Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
Local recycling of synaptic vesicles is required to maintain neurotransmission via clathrin-mediated endocytosis and clathrin-independent ultrafast endocytosis. Clathrin also plays a role in ultrafast endocytosis to regenerate vesicles from a recycling endosome. Here the authors have further tested the role of clathrin and clathrin adaptors in synaptic vesicle endocytosis. This paper raises the interesting possibility that adaptor protein AP-2 but not clathrin contributes to the endocytosis of synaptic vesicle proteins. There are some concerns about differential knockdown of clathrin and AP-2 but if the authors can resolve these, this would be an important result.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.
Article activity feed
-
-
Evaluation Summary:
Local recycling of synaptic vesicles is required to maintain neurotransmission via clathrin-mediated endocytosis and clathrin-independent ultrafast endocytosis. Clathrin also plays a role in ultrafast endocytosis to regenerate vesicles from a recycling endosome. Here the authors have further tested the role of clathrin and clathrin adaptors in synaptic vesicle endocytosis. This paper raises the interesting possibility that adaptor protein AP-2 but not clathrin contributes to the endocytosis of synaptic vesicle proteins. There are some concerns about differential knockdown of clathrin and AP-2 but if the authors can resolve these, this would be an important result.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
Local recycling of synaptic vesicles is required to maintain neurotransmission via clathrin-mediated endocytosis and clathrin-independent ultrafast endocytosis. Clathrin also plays a role in ultrafast endocytosis to regenerate vesicles from a recycling endosome. Here the authors have further tested the role of clathrin and clathrin adaptors in synaptic vesicle endocytosis. This paper raises the interesting possibility that adaptor protein AP-2 but not clathrin contributes to the endocytosis of synaptic vesicle proteins. There are some concerns about differential knockdown of clathrin and AP-2 but if the authors can resolve these, this would be an important result.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
In this project, the Takamori and the Haucke lab joined forces to investigate the mechanisms of endocytosis at central synapses. Particularly, the authors aimed to resolve the role of clathrin and AP-2 for endocytosis. To this end, they systematically investigated the kinetics of endocytosis with pH sensitive fluorophores (pHluorins) coupled to six major synaptic vesicle proteins: Synaptotagmin 1 (Syt1), the synaptic vesicle glycoprotein 2A, VAMP/Synaptobrevin 2 (Syb2), Synaptophysin (Syp), the vesicular glutamate transporter 1 (VGLUT1), and the vesicular GABA transporter (VGAT). The function of clathrin and AP-2 were perturbed by knock-down experiments using lentivirus based expression of shRNA validated by immunohistochemistry and rescue experiments. In addition, pharmacological tools were tested (pitstop …
Reviewer #1 (Public Review):
In this project, the Takamori and the Haucke lab joined forces to investigate the mechanisms of endocytosis at central synapses. Particularly, the authors aimed to resolve the role of clathrin and AP-2 for endocytosis. To this end, they systematically investigated the kinetics of endocytosis with pH sensitive fluorophores (pHluorins) coupled to six major synaptic vesicle proteins: Synaptotagmin 1 (Syt1), the synaptic vesicle glycoprotein 2A, VAMP/Synaptobrevin 2 (Syb2), Synaptophysin (Syp), the vesicular glutamate transporter 1 (VGLUT1), and the vesicular GABA transporter (VGAT). The function of clathrin and AP-2 were perturbed by knock-down experiments using lentivirus based expression of shRNA validated by immunohistochemistry and rescue experiments. In addition, pharmacological tools were tested (pitstop 2). To control for overexpression of the pHluorin-linked synaptic proteins, antibodies directed against luminal domain of VGAT coupled to the pH-sensitive fluorophore cypHer 5E were used.
The data show that clathrin is dispensable for endocytosis of all six tested proteins when tested at near-physiological temperatures (33-37 degree celsius) but clathrin can have effects on endocytosis kinetics in experiments performed at room temperature. The data support recent evidence for clathrin-independent endocytosis and resolve discrepancies with previous studies performed at room temperature. The most interesting findings of the study is that AP-2 is required for retrieval of some of the tested proteins (VGAT and VGLUT1) and this function is independent of clathrin. The study therefore reveals a clathrin-independent function of AP-2 in the endocytic sorting of a subset of synaptic vesicle proteins at central synapses. The study is of high technical quality and carefully designed. The manuscript is carefully written and the conclusions are supported by the data. The results provide exciting insights into the mechanisms of endocytosis.
-
Reviewer #2 (Public Review):
Previous work has shown that clathrin and AP-2 operate to form synaptic vesicles from an endosomal intermediate that follows clathrin-independent endocytosis. The current study corroborates this finding and its importance at physiological rather than room temperature. However, this study goes one step further, to suggest a role for AP-2 but not clathrin in endocytosis of particular SV proteins, the neurotransmitter transporters. This raises the possibility of a novel mechanism by which AP-2, generally thought to depend on clathrin, can promote endocytosis independent of clathrin.
-
Reviewer #3 (Public Review):
Here the authors have further tested the role of clathrin and clathrin adaptors in synaptic vesicle endocytosis. What is known is that endocytosis of membrane is fast and appears to be clathrin independent at synapses, at least during low frequency stimulation. Oddly, the clathrin adaptor complex AP-2 plays some role at synapses, potentially cargo recruitment to endocytic sites, or assembly of a clathrin coat to execute endocytosis. What was not known was whether synaptic vesicle proteins are retrieved by a clathrin-independent mechanism, and whether the roles for AP-2 are separable: specifically, does AP-2 recruit cargo for ultrafast endocytosis?
The authors claim:
1. Knockdown of clathrin heavy chain or application of the clathrin inhibitor Pitstop2 do not change re-acidification time constants of six …
Reviewer #3 (Public Review):
Here the authors have further tested the role of clathrin and clathrin adaptors in synaptic vesicle endocytosis. What is known is that endocytosis of membrane is fast and appears to be clathrin independent at synapses, at least during low frequency stimulation. Oddly, the clathrin adaptor complex AP-2 plays some role at synapses, potentially cargo recruitment to endocytic sites, or assembly of a clathrin coat to execute endocytosis. What was not known was whether synaptic vesicle proteins are retrieved by a clathrin-independent mechanism, and whether the roles for AP-2 are separable: specifically, does AP-2 recruit cargo for ultrafast endocytosis?
The authors claim:
1. Knockdown of clathrin heavy chain or application of the clathrin inhibitor Pitstop2 do not change re-acidification time constants of six different synaptic vesicle proteins at physiological temperature (200 AP at 40 Hz). The clathrin-independent mechanism is apparently universal for synaptic vesicle proteins and not exhausted by intense stimulation.
2. Knockdown of the AP-2 mu subunit slows down the re-acidification of the vesicular transporters (VGLUT1 and VGAT), synaptophysin, and synaptobrevin, but has no effect on synaptotagmin or SV2 (200 AP at 40 Hz). There is a differential requirement by synaptic vesicle proteins for AP-2 function during endocytosis.
3. The requirement for AP-2 mu is not simply due to fatigue caused by intense stimulation, but is required under less intense stimulations to retrieve proteins from the readily releasable pool of synaptic vesicles (50 AP at 20 Hz).
4. The requirement for AP-2u is not caused by overexpression of tagged proteins, demonstrated by using fluorescently tagged antibodies and an acid-dependent dye.
5. The transporters are stranded on the surface in the AP-2 mu knockdown as determined by biotin labeling of membrane proteins, and by immunofluorescence.
6. Mutations of VGLUT1 and VGAT AP-2-binding sites slow recycling and acidification.
There are two major conclusions from these data:
1- Recycling of synaptic vesicle proteins is fast and clathrin-independent at physiological temperatures. These results complement previous morphological and electrophysiological studies tracking ultrafast membrane endocytosis at the synapse, and fundamentally change our understanding of protein recycling at the synapse.
2- Despite the absence of a role for clathrin, the clathrin adaptor AP-2 is required for retrieval of some proteins. The role for AP-2 is not to assemble clathrin or some other membrane-bending protein to execute membrane invagination, because endocytosis of synaptotagmin and SV2 is independent of AP-2. Rather, AP-2 is likely to be recruiting proteins to the endocytic site.
These conclusions will be of great interest to both the neuroscience community and broader cell biology community. The data are largely convincing, but because they are at odds with the accepted conclusions about both clathrin and AP-2, the results will be disputed.
-
