scholarly journals Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Zohreh Farsi ◽  
Sindhuja Gowrisankaran ◽  
Matija Krunic ◽  
Burkhard Rammner ◽  
Andrew Woehler ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Synaptic Vesicle ◽  
Mouse Brain ◽  
Synaptic Vesicles ◽  
Vacuolar Atpase ◽  
Coated Vesicles ◽  
Electrochemical Gradient ◽  
Proper Timing ◽  
Adenosine Triphosphatases ◽  
Single Vesicle

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.

scholarly journals The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

1996 ◽  
Vol 133 (6) ◽  
pp. 1237-1250 ◽  
Author(s):  
K Takei ◽  
O Mundigl ◽  
L Daniell ◽  
P De Camilli
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Nerve Terminals ◽  
Vesicle Budding ◽  
Vesicle Cycle ◽  
Single Vesicle

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

scholarly journals Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Jiajie Diao ◽  
Jacqueline Burré ◽  
Sandro Vivona ◽  
Daniel J Cipriano ◽  
Manu Sharma ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Lipid Binding ◽  
Snare Complex ◽  
Loss Of Function ◽  
Related Point ◽  
Molecular Mechanism Of Action ◽  
Presynaptic Protein ◽  
Single Vesicle

α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself.

scholarly journals EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

1973 ◽  
Vol 57 (2) ◽  
pp. 315-344 ◽  
Author(s):  
J. E. Heuser ◽  
T. S. Reese
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Motor Nerve ◽  
Coated Vesicles ◽  
Synaptic Membrane ◽  
Nerve Terminals ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Intracellular Compartments

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

scholarly journals Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jung-Hwa Tao-Cheng
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Immunogold Labeling ◽  
Coated Vesicles ◽  
Presynaptic Terminals ◽  
Neuronal Soma ◽  
High K ◽  
Neuronal Stimulation ◽  
Transmitter Receptors ◽  
Neuronal Compartments

Abstract Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~ 40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~ 90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon depolarization with high K+, label for clathrin became scattered among de-clustered synaptic vesicles and moved closer to the presynaptic active zone. In contrast to axons, clathrin-labeled CCVs and CCPs were prominent in soma/dendrites under resting conditions, and became inconspicuous upon depolarization with high K+. Thus, EM examination suggests that the regulation and mechanism of clathrin-mediated endocytosis differ between axon and dendrite, and that clathrin redistributes differently in these two neuronal compartments upon depolarization.

scholarly journals Membrane recycling in the cone cell endings of the turtle retina.

1978 ◽  
Vol 79 (3) ◽  
pp. 802-825 ◽  
Author(s):  
S F Schaeffer ◽  
E Raviola
Keyword(s):  
Synaptic Vesicle ◽  
Cold Exposure ◽  
Synaptic Vesicles ◽  
Surface Density ◽  
Room Temperature ◽  
Coated Vesicles ◽  
Vesicle Membrane ◽  
Cone Cell ◽  
Dark Light ◽  
Thin Sections

The ultrastructural effects of dark, light, and low temperature were investigated in the cone cell endings of the red-eared turtle (Pseudemys scripta elegans). Thin sections revealed that in dark-adapted retinas maintained at 22 degrees C, the neural processes which contact the cone cells at the invaginating synapses penetrated deeply into the photoreceptor endings. When dark-adapted retinas were illuminated for 1 h at 22 degrees C, the invaginating processes were apparently extruded from the synaptic endings. On the other hand, 1-h exposure to a temperature of 4 degrees C in the dark caused the invaginating processes to become much more strikingly inserted than at room temperature. A morphometric analysis showed that the ratio between the synaptic surface density of the endings and their total surface density decreased in the light and increased in the dark and cold. Freeze-fracturing documented fusion of synaptic vesicles with the presynaptic membrane in all conditions tested. These observations suggest that the changes in configuration of the pedicles in the light, dark, and cold reflect a different balance between addition and retrieval of synaptic vesicle membrane from the plasmalemma; in the dark, the rate of vesicle fusion is increased, whereas in the cold, membrane retrieval is blocked. When the eyecups were warmed up and illuminated for 30-45 min after cold exposure, a striking number of vacuoles and cisterns appeared in the cytoplasm and coated vesicles were commonly seen budding from the plasmalemma. 60-90 min after returning to room temperature, the endings had reverted to their normal configuration, and the vast majority of vacuoles, cisterns, and coated vesicles had disappeared. When horseradish peroxidase was included in the incubation medium, very few synaptic vesicles were labeled at the end of the period of cold exposure. 30-45 min after returning to 22 degrees C, vacuoles and cisterns contained peroxidase, whereas most synaptic vesicles were devoid of reaction product. 2 h after returning to 22 degrees C, coated vesicles, vacuoles, and cisterns had disappeared and a number of synaptic vesicles were labeled. These experiments suggest that vacuoles, cisterns, and coated vesicles mediate the retrieval of the synaptic vesicle membrane that has been added to the plasmalemma during cold exposure.

scholarly journals Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites

2020 ◽  
Author(s):  
Jung-Hwa Tao-Cheng
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Immunogold Labeling ◽  
Coated Vesicles ◽  
Presynaptic Terminals ◽  
Neuronal Soma ◽  
High K ◽  
Neuronal Stimulation ◽  
Transmitter Receptors ◽  
Neuronal Compartments

Abstract Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon depolarization with high K+, label for clathrin became scattered among de-clustered synaptic vesicles and moved closer to the presynaptic active zone. In contrast to axons, clathrin-labeled CCVs and CCPs were prominent in soma/dendrites under resting conditions, and became inconspicuous upon depolarization with high K+. Thus, EM examination suggests that the regulation and mechanism of clathrin-mediated endocytosis differ between axon and dendrite, and that clathrin redistributes differently in these two neuronal compartments upon depolarization.

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

1992 ◽  
Vol 118 (6) ◽  
pp. 1379-1388 ◽  
Author(s):  
P R Maycox ◽  
E Link ◽  
A Reetz ◽  
S A Morris ◽  
R Jahn
Keyword(s):  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Synaptic Vesicles ◽  
Nervous Tissue ◽  
Coated Vesicles ◽  
Nerve Terminals ◽  
Coat Proteins ◽  
Synaptic Vesicle Recycling ◽  
Vesicle Recycling ◽  
Vesicle Proteins

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.

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