗
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↗
PubMed1993

Biogenesis of synaptic vesicles.

The basic endosomal recycling pathway can be modified to generate transcytotic vesicles, storage vesicles and synaptic vesicles. Sorting into synaptic vesicles requires specialized sorting information not present in the transcytotic and storage vesicle proteins. Using mutagenesis we have distinguished the signals for rapid endocytosis and SV targeting in synaptobrevin. Finally, we have evidence that synaptic vesicles can be generated from an endosomal compartment in vitro.

Animals↗
PubMed1990

Biogenesis of synaptic vesicle-like structures in a pheochromocytoma cell line PC-12.

The presence of unique proteins in synaptic vesicles of neurons suggests selective targeting during vesicle formation. Endocrine, but not other cells, also express synaptic vesicle membrane proteins and target them selectively to small intracellular vesicles. We show that the rat pheochromocytoma cell line, PC12, has a population of small vesicles with sedimentation and density properties very similar to those of rat brain synaptic vesicles. When synaptophysin is expressed in nonneuronal cells, it is found in intracellular organelles that are not the size of synaptic vesicles. The major protein in the small vesicles isolated from PC12 cells is found to be synaptophysin, which is also the major protein in rat brain vesicles. At least two of the minor proteins in the small vesicles are also known synaptic vesicle membrane proteins. Synaptic vesicle-like structures in PC12 cells can be shown to take up an exogenous bulk phase marker, HRP. Their proteins, including synaptophysin, are labeled if the cells are surface labeled and subsequently warmed. Although the PC12 vesicles can arise by endocytosis, they seem to exclude the receptor-mediated endocytosis marker, transferrin. We conclude that PC12 cells contain synaptic vesicle-like structures that resemble authentic synaptic vesicles in physical properties, protein composition and endocytotic origin.

Animals↗
PubMed1983

A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft.

Animals↗