Limits...
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.

Show MeSH

Related in: MedlinePlus

Synaptic vesicles are regenerated from endosomes at 37°C.Hippocampal synapses were stimulated once and frozen at the indicated times.The experiments were performed at 37°C in the presence of 4 mMexternal Ca2+. (a) Electron micrographs showing invaginations andlarge endocytic vesicles (arrowheads) recovered via ultrafast endocytosis.(b) Micrographs showing single coated buds (left, middle) or multiple buds(right) forming on an endosome. (c) Virtual section from an electrontomogram (left) and reconstruction (middle) showing a synaptic endosome withbuds following a single stimulus. The average intensity of coated buds fromthe top 20 nm (right, top) and the bottom 40 nm (right, bottom) is shown.Clathrin-cages can be preserved in our fixation and are visible in thetomogram. We found a total of 32 endosomes in these reconstructions (14endosomes in the unstimulated control and 28 endosomes 3 seconds afterstimulation). Of the total 32 endosomes, none were connected to the plasmamembrane or showed evidence of a truncated tubule extending from theendosomal membrane. Of the 14 unstimulated endosomes, 8 were containedwithin single tomograms, and are therefore unambiguously closed on bothends. Of the 28 endosomes in stimulated synapses, 16 endosomes werecontained within single tomograms so that it was clear that no attachment tothe plasma membrane was possible. (d) Examples of a coated pit on the plasmamembrane (top) and a coated bud on an endosome (bottom). Note that themorphology of the coats is similar. (e) Increase in the number of eachendocytic structure per synaptic profile after a single stimulus at37°C. The prevalence of large endocytic vesicles and endosomes isfollowed by an increase in the number of clathrin-coated vesicles. Coatedpits were not observed on the plasma membrane (PM). (f) Frequency ofprofiles or tomograms that contain endosomal structures at 37°C.Roughly, 60% of unstimulated synapses contained one endosome. One secondafter stimulation, 60% of the synapses contained at least one endosome, andhalf of those synapses contained two endosomes. Three seconds afterstimulation, ~30% of the synapses contained budded endosomes andclathrin-coated vesicles, suggesting that those synapses were active. Thestandard error of the mean is shown in each graph. For N values, detailednumbers and statistical analysis, seeSupplementary Table1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4291189&req=5

Figure 7: Synaptic vesicles are regenerated from endosomes at 37°C.Hippocampal synapses were stimulated once and frozen at the indicated times.The experiments were performed at 37°C in the presence of 4 mMexternal Ca2+. (a) Electron micrographs showing invaginations andlarge endocytic vesicles (arrowheads) recovered via ultrafast endocytosis.(b) Micrographs showing single coated buds (left, middle) or multiple buds(right) forming on an endosome. (c) Virtual section from an electrontomogram (left) and reconstruction (middle) showing a synaptic endosome withbuds following a single stimulus. The average intensity of coated buds fromthe top 20 nm (right, top) and the bottom 40 nm (right, bottom) is shown.Clathrin-cages can be preserved in our fixation and are visible in thetomogram. We found a total of 32 endosomes in these reconstructions (14endosomes in the unstimulated control and 28 endosomes 3 seconds afterstimulation). Of the total 32 endosomes, none were connected to the plasmamembrane or showed evidence of a truncated tubule extending from theendosomal membrane. Of the 14 unstimulated endosomes, 8 were containedwithin single tomograms, and are therefore unambiguously closed on bothends. Of the 28 endosomes in stimulated synapses, 16 endosomes werecontained within single tomograms so that it was clear that no attachment tothe plasma membrane was possible. (d) Examples of a coated pit on the plasmamembrane (top) and a coated bud on an endosome (bottom). Note that themorphology of the coats is similar. (e) Increase in the number of eachendocytic structure per synaptic profile after a single stimulus at37°C. The prevalence of large endocytic vesicles and endosomes isfollowed by an increase in the number of clathrin-coated vesicles. Coatedpits were not observed on the plasma membrane (PM). (f) Frequency ofprofiles or tomograms that contain endosomal structures at 37°C.Roughly, 60% of unstimulated synapses contained one endosome. One secondafter stimulation, 60% of the synapses contained at least one endosome, andhalf of those synapses contained two endosomes. Three seconds afterstimulation, ~30% of the synapses contained budded endosomes andclathrin-coated vesicles, suggesting that those synapses were active. Thestandard error of the mean is shown in each graph. For N values, detailednumbers and statistical analysis, seeSupplementary Table1.

Mentions: Clathrin polyhedral coats on endocytic membranes are distinctive inelectron micrographs (Fig. 1, Extended Data Fig 2). In our preparations,these coats are observed on pits on the cell body (Extended Data Fig. 2d) but rarely observed on the plasma membrane atsynapses. To test the requirement of clathrin after stimulation, a singlestimulus was applied to hippocampal cells expressing a variant ofchannelrhodopsin (ChetaTC)13.The experiments were performed in the presence of 4 mM Ca2+ in theexternal solution at 34°C (Fig. 1)and 37°C (Extended Data Fig. 2).After a single stimulus, endocytic pits that lack distinctive clathrin coatsformed 50-100 ms after fusion (Fig. 1a,left panel; Extended Data Fig. 2a, leftpanel)10. Theseinvaginations resolved into large vesicles about 80 nm in diameter at thelateral edges of the active zone (Fig. 1a;Extended Data Fig. 2a). The number oflarge endocytic vesicles adjacent to the plasma membrane peaked at 100 ms (Fig. 1d; Extended Data Fig. 2e). Thereafter, endosome-like structures beganaccumulating in the bouton and peaked at 1 s (Fig.1b,d; Extended Data Fig. 2b,e).These organelles are larger in diameter (116.4 ± 2.5 nm, 8 synapticvesicle equivalents) than the endocytic vesicles at the periphery (80.6 ±0.7 nm; 4 synaptic vesicle equivalents; ExtendedData Fig. 3a, see Methods), suggesting that thelarge endocytic vesicles fuse to form a synaptic endosome14. Tomographic reconstructionsof these endosomes demonstrated that they are not connected to the plasmamembrane (Extended Data Fig. 2c and Supplementary Video 1).Clathrin-like coats were visible on some of these endosomes (Fig. 1b,c; Extended Data Fig. 2b,c,d), and budded endosomes peaked 3 s afterstimulation (Fig. 1c,d; Extended Data Fig. 2b,c,e). The decline inendosomes was accompanied by an accumulation of clathrin-coated vesicles insidethe bouton at 3 s (Fig. 1d; Extended Data Fig. 2e, f). By contrast,clathrin-coated pits on the plasma membrane were only observed in 0.4% ofsynaptic profiles (4/907) between 1 to 10 s after stimulation (1s 2/332; 3s2/345; 10 s 0/330) (Fig. 1d; Extended Data Fig. 2e). These results suggestthat clathrin does not regenerate synaptic vesicles via endocytosis at theplasma membrane but rather by budding vesicles from an endosome.


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)

Synaptic vesicles are regenerated from endosomes at 37°C.Hippocampal synapses were stimulated once and frozen at the indicated times.The experiments were performed at 37°C in the presence of 4 mMexternal Ca2+. (a) Electron micrographs showing invaginations andlarge endocytic vesicles (arrowheads) recovered via ultrafast endocytosis.(b) Micrographs showing single coated buds (left, middle) or multiple buds(right) forming on an endosome. (c) Virtual section from an electrontomogram (left) and reconstruction (middle) showing a synaptic endosome withbuds following a single stimulus. The average intensity of coated buds fromthe top 20 nm (right, top) and the bottom 40 nm (right, bottom) is shown.Clathrin-cages can be preserved in our fixation and are visible in thetomogram. We found a total of 32 endosomes in these reconstructions (14endosomes in the unstimulated control and 28 endosomes 3 seconds afterstimulation). Of the total 32 endosomes, none were connected to the plasmamembrane or showed evidence of a truncated tubule extending from theendosomal membrane. Of the 14 unstimulated endosomes, 8 were containedwithin single tomograms, and are therefore unambiguously closed on bothends. Of the 28 endosomes in stimulated synapses, 16 endosomes werecontained within single tomograms so that it was clear that no attachment tothe plasma membrane was possible. (d) Examples of a coated pit on the plasmamembrane (top) and a coated bud on an endosome (bottom). Note that themorphology of the coats is similar. (e) Increase in the number of eachendocytic structure per synaptic profile after a single stimulus at37°C. The prevalence of large endocytic vesicles and endosomes isfollowed by an increase in the number of clathrin-coated vesicles. Coatedpits were not observed on the plasma membrane (PM). (f) Frequency ofprofiles or tomograms that contain endosomal structures at 37°C.Roughly, 60% of unstimulated synapses contained one endosome. One secondafter stimulation, 60% of the synapses contained at least one endosome, andhalf of those synapses contained two endosomes. Three seconds afterstimulation, ~30% of the synapses contained budded endosomes andclathrin-coated vesicles, suggesting that those synapses were active. Thestandard error of the mean is shown in each graph. For N values, detailednumbers and statistical analysis, seeSupplementary Table1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4291189&req=5

Figure 7: Synaptic vesicles are regenerated from endosomes at 37°C.Hippocampal synapses were stimulated once and frozen at the indicated times.The experiments were performed at 37°C in the presence of 4 mMexternal Ca2+. (a) Electron micrographs showing invaginations andlarge endocytic vesicles (arrowheads) recovered via ultrafast endocytosis.(b) Micrographs showing single coated buds (left, middle) or multiple buds(right) forming on an endosome. (c) Virtual section from an electrontomogram (left) and reconstruction (middle) showing a synaptic endosome withbuds following a single stimulus. The average intensity of coated buds fromthe top 20 nm (right, top) and the bottom 40 nm (right, bottom) is shown.Clathrin-cages can be preserved in our fixation and are visible in thetomogram. We found a total of 32 endosomes in these reconstructions (14endosomes in the unstimulated control and 28 endosomes 3 seconds afterstimulation). Of the total 32 endosomes, none were connected to the plasmamembrane or showed evidence of a truncated tubule extending from theendosomal membrane. Of the 14 unstimulated endosomes, 8 were containedwithin single tomograms, and are therefore unambiguously closed on bothends. Of the 28 endosomes in stimulated synapses, 16 endosomes werecontained within single tomograms so that it was clear that no attachment tothe plasma membrane was possible. (d) Examples of a coated pit on the plasmamembrane (top) and a coated bud on an endosome (bottom). Note that themorphology of the coats is similar. (e) Increase in the number of eachendocytic structure per synaptic profile after a single stimulus at37°C. The prevalence of large endocytic vesicles and endosomes isfollowed by an increase in the number of clathrin-coated vesicles. Coatedpits were not observed on the plasma membrane (PM). (f) Frequency ofprofiles or tomograms that contain endosomal structures at 37°C.Roughly, 60% of unstimulated synapses contained one endosome. One secondafter stimulation, 60% of the synapses contained at least one endosome, andhalf of those synapses contained two endosomes. Three seconds afterstimulation, ~30% of the synapses contained budded endosomes andclathrin-coated vesicles, suggesting that those synapses were active. Thestandard error of the mean is shown in each graph. For N values, detailednumbers and statistical analysis, seeSupplementary Table1.
Mentions: Clathrin polyhedral coats on endocytic membranes are distinctive inelectron micrographs (Fig. 1, Extended Data Fig 2). In our preparations,these coats are observed on pits on the cell body (Extended Data Fig. 2d) but rarely observed on the plasma membrane atsynapses. To test the requirement of clathrin after stimulation, a singlestimulus was applied to hippocampal cells expressing a variant ofchannelrhodopsin (ChetaTC)13.The experiments were performed in the presence of 4 mM Ca2+ in theexternal solution at 34°C (Fig. 1)and 37°C (Extended Data Fig. 2).After a single stimulus, endocytic pits that lack distinctive clathrin coatsformed 50-100 ms after fusion (Fig. 1a,left panel; Extended Data Fig. 2a, leftpanel)10. Theseinvaginations resolved into large vesicles about 80 nm in diameter at thelateral edges of the active zone (Fig. 1a;Extended Data Fig. 2a). The number oflarge endocytic vesicles adjacent to the plasma membrane peaked at 100 ms (Fig. 1d; Extended Data Fig. 2e). Thereafter, endosome-like structures beganaccumulating in the bouton and peaked at 1 s (Fig.1b,d; Extended Data Fig. 2b,e).These organelles are larger in diameter (116.4 ± 2.5 nm, 8 synapticvesicle equivalents) than the endocytic vesicles at the periphery (80.6 ±0.7 nm; 4 synaptic vesicle equivalents; ExtendedData Fig. 3a, see Methods), suggesting that thelarge endocytic vesicles fuse to form a synaptic endosome14. Tomographic reconstructionsof these endosomes demonstrated that they are not connected to the plasmamembrane (Extended Data Fig. 2c and Supplementary Video 1).Clathrin-like coats were visible on some of these endosomes (Fig. 1b,c; Extended Data Fig. 2b,c,d), and budded endosomes peaked 3 s afterstimulation (Fig. 1c,d; Extended Data Fig. 2b,c,e). The decline inendosomes was accompanied by an accumulation of clathrin-coated vesicles insidethe bouton at 3 s (Fig. 1d; Extended Data Fig. 2e, f). By contrast,clathrin-coated pits on the plasma membrane were only observed in 0.4% ofsynaptic profiles (4/907) between 1 to 10 s after stimulation (1s 2/332; 3s2/345; 10 s 0/330) (Fig. 1d; Extended Data Fig. 2e). These results suggestthat clathrin does not regenerate synaptic vesicles via endocytosis at theplasma membrane but rather by budding vesicles from an endosome.

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