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Clathrin regenerates synaptic vesicles from endosomes.

Watanabe S, Trimbuch T, Camacho-Pérez M, Rost BR, Brokowski B, Söhl-Kielczynski B, Felies A, Davis MW, Rosenmund C, Jorgensen EM - Nature (2014)

Bottom Line: Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature.In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis.These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112-0840, USA.

ABSTRACT
Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 s, which in turn become small-diameter synaptic vesicles 5-6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.

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Following a single stimulus, clathrin is required at endosomes toregenerate synaptic vesicles. (a) Virtual section from an electron tomogram(left) and a reconstruction (right) showing a budded synaptic endosomecontaining ferritin particles in the scrambled shRNA control cell. We founda total of 33 endosomes in these reconstructions. Of these 33 endosomes,none were connected to the plasma membrane or showed evidence of a tubuleextending from the endosomal membrane. 17 of these 33 total endosomes werefully contained within the 200 nm tomogram. Of these 33 endosomes, 16 wereferritin-positive, and 8 of these16 endosomes were fully contained in thetomogram. (b) Micrographs showing ferritin-positive synaptic vesicles dockedto active zone 10-20 s after stimulation. (c) Average number of ferritinparticles in large endocytic vesicles, clathrin-coated vesicles, andsynaptic vesicles per synaptic profile examined. At least 134 synapses wereanalyzed per time point. Ferritin progresses to synaptic vesicles in thecontrol, but is trapped in large endocytic vesicles or endosomes in theclathrin knock-down. The fraction of synaptic profiles with ferritin was 27%for the control and 31% in the knockdown, suggesting that 70% of thesynapses were silent. The mean number of ferritin particles found in anindividual endosome, clathrin-coated vesicle, and synaptic vesicle, are 9.3± 1.0, 2.0 ± 0.2, and 1.9 ± 0.2, respectively. Thetotal number of ferritin particles (indicated above), declined by 40% in thecontrol relative to the 1 s time point but not in the knockdown, either dueto the fusion of the newly formed synaptic vesicles or by return of excessmembrane to the surface of the synapse. The standard error of the mean isshown. (d) Distribution of ferritin-positive clathrin-coated vesicles(yellow) and synaptic vesicles (blue) relative to the active zone at definedtime points after stimulation in the control cells. Numbers are binned by 50nm. The first bin ‘0 nm’ means vesicles are docked in activezone. Endosomes are found at 285 ± 38 nm from the active zone. Notethat the data in this figure represent further analysis of the data shown inFig. 3.
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Figure 11: Following a single stimulus, clathrin is required at endosomes toregenerate synaptic vesicles. (a) Virtual section from an electron tomogram(left) and a reconstruction (right) showing a budded synaptic endosomecontaining ferritin particles in the scrambled shRNA control cell. We founda total of 33 endosomes in these reconstructions. Of these 33 endosomes,none were connected to the plasma membrane or showed evidence of a tubuleextending from the endosomal membrane. 17 of these 33 total endosomes werefully contained within the 200 nm tomogram. Of these 33 endosomes, 16 wereferritin-positive, and 8 of these16 endosomes were fully contained in thetomogram. (b) Micrographs showing ferritin-positive synaptic vesicles dockedto active zone 10-20 s after stimulation. (c) Average number of ferritinparticles in large endocytic vesicles, clathrin-coated vesicles, andsynaptic vesicles per synaptic profile examined. At least 134 synapses wereanalyzed per time point. Ferritin progresses to synaptic vesicles in thecontrol, but is trapped in large endocytic vesicles or endosomes in theclathrin knock-down. The fraction of synaptic profiles with ferritin was 27%for the control and 31% in the knockdown, suggesting that 70% of thesynapses were silent. The mean number of ferritin particles found in anindividual endosome, clathrin-coated vesicle, and synaptic vesicle, are 9.3± 1.0, 2.0 ± 0.2, and 1.9 ± 0.2, respectively. Thetotal number of ferritin particles (indicated above), declined by 40% in thecontrol relative to the 1 s time point but not in the knockdown, either dueto the fusion of the newly formed synaptic vesicles or by return of excessmembrane to the surface of the synapse. The standard error of the mean isshown. (d) Distribution of ferritin-positive clathrin-coated vesicles(yellow) and synaptic vesicles (blue) relative to the active zone at definedtime points after stimulation in the control cells. Numbers are binned by 50nm. The first bin ‘0 nm’ means vesicles are docked in activezone. Endosomes are found at 285 ± 38 nm from the active zone. Notethat the data in this figure represent further analysis of the data shown inFig. 3.

Mentions: To determine whether clathrin is required to resolve endosomes intosynaptic vesicles, control and clathrin knock-down cells were stimulated in thepresence of cationized ferritin (4 mM Ca2+, 34°C). In controlcultures 1 s after a single stimulus (Fig.3a,b), ferritin molecules were found in endosomes but not inclathrin-coated vesicles. At 3 s endosomes were budding and ferritin began toappear in clathrin-coated vesicles. The size of endosomes was larger thanendocytic vesicles, again suggesting that endocytic vesicles carrying ferritinfuse to form synaptic endosomes (Extended DataFig. 3b,c). Sixteen ferritin-positive endosomes were reconstructedfrom tomograms, none of them extended a tubule to the plasma membrane (Extended Data Fig. 6a). However, half ofthose endosomes were not fully contained within the 200 nm tomogram. Therefore,we reconstructed 11 complete synapses by assembling serial tomograms of synapses3 s after stimulation. None of the 17 complete end-to-end endosomes wereconnected to the plasma membrane (6 of which contained ferritin), suggestingthat they are true intracellular organelles, not extensions of the plasmamembrane (Supplementary Videos1-5). Thedecline in the number of endosomes is followed by an accumulation offerritin-positive coated vesicles and synaptic vesicles (Fig. 3b). Some clathrin-coated vesicles were observed before3 s after stimulation (1 s, 4/149 synaptic profiles and Fig. 1d), but they did not contain ferritin molecules,suggesting that they were derived from pre-existing endosomes. The total numberof ferritin granules per synaptic profile does not increase suggesting thatthere is not an additional wave of endocytic events that occurs during these 20s (Extended Data Fig. 6c). Some of theferritin-filled small vesicles docked to the active zone (Fig. 3a, right panel; Extended Data Fig. 6b,d), suggesting that these vesicles aresynaptic vesicles. These results indicate that recently endocytosed membranepasses through endosomes to regenerate synaptic vesicles within about 5-6 safter fusion.


Clathrin regenerates synaptic vesicles from endosomes.

Watanabe S, Trimbuch T, Camacho-Pérez M, Rost BR, Brokowski B, Söhl-Kielczynski B, Felies A, Davis MW, Rosenmund C, Jorgensen EM - Nature (2014)

Following a single stimulus, clathrin is required at endosomes toregenerate synaptic vesicles. (a) Virtual section from an electron tomogram(left) and a reconstruction (right) showing a budded synaptic endosomecontaining ferritin particles in the scrambled shRNA control cell. We founda total of 33 endosomes in these reconstructions. Of these 33 endosomes,none were connected to the plasma membrane or showed evidence of a tubuleextending from the endosomal membrane. 17 of these 33 total endosomes werefully contained within the 200 nm tomogram. Of these 33 endosomes, 16 wereferritin-positive, and 8 of these16 endosomes were fully contained in thetomogram. (b) Micrographs showing ferritin-positive synaptic vesicles dockedto active zone 10-20 s after stimulation. (c) Average number of ferritinparticles in large endocytic vesicles, clathrin-coated vesicles, andsynaptic vesicles per synaptic profile examined. At least 134 synapses wereanalyzed per time point. Ferritin progresses to synaptic vesicles in thecontrol, but is trapped in large endocytic vesicles or endosomes in theclathrin knock-down. The fraction of synaptic profiles with ferritin was 27%for the control and 31% in the knockdown, suggesting that 70% of thesynapses were silent. The mean number of ferritin particles found in anindividual endosome, clathrin-coated vesicle, and synaptic vesicle, are 9.3± 1.0, 2.0 ± 0.2, and 1.9 ± 0.2, respectively. Thetotal number of ferritin particles (indicated above), declined by 40% in thecontrol relative to the 1 s time point but not in the knockdown, either dueto the fusion of the newly formed synaptic vesicles or by return of excessmembrane to the surface of the synapse. The standard error of the mean isshown. (d) Distribution of ferritin-positive clathrin-coated vesicles(yellow) and synaptic vesicles (blue) relative to the active zone at definedtime points after stimulation in the control cells. Numbers are binned by 50nm. The first bin ‘0 nm’ means vesicles are docked in activezone. Endosomes are found at 285 ± 38 nm from the active zone. Notethat the data in this figure represent further analysis of the data shown inFig. 3.
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Related In: Results  -  Collection

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Figure 11: Following a single stimulus, clathrin is required at endosomes toregenerate synaptic vesicles. (a) Virtual section from an electron tomogram(left) and a reconstruction (right) showing a budded synaptic endosomecontaining ferritin particles in the scrambled shRNA control cell. We founda total of 33 endosomes in these reconstructions. Of these 33 endosomes,none were connected to the plasma membrane or showed evidence of a tubuleextending from the endosomal membrane. 17 of these 33 total endosomes werefully contained within the 200 nm tomogram. Of these 33 endosomes, 16 wereferritin-positive, and 8 of these16 endosomes were fully contained in thetomogram. (b) Micrographs showing ferritin-positive synaptic vesicles dockedto active zone 10-20 s after stimulation. (c) Average number of ferritinparticles in large endocytic vesicles, clathrin-coated vesicles, andsynaptic vesicles per synaptic profile examined. At least 134 synapses wereanalyzed per time point. Ferritin progresses to synaptic vesicles in thecontrol, but is trapped in large endocytic vesicles or endosomes in theclathrin knock-down. The fraction of synaptic profiles with ferritin was 27%for the control and 31% in the knockdown, suggesting that 70% of thesynapses were silent. The mean number of ferritin particles found in anindividual endosome, clathrin-coated vesicle, and synaptic vesicle, are 9.3± 1.0, 2.0 ± 0.2, and 1.9 ± 0.2, respectively. Thetotal number of ferritin particles (indicated above), declined by 40% in thecontrol relative to the 1 s time point but not in the knockdown, either dueto the fusion of the newly formed synaptic vesicles or by return of excessmembrane to the surface of the synapse. The standard error of the mean isshown. (d) Distribution of ferritin-positive clathrin-coated vesicles(yellow) and synaptic vesicles (blue) relative to the active zone at definedtime points after stimulation in the control cells. Numbers are binned by 50nm. The first bin ‘0 nm’ means vesicles are docked in activezone. Endosomes are found at 285 ± 38 nm from the active zone. Notethat the data in this figure represent further analysis of the data shown inFig. 3.
Mentions: To determine whether clathrin is required to resolve endosomes intosynaptic vesicles, control and clathrin knock-down cells were stimulated in thepresence of cationized ferritin (4 mM Ca2+, 34°C). In controlcultures 1 s after a single stimulus (Fig.3a,b), ferritin molecules were found in endosomes but not inclathrin-coated vesicles. At 3 s endosomes were budding and ferritin began toappear in clathrin-coated vesicles. The size of endosomes was larger thanendocytic vesicles, again suggesting that endocytic vesicles carrying ferritinfuse to form synaptic endosomes (Extended DataFig. 3b,c). Sixteen ferritin-positive endosomes were reconstructedfrom tomograms, none of them extended a tubule to the plasma membrane (Extended Data Fig. 6a). However, half ofthose endosomes were not fully contained within the 200 nm tomogram. Therefore,we reconstructed 11 complete synapses by assembling serial tomograms of synapses3 s after stimulation. None of the 17 complete end-to-end endosomes wereconnected to the plasma membrane (6 of which contained ferritin), suggestingthat they are true intracellular organelles, not extensions of the plasmamembrane (Supplementary Videos1-5). Thedecline in the number of endosomes is followed by an accumulation offerritin-positive coated vesicles and synaptic vesicles (Fig. 3b). Some clathrin-coated vesicles were observed before3 s after stimulation (1 s, 4/149 synaptic profiles and Fig. 1d), but they did not contain ferritin molecules,suggesting that they were derived from pre-existing endosomes. The total numberof ferritin granules per synaptic profile does not increase suggesting thatthere is not an additional wave of endocytic events that occurs during these 20s (Extended Data Fig. 6c). Some of theferritin-filled small vesicles docked to the active zone (Fig. 3a, right panel; Extended Data Fig. 6b,d), suggesting that these vesicles aresynaptic vesicles. These results indicate that recently endocytosed membranepasses through endosomes to regenerate synaptic vesicles within about 5-6 safter fusion.

Bottom Line: Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature.In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis.These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112-0840, USA.

ABSTRACT
Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 s, which in turn become small-diameter synaptic vesicles 5-6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis.

Show MeSH
Related in: MedlinePlus