Stonin 2 Is a Major Adaptor Protein for Clathrin-Mediated Synaptic Vesicle Retrieval

Abstract

At small synapses in the brain, clathrin-mediated endocytosis (CME) is the dominant mode of synaptic vesicle retrieval following weak stimulation [1Dittman J. Ryan T.A. Molecular circuitry of endocytosis at nerve terminals.Annu. Rev. Cell Dev. Biol. 2009; 25: 133-160Crossref PubMed Scopus (183) Google Scholar, 2Granseth B. Odermatt B. Royle S.J. Lagnado L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.Neuron. 2006; 51: 773-786Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 3Zhu Y. Xu J. Heinemann S.F. Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging.Neuron. 2009; 61: 397-411Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 4Royle S.J. Lagnado L. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules.Traffic. 2010; 11: 1489-1497Crossref PubMed Scopus (39) Google Scholar]. Clathrin cannot bind to membranes or cargo directly and instead uses adaptor proteins to do so [5Reider A. Wendland B. Endocytic adaptors—social networking at the plasma membrane.J. Cell Sci. 2011; 124: 1613-1622Crossref PubMed Scopus (84) Google Scholar]. Although the involvement of clathrin and dynamin in synaptic vesicle retrieval is clear, it is unknown which adaptor proteins are used to sort the essential components into the vesicle [1Dittman J. Ryan T.A. Molecular circuitry of endocytosis at nerve terminals.Annu. Rev. Cell Dev. Biol. 2009; 25: 133-160Crossref PubMed Scopus (183) Google Scholar, 4Royle S.J. Lagnado L. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules.Traffic. 2010; 11: 1489-1497Crossref PubMed Scopus (39) Google Scholar, 6Newton A.J. Kirchhausen T. Murthy V.N. Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis.Proc. Natl. Acad. Sci. USA. 2006; 103: 17955-17960Crossref PubMed Scopus (192) Google Scholar]. In nonneuronal cells, CME of the majority of transmembrane receptors is either directly or indirectly via the heterotetrameric AP-2 complex [5Reider A. Wendland B. Endocytic adaptors—social networking at the plasma membrane.J. Cell Sci. 2011; 124: 1613-1622Crossref PubMed Scopus (84) Google Scholar]. In neurons, RNAi of the μ2 subunit of AP-2 resulted in only minor inhibition of synaptic vesicle retrieval [7Kim S.H. Ryan T.A. A distributed set of interactions controls mu2 functionality in the role of AP-2 as a sorting adaptor in synaptic vesicle endocytosis.J. Biol. Chem. 2009; 284: 32803-32812Crossref PubMed Scopus (18) Google Scholar, 8Kim S.H. Ryan T.A. Synaptic vesicle recycling at CNS snapses without AP-2.J. Neurosci. 2009; 29: 3865-3874Crossref PubMed Scopus (97) Google Scholar], a result echoed in C. elegans [9Gu M. Schuske K. Watanabe S. Liu Q. Baum P. Garriga G. Jorgensen E.M. Mu2 adaptin facilitates but is not essential for synaptic vesicle recycling in Caenorhabditis elegans.J. Cell Biol. 2008; 183: 881-892Crossref PubMed Scopus (42) Google Scholar]. These results suggest that alternative adaptors may be employed for vesicle retrieval. Here, we tested which adaptors are required for vesicle retrieval at hippocampal synapses using a targeted RNAi screen coupled with optical measurements. Stonin 2 emerged as a major adaptor, whereas AP-2 played only a minor role in endocytosis at the synapse. Moreover, using chemically induced rerouting of stonin 2 to mitochondria it was possible to switch endocytically competent synapses to an impaired state on a timescale of minutes.