Studies of membrane-binding proteins involved in synaptic vesicle recycling

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From the DEPARTMENT OF NEUROSCIENCE Karolinska Institutet, Stockholm, Sweden

STUDIES OF MEMBRANE-BINDING PROTEINS INVOLVED IN SYNAPTIC VESICLE RECYCLING

Fredrik Andersson

Stockholm 2009

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All previously published papers were reproduced with permission from the publishers.

Published by Karolinska Institutet Printed by E-PRINT AB

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ABSTRACT

Signaling between neurons occurs at synapses. A presynaptic nerve terminal releases neurotransmitter, which diffuses over the synaptic cleft, and binds to postsynaptic receptors. Release occurs by fusion of neurotransmitter-containing vesicles with the plasma membrane. To support sustained transmitter release the synaptic vesicles are recycled locally at the nerve terminal. After the synaptic vesicle has collapsed into the plasma membrane synaptic vesicles can be retrieved by two different mechanisms, clathrin-mediated endocytosis and bulk endocytosis. Both recycling pathways depend on interplay between many proteins, several of which bind to and can rearrange lipid membranes.

The aim with this thesis is to investigate the role of three different proteins;

epsin, endophilin and syndapin, each containing a lipid binding domain known to induce membrane curvature in vitro, in synaptic vesicle recycling at a living synapse.

The model system used is synapses located in the giant reticulospinal axons in lamprey that permits acute perturbation of protein-protein and protein-lipid interactions by micro-injection of reagents. These synapses have an organization that facilitates quantitative morphological analysis.

Perturbation of each of the three different proteins caused a stimulus-dependent decrease in the number of synaptic vesicles in the synaptic vesicle cluster suggesting an involvement in synaptic vesicle recycling. Epsin appears to be involved in recruitment or assembly of the clathrin coat since perturbation of the membrane-binding ENTH domain of epsin resulted in a decrease of coated intermediates. In further support of this possibility, coated intermediates with distorted structure was observed after perturbation of the clathrin and AP2 binding region of epsin.

Perturbation of the membrane-binding BAR domain of endophilin with IgG and Fab fragments resulted in accumulation of shallow coated pits at the plasma membrane.

Injection of the BAR domain also resulted in accumulation of shallow coated pits.

However, similar coated pits occurred after injection of an F-BAR domain with different curvature indicating that the curvature-inducing properties of the BAR domain is not directly involved in the progression of coated pit invagination. Therefore the accumulation of shallow coated pits is probably linked to the binding partners of endophilin.

Clathrin-mediated endocytosis did not show any detectable change in synapses at which syndapin had been perturbed. Instead there was an accumulation of membranous cisternae containing synaptic vesicle membrane and with associated coated pits, indicating an involvement of syndapin in bulk endocytosis and/or stabilization of the plasma membrane at high rates of synaptic activity.

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LIST OF PUBLICATIONS

I. Jakobsson J, Gad H, Andersson F, Löw P, Shupliakov O, Brodin L. (2008) Role of epsin 1 in synaptic vesicle endocytosis. Proc Natl Acad Sci U S A.

29;105(17):6445-50.

II. Andersson F, Gad H, Löw P, Brodin L. The BAR domain of endophilin is essential for the progression of synaptic vesicle endocytosis. Manuscript III. Andersson F, Jakobsson J, Löw P, Shupliakov O, Brodin L. (2008)

Perturbation of syndapin/PACSIN impairs synaptic vesicle recycling evoked by intense stimulation. J Neurosci. 9;28(15):3925-33.

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LIST OF ABBREVIATIONS

AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate AP adaptor protein or assembly protein

Arp 2/3 actin-related protein 2/3

BAR Bin/Amphiphysin/RVS

cdk5 cyclin-dependent kinase 5 CIP4 cdc42-interacting protein 4 CK2 casein kinase 2

CLAP clathrin/AP2 binding region DPW aspartate-proline-tryptophan EGF epidermal growth factor

EH Eps15 homology

ENTH Epsin N-terminal homology

Eps15 Epidermal growth factor receptor pathway substrate 15 EPSP excitatory postsynaptic potential

F-BAR FCH-BAR

FBP17 formin binding protein

FCH FER-CIP4 homology

FER Fes related protein GED GTPase effector domain hsp70 heat shock protein 70 LDL low-density lipoprotein N-BAR N-terminal BAR

NMDA N-methyl-D-aspartic acid NPF aspargine-proline-phenylanaline

N-WASP neuronal Wiskott-Aldrich syndrome protein

PH pleckstrin homology

PIP phosphatidylinositol phosphate PKC protein kinase C

PRD proline-rich domain

SAC1 suppressor of actin mutations 1

SH3 Src homology 3

SNARE soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors

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VAMP vesicle associated membrane protein

WH WASP-Homology

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INTRODUCTION

THE SYNAPSE AND SYNAPTIC VESICLE TURNOVER

Signalling between neurons occurs via chemical synapses at which neurotransmitter is released from the presynaptic compartment to act at receptors at the postsynaptic compartment. Neurotransmitter is stored in synaptic vesicles which fuse with the plasma membrane at active zones in response to Ca²⁺ entry. Fusion of vesicles is mediated by SNARE proteins and synaptotagmin acts as Ca²⁺ sensor (Wojcik and Brose, 2007). To ensure a reliable synaptic signalling an efficient supply of synaptic vesicles is essential, which can only be accomplished if synaptic vesicles recycle locally at the presynaptic terminal. Recycling of synaptic vesicles has been suggested to occur by different mechanisms (Figure 1). Clathrin mediated endocytosis is by far the most studied and well-characterized pathway. In this pathway clathrin coats assemble at membrane patches and participates in the formation of a synaptic vesicle (Jung and Haucke, 2007). Another pathway, bulk endocytosis, involves invagination and retrieval of large membrane portions. Further processing into synaptic vesicles is likely to be mediated by the clathrin machinery since clathrin coated pits occur on these membrane structures (Clayton et al., 2007). Bulk endocytosis and clathrin mediated endocytosis primarily take place at the periactive zone, adjacent to the active zone.

Finally, it is proposed that release of transmitter can occur without full collapse of the synaptic vesicle into the plasma membrane. This mechanism is called “kiss and run”

and its importance for synaptic transmission is much debated.

Figure 1. Illustration of different pathways suggested to retrieve synaptic vesicles. A. Clathrin mediated endocytosis from the plasma membrane. B. Retrieval of large membranous structures.

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CLATHRIN MEDIATED ENDOCYTOSIS

Clathrin mediated synaptic vesicle endocytosis involves several distinct steps in which different protein-protein and protein-lipid interactions have been implicated (Figure 2).

The process starts with assembly of adaptors and clathrin at the plasma membrane. The second step involves invagination and generation of a clathrin coated pit with an initial shallow curvature. The curvature will continue to increase until the coated pit has obtained the shape of a bucket. The base is then constricted such that a deeply invaginated clathrin coated pit with narrow neck has formed. Fission of the neck will result in a free coated vesicle. The coated vesicle must shed its coat before it can be used in a new round of release.

Figure 2. Illustration of the formation of a clathrin coated pit. Assembly of the clathrin coat occurs along with invagination of the clathrin coated pit. After fission the coated bud is released from the plasma membrane.

Clathrin and adaptor proteins

Clathrin forms as a cage-like lattice consisting of pentagons and hexagons (Pearse, 1976).The hexagon and pentagons consist of heavy chains (180 kDa) and light chains (33-36 kDa), which assemble in the form of triskelions (Kirchhausen and Harrison, 1981; Ungewickell and Branton, 1981).

Clathrin is associated with the membrane via adaptors, such as AP2 (Vigers et al., 1986; Robinson, 1989). The neuronal form of AP2 consists of two larger subunits, α and β2, one medium subunit, µ2, and one small subunit, δ2 (reviewed in (Robinson, 2004)). A hinge domain in the β2 subunit and the α subunit mediates the binding to clathrin (Goodman and Keen, 1995; Shih et al., 1995). The binding to clathrin is phosphorylation dependent; phosphorylation of AP2 decreases binding to clathrin (Wilde and Brodsky, 1996).

AP2 binds to PI(4,5)P2 (Di Paolo et al., 2004) in the plasma membrane (Beck and Keen, 1991; Jost et al., 1998) via its α subunit as shown by mutation studies and structural studies (Gaidarov and Keen, 1999; Rohde et al., 2002). Structural studies also

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suggest an additional PIP2 interacting site on the µ2 subunit (Collins et al., 2002).

Antibodies against the α subunit of AP2 label presynaptic terminals in Drosophila melanogaster (Gonzalez-Gaitan and Jäckle, 1997). Terminals lacking the α subunit show depletion of synaptic vesicles and a decrease of coated pits at the plasma membrane (Gonzalez-Gaitan and Jäckle, 1997), consistent with a deficiency in coated pit formation.

After mutation of the α subunit in C. elegans, clathrin is mislocalized and the number of coated intermediates at synapses is reduced, however a complete block in endocytosis was not observed (Gu et al., 2008). Knock down of the µ2 subunit of AP2 in HeLa cell cultures reduced receptor-mediated endocytosis of transferrin and decreased the amount of coated structures. Uptake of LDL receptor and EGF receptor was, however, unaffected (Motley et al., 2003), indicating that alternative adaptors exist. An alternative adaptor for AP2 at the synapse is AP180. AP180 is enriched at synaptic release sites (Sousa et al., 1992). In vitro experiments have shown that AP180 binds to PIP2 and is important for membrane assembly of clathrin (Ford et al., 2001).

Mutation of AP180 in Drosophila and C. elegans results in a decrease in the number of synaptic vesicles. The remaining vesicles were enlarged, suggesting a role for AP180 in determining the size of synaptic vesicles (Zhang et al., 1998; Nonet et al., 1999). Acute perturbation studies in the giant presynaptic terminal in squid showed similar results (Morgan et al., 1999). Other proteins suggested to act as adaptors for clathrin are amphiphysin and epsin, both of which contain membrane-binding domains as well as clathrin-binding motifs (see below).

Dynamin

Dynamin is a large GTPase of 100 kDa, which besides a GTPase domain and a GTPase effector domain (GED) (reviewed in (Praefcke and McMahon, 2004) contains a pleckstrin homology (PH) domain that mediates binding to membranes (Ferguson et al., 1994; Zheng et al., 1996). The C-terminal consists of a proline-rich domain (PRD). The PRD mediates binding to several SH3 domain-containing proteins, including syndapin, endophilin and amphiphysin (Simpson et al., 1999). Microinjection of the SH3 domain of amphiphysin in lamprey (Shupliakov et al., 1997) and overexpression of this SH3 domain in COS7 cells (Wigge et al., 1997) inhibit clathrin mediated endocytosis at a late stage. Interactions with the SH3 domains of intersectin and endophilin have also

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been implicated at a step preceding fission (Simpson et al., 1999; Gad et al., 2000;

Evergren et al., 2007).

The temperature-sensitive Drosophila mutant Shibire carries a mutation in the dynamin gene (Shpetner and Vallee, 1989; van der Bliek and Meyerowitz, 1991). When mutants are maintained at the restrictive temperature, nerve terminals show depletion of synaptic vesicles along with accumulation of deeply invaginated membrane pits with electron dense collars surrounding the neck (Koenig and Ikeda, 1989). Overexpression in HeLa cells of mutant dynamin deficient in GTP binding, inhibits clathrin mediated endocytosis with failure of coated vesicles to bud off into cells (Damke et al., 1994).

Incubation of synaptic membranes with cytosol and the non-hydrolysable GTP analog GTPγS resulted in coated pits with abnormally long necks surrounded by dynamin- containing spirals (Takei et al., 1995). Dynamin knockout mice die soon after birth and a subset of synapses are filled with a membranous network containing coated pits seemingly unable to undergo fission (Ferguson et al., 2007; Hayashi et al., 2008).

Although dynamin is widely assumed to function as a mechanoenzyme its precise role in membrane fission remains unclear (Sweitzer and Hinshaw, 1998; Sever et al., 1999;

Stowell et al., 1999; Roux et al., 2006). It was recently suggested that dynamin is important for stabilizing a narrow membranous neck, which, when the dynamin spiral is disassembled, is destabilized causing fission to occur (Pucadyil and Schmid, 2008).

Synaptojanin

After the clathrin coated vesicle has been internalized the clathrin coat is disassembled.

One important factor in this process is synaptojanin, an endophilin-binding protein with two catalytic domains, a 5-phosphatase domain and a SAC1 homology domain (McPherson et al., 1996). Synaptojanin dephosphorylates PIPs at the 4 and 5 positions and thereby reduces the binding affinity for endocytic proteins (see below). The coat- associated protein auxilin acting together with the chaperone hsp70 also participate in the uncoating reaction (Ungewickell et al., 1995; Morgan et al., 2001).

Synaptojanin knockout mice show impaired metabolism of phosphatidylinositols with increased PIP2 levels in the brain and an increased binding of coat proteins to membranes (Cremona et al., 1999). In nerve terminals of these mice free coated

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vesicles are present, an intermediate rarely seen at synapses, and a dense actin-like cytomatrix surround the synaptic vesicles (Kim et al., 2002). Electrophysiological analysis revealed that synapses showed enhanced synaptic depression after prolonged stimulation. A reduction in the size of the recycling synaptic vesicle pool was also detected in FM dye experiments (Kim et al., 2002). Acute perturbation studies in the lamprey reticulospinal axon using an antibody against the PRD of synaptojanin results in accumulation of actin rich matrix (see Bloom et al., 2003) and free coated vesicles (Gad et al., 2000). Inhibition of interactions with the SH3 domain of endophilin using a peptide (see below) also results in appearance of free coated vesicles (Gad et al., 2000).

Mutation of the C. elegans gene encoding synaptojanin showed similar results, and in addition coated pits (Harris et al., 2000).

In COS7 cells a long isoform of synaptojanin containing binding motifs to clathrin and AP2 is recruited along with clathrin early in the endocytic process. The short isoform, lacking the clathrin and AP2 binding motifs, is recruited at the same time as endophilin and dynamin late in the endocytic process (Perera et al., 2006). In cortical neurons in culture both phosphatase domains of synaptojanin have been shown to be of importance for endocytosis. The PRD interaction with endophilin is also critical for endocytosis but only during prolonged stimulation suggesting that synaptojanin can be recruited to endocytic sites by other factors then endophilin during low levels of activity (Mani et al., 2007).

The accumulation of actin observed at synapses after perturbation of synaptojanin is consistent with the binding of actin regulating proteins to PIP2 (for review see Takenawa and Itoh, 2001). One example is N-WASP that binds to PIP2 via a WH1 domain (Miki et al., 1996).

Epsin

In a screening for binding partners for the EH domain of the clathrin-coat associated protein Eps15 a protein of 94 kD was found (Chen et al., 1998). The protein was referred to as epsin for Eps15 interacting protein. Cloning of rat epsin showed that it contains C-terminal aspargine-proline-phenylanaline (NPF) motifs which interact with the EH domain of Eps15. In the central part of the protein several aspartate-proline- tryptophan (DPW) motif are located which bind to the α-appendage domain of AP2

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(Wang et al., 1995). Injection of IgG and overexpression of a GST-DPW construct of epsin was shown to inhibit endocytosis in cell cultures (Chen et al., 1998).

The N-terminal part of epsin, named N-terminal Epsin homology (ENTH) domain is the most conserved part of the protein (Chen et al., 1998). The structure of epsin has been determined which revealed that the ENTH domain consists of 4 helices connected with loops and an N-terminal unstructured region which becomes ordered upon membrane binding and fold into a helix oriented in a way so that it can interact with the plasma membrane (Itoh et al., 2001; Ford et al., 2002). The ENTH domain of epsin binds liposomes containing phosphoinositides with the highest affinity for PI(4,5)P2

(Itoh et al., 2001).The ENTH domain of epsin is also known to convert liposomes into narrow tubular structures (Ford et al., 2002), and epsin can recruit clathrin to lipid monolayers and induce invagination (Ford et al., 2002). Overexpression of mutant ENTH domain in cells is sufficient to inhibit endocytosis of the EGF receptor (Itoh et al., 2001).

Epsin also contains ubiquitin interacting motifs and distinct sites which can be mono- ubiquitinated (Polo et al., 2002). The function of mono-ubiqutination is still not known however epsin has been shown to be deubiquitinated in a Ca²⁺ dependent manner (Chen et al., 2003). Ubiquitination of epsin has been suggested to decrease the binding between epsin and adaptor proteins (Chen and De Camilli, 2005).

Endophilin and other BAR domain proteins

The BAR (Bin/Amphiphysin/RVS) domain is a protein module which is conserved from yeast to mammals. The first BAR domain to be crystallized was that of Drosophila amphiphysin which revealed that it forms a banana-shaped dimer (Peter et al., 2004). The concave surface of the dimer has a positively charged surface that mediates binding to membranes. Amphiphysin is also known to tubulate liposomes (Takei et al., 1999; Peter et al., 2004). The very N-terminal part of the dimer is disordered but form an amphiphatic helix upon binding to lipids. The predicted curvature diameter of the amphiphysin BAR dimer is 22 nm (Peter et al., 2004).

BAR domains are found in a variety of proteins (Peter et al., 2004), including endophilin. Like in amphipysin, the BAR domain in endophilin forms dimers and can

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tubulate liposomes (Farsad et al., 2001; Gallop et al., 2006). The structure of the BAR domain in endophilin resembles that in amphiphysin, including the presence of a N- terminal domain that gets ordered upon lipid binding. However, the BAR dimer of endophilin contains an extra loop in the concave surface (Weissenhorn, 2005; Gallop et al., 2006). Removal of the loop reduces the BAR domains ability to tubulate membrane, possible due to decreased tendency to dimerize (Gallop et al., 2006).

Endophilin was first identified in a screening for SH3-containing protein (Sparks et al., 1996). Endophilin 1, the brain enriched isoform, have a size of 40 kD and interacts via an SH3 domain with dynamin and synaptojanin (Ringstad et al., 1997). Endophilin has been studied in several model systems and it has been proposed to participate in different stages of clathrin mediated synaptic vesicle endocytosis. Studies in the lamprey reticulospinal axon suggest that endophilin is important for early invagination since injection of antibodies against the SH3 domain of endophilin resulted in accumulation of shallow coated pits (Ringstad et al., 1999). In the same study, in vitro experiments were conducted in which endophilin was shown to localize in GTP γ-S induced dynamin spirals, suggesting a role at late endocytic stages together with dynamin. This possibility is also supported by the accumulation of late stage coated pits after injection of a peptide specifically blocking the interaction with the SH3 domain of endophilin (Gad et al., 2000). In Drosophila neuromuscular junction endophilin mutants results in accumulation of coated pits at an early stage (Guichet et al., 2002;

Verstreken et al., 2002).

The major binding partner for endophilin is the phosphatidyl inositol phosphatase synaptojanin (Micheva et al., 1997) (see above). The effects of perturbing endophilin and synaptojanin (see above) are in many ways similar. Drosophila photoreceptor terminals in endophilin null mutant terminals showed electron dense vesicles resembling free coated vesicles (Fabian-Fine et al., 2003). Similar changes were also seen in Drosophila and C. elegans synaptojanin mutants, suggesting that the two proteins act in the same pathway. Moreover endophilin appears to be important to localize synaptojanin at nerve terminals in Drosophila (Verstreken et al., 2003) and C.

elegans (Schuske et al., 2003) (see also Dickman et al., 2005).

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Syndapin and other F-BAR domain proteins

Following the crystallization of BAR domains, the N-terminal of proteins of the FER- CIP4 homology (FCH) domain family was proposed to have a similar structure (Peter et al., 2004). This suggestion was confirmed by crystallization of the FCH-BAR (F- BAR) domain of formin binding protein 17 (FBP 17) which like the BAR domain was shown to form banana-shaped dimers with a concave surface (Shimada et al., 2007).

The curvature diameter of the dimer was larger than that reported for BAR domains.

The F-BAR dimer of FBP 17 was also shown to bind another dimer in a tip to tip manner and also via lateral interactions (Shimada et al., 2007; Frost et al., 2008).

Unlike the N-BAR domain (Peter et al., 2004) and the ENTH domain (Ford et al., 2002), the F-BAR domain lacks amphiphatic helix that can be inserted into the membrane and drive curvature formation (Frost et al., 2008). The F-BAR domain has been suggested to induce curvature by coordinated tilting of connected sheets of F- BAR domains (Frost et al., 2008).

Syndapin/PACSIN was first discovered as an SH3 domain containing brain enriched protein phosphorylated by casein kinase 2 (CK2) and protein kinase C (PKC) (therefore the name PACSIN for PKC and CK2 substrate in neurons) (Plomann et al., 1998).

Further studies revealed that the SH3 domain interacts with dynamin (therefore the name syndapin for synaptic dynamin interacting protein) (Qualmann et al., 1999). In GST pulldown assays the SH3 domain was also shown to bind synaptojanin, N-WASP and synapsin. Immunocytochemical labelling showed that syndapin is colocalized with dynamin and synaptophysin in cultured neurons (Qualmann et al., 1999), although it was found to co-localize poorly with clathrin (Modregger et al., 2002). Syndapin has also been shown to regulate actin polymerization and, overexpression of syndapin results in formation of filopodia in cultured cells. The induction of actin polymerization is linked to N-WASP and Arp 2/3 (for review see Miki and Takenawa, 2003) since it can be rescued by co-overexpression of the carboxy terminal of N-WASP (Qualmann and Kelly, 2000). Syndapin and N-WASP have been shown to co-localize in growth cones in primary hippocampal neuron cultures (Kessels and Qualmann, 2004).

Syndapin has also been shown to regulate actin via its interaction with Cordon-Blue (cobl), an actin nucleating factor found in neurons (Ahuja et al., 2007). Syndapin and PACSIN can thus mediate actin polymerisation in two different ways, by the interaction with the N-WASP-Arp 2/3 actin-regulating pathway or by Cobl. Oligomer

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formation of syndapin was shown to be mediated by the F-BAR domain (Kessels and Qualmann, 2006; Henne et al., 2007) indicating a similar mechanism for membrane tubulation as other F-BAR containing proteins (Shimada et al., 2007; Frost et al., 2008).

Syndapin has been suggested to participate in clathrin-mediated endocytosis. In HeLa cells overexpression of the SH3 domains of syndapin I and II inhibits transferrin receptor uptake (Simpson et al., 1999; Qualmann and Kelly, 2000). Accumulation of clathrin coated pits has also been observed in primary rabbit lacrimal acini after overexpression of syndapin II (Da Costa et al., 2003). Finally in neurons syndapin has been linked to uptake of the NMDA receptor in postsynaptic dendrites (Perez-Otano et al., 2006). Recently, syndapin was implicated in synaptic vesicle endocytosis by the finding that calcium-induced dephosphorylation of dynamin enhances the interaction between syndapin and dynamin (Anggono et al., 2006). It was further suggested that this interaction is used in bulk endocytosis during intense synaptic activity (Clayton et al., 2007).

BULK ENDOCYTOSIS

Bulk endocytosis is a form of endocytosis by which large pieces of synaptic vesicle membrane can be retrieved from the plasma membrane. Such endocytosis is generally activated during more intense levels of stimulations (Heuser and Reese, 1973; Leenders et al., 2002; de Lange et al., 2003; Teng et al., 2007; Clayton et al., 2008). It involves invagination of membrane which form large endosome-like structures, “cisternae”, from which synaptic vesicles form by clathrin-mediated budding. Kinetic studies using both capacitance measurements and measurement of dye uptake have shown that bulk endocytosis can occur rapidly (Wu and Wu, 2007; Clayton et al., 2008). However, vesicle budding may be slower and cisternae can remain for minutes after stimulation (Heuser and Reese, 1973). The molecular components of bulk endocytosis have not been well characterized. Involvement of dynamin has been suggested by accumulation of endosome-like structures after mutation or knockout of dynamin genes (Koenig and Ikeda, 1996; Ferguson et al., 2007). Studies in both goldfish bipolar terminals (Holt et al., 2003) and the frog neuromuscular junction indicate an important role of actin in bulk endocytosis. In the latter model perturbation of actin causes a reduction in the amount of synaptic vesicles and induces accumulation of cisternae (Richards et al., 2004). Rolling blackout is another protein suggested to participate in bulk endocytosis.

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Mutation of Rolling blackout in Drosophila results in an inhibition of endosome formation, without apperent effects on clathrin mediated endocytosis (Vijayakrishnan et al., 2009). Cyclin-dependent kinase 5 (cdk5), a kinase known to rephosphorylate, among other proteins dynamin, has also been linked with bulk retrieval of synaptic vesicle membrane (Evans and Cousin, 2007).

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AIMS

The aim of this thesis is to investigate mechanisms in synaptic vesicle recycling at a living synapse, the giant reticulospinal synapse in the lamprey. Three different proteins are in focus; epsin, endophilin and syndapin. These proteins all contain lipid–binding domains; ENTH domain, BAR domain and F-BAR domain, respectively. Each of these different proteins induces tubular structures of membranes both in vitro and after overexpression in cell cultures.

Here the specific aims are:

To investigate the role of epsin in synaptic vesicle recycling.

To investigate the role of the BAR domain of endophilin in synaptic vesicle recycling.

To investigate the role of syndapin in synaptic vesicle recycling.

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METHODS

A detailed description of the different methods used in this thesis is provided in each paper. Some aspects of the methodology are discussed below.

THE LAMPREY RETICULOSPINAL MODEL SYSTEM

The giant reticulospinal axons in the river lamprey (Lampetra fluviatilis) can be up to 100 µm in diameter and run unbranched along the spinal cord. The soma of the large cells are located in the brainstem. Along the axon, glutamatergic synapses form with motoneurons and interneurons (Figure 3). Many of the synapses also contain gap junctions (Rovainen, 1979). The synapses exhibit a distinct synaptic vesicle cluster which contains 4000–12000 synaptic vesicles (Brodin and Shupliakov, 2006). Synaptic vesicle recycling occurs around the synaptic vesicle cluster in the periactive zone (Gad et al., 1998).

Figure 3. Synapse located in a reticulospinal axon. Electron micrograph of a synapse stimulated at 5 Hz for 30 min. The stimulation period was ended with fixation. Arrows indicate coated pits, ax=

axoplasmic matrix, d=dendrite, sv=synaptic vesicles (scalebar=200 nm).

Ultrastructural analysis has shown that exocytosis and endocytosis are in balance at 5 Hz stimulation and there is no evident depletion of synaptic vesicle in the synaptic vesicle cluster at this rate. Prolonged intense stimulation, e.g. 20 Hz for 30 min, leads to a decrease in the number of synaptic vesicles, which recovers upon rest (Wickelgren et al., 1985). In the living animal the reticulospinal axons are typically active at rates

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around 5 Hz but occasional brief bursts at rates up to 20 Hz have been reported (Zelenin, 2005).

The reticulospinal axon permits microinjection of peptides, protein domains and antibodies (Pieribone et al., 1995). Binding of microinjected fluorescent compounds at identified release site can be visualized by co-microinjection with antibodies to a synaptic vesicle marker (e.g. VAMP or synaptotagmin). Depending on the compound microinjections can be performed using either pressure pulses or current.

In paper II the amount of injected BAR domain was estimated. The fluorescent BAR domain could be reliably injected with a fixed negative current making the injection repeatable by using the same current. The fluorescence was evenly distributed in the axon. Its intensity was measured using a confocal microscope. For comparison the fluorescence intensity in microinjection electrodes filled with known concentrations of fluorescent BAR domain were measured.

In paper III the aim of one set of experiments was to study endocytosis separated from exocytosis. Here 20 Hz stimulation was applied for 30 min. The temperature was then rapidly lowered from 8º C (normal condition) to 1º C to inhibit endocytosis and thus trap synaptic vesicle membrane in the plasma membrane. At this point microinjection of IgG (active or control) was performed. When the temperature was raised to 8º C the synaptic vesicle clusters in axons injected with control IgG recovered to their normal size. This procedure thus permits analysis of the effect of protein perturbation on endocytosis temporally isolated from exocytosis.

Visualization of actin in the lamprey reticulospinal axon can be performed by microinjection of fluorescence-labeled actin monomers (Bourne et al., 2006). In paper III the effect of stimulation on fluorescent actin at synapses in syndapin antibody- injected axons was investigated. Here actin was co-injected together with either syndapin IgG or control IgG in the reticulospinal axon. The fluorescence intensity of actin at spots corresponding to synapses was measured in a confocal microscope before and after a stimulation period (5 Hz for 30 min). To avoid fading, scanning was not performed during the stimulation period.

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Many of the proteins involved in membrane trafficking are fairly well conserved between lamprey and mammals. To generate specific antibodies with high affinity for perturbation studies cloning of the lamprey ortholouge is, however, in most cases necessary. Cloning was performed with PCR using degenerated primers corresponding to conserved regions. An important aspect of the cloning procedure is to obtain a high quality cDNA (complementary DNA) from lamprey CNS to use as template. In general cloning in lamprey is often more difficult than in mammals due to a higher CG content in the lamprey genome compared to mammalian genomes (Löw, Jakobsson, Andersson unpublished observation). A genome sequencing project of the sea lamprey (Petromyzon marinus) has been started, and the high degree of sequence similarity with proteins cloned in the river lamprey has greatly facilitated the design of primers for PCR.

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RESULTS AND DISCUSSION

EPSIN IN SYNAPTIC VESICLE RECYCLING (PAPER I)

In paper I the role of epsin in synaptic vesicle recycling was investigated. A lamprey orthologue of epsin was cloned to generate specific reagents. The ortologue found in lamprey showed about 50% amino acid similarity to mammalian epsin 1, the neuronally expressed isoform. Lamprey epsin contains a highly conserved ENTH domain (about 80% similarity) and a region that binds clathrin and AP2 (CLAP region). The ENTH domain was expressed in bacteria and antibodies were generated in rabbit. The antibodies recognized a single band of 75 kDa on Western blot.

Fluorescence-labeled antibodies accumulated in spots following microinjection into the reticulospinal axon. These spots were identified as synaptic release sites (see below).

The IgG raised against the ENTH domain efficiently inhibited tubulation of lipids induced by the ENTH domain in vitro, suggesting that they inhibit the binding between epsin and membranes. Ultrastructural analysis of synapses located in microinjected axons subjected to repetitive stimulations showed that the number of synaptic vesicles in the synaptic vesicle cluster was reduced, indicating a deficiency in synaptic vesicle recycling. Clathrin mediated endocytosis appeared to be affected since there was a decrease in the number of coated intermediates in the periactive zone. This observation further suggests that the ENTH domain of epsin is involved at early stages of coat formation. The data are consistant with the hypothesis that the interaction between the ENTH domain of epsin and the plasma membrane is important for membrane curvature and clathrin assembly (Ford et al., 2002). To test this possibility further we performed immunogold labeling to detect a coat component, amphiphysin (Evergren et al., 2004), in axons injected with ENTH IgG. Amphiphysin labeling was decreased in the periactive zone in ENTH perturbed synapses compared to control synapses consistent with a mislocalization of coat components.

IgG were also generated against the CLAP region of epsin to perturb epsins binding with AP2 and clathrin. The antibodies efficiently inhibited the binding between these proteins in in vitro pull-down experiments. These antibodies also co-localized with antibodies against the synaptic vesicle marker VAMP, and also with antibodies against

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experiments they labelled coated pits. Synapses in axons microinjected with CLAP IgG showed a reduced number of synaptic vesicles further supporting the involvement of epsin in synaptic vesicle recycling. Analysis of coated pits showed a shift in distribution towards earlier stages, i.e. a higher proportion of shallow and bucket shaped coated pits. These intermediates were also significantly larger than those seen in control synapses and could probably not proceed in the invagination process since the synaptic vesicles in the synaptic vesicle cluster was of similar size as in control synapses. The increase in curvature diameter might be a result of prevention of epsin to coordinate curvature formation with assembly of AP2 and clathrin. Another striking difference between control injected synapses and synapses injected with CLAP IgG was the occurrence of clathrin coats on internal endosome-like membrane structures and clathrin patches at the plasma membrane (Figure 4). Similar patches are seen if the CLAP region of amphiphysin is perturbed suggesting that precise composition of clathrin adaptors are required to prevent unorganized clathrin assembly (Evergren et al., 2004).

Figure 4. Schematic picture of the effects of perturbation of epsins CLAP region. The number of synaptic vesicles in the synaptic vesicle cluster is reduced and coated pits are enlarged or show a distorted shape. Clathrin coats also occur on cisterna and on flat plasma membrane regions.

In summary this study has shown that epsin is important for clathrin coated pit formation during synaptic vesicle recycling. It supports the hypothesis that epsin via its ENTH domain induces curvature and via the CLAP region coordinates curvature formation with coat assembly.

ENDOPHILIN BAR DOMAIN IS ESSENTIAL FOR COATED PIT PROGRESSION (PAPER II)

To investigate the importance of a BAR domain in a living synapse antibodies against the BAR domain of lamprey endophilin were generated. Fab fragments produced from these IgG efficiently blocked membrane binding between the N-BAR domain of endophilin and liposomes in an in vitro sedimentation assay. When microinjected IgG against endophilin co-localized with VAMP in spots in the reticulospinal axon.

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Microinjection of IgG against the BAR domain of endophilin followed by stimulation interrupted synaptic vesicle recycling as suggested by a reduction in the number of synaptic vesicles in the vesicle cluster. Along with the reduction of synaptic vesicles the number of coated intermediates in the periactive zone was increased and consisted mainly of shallow coated pits (Figure 5). This result resembles the effect seen in microinjection experiment using IgG against the SH3 domain of mammalian endophilin (Ringstad et al., 1999). To investigate if accumulation of shallow coated pits could be due to precipitation of endophilin and its binding partners BAR-directed Fab fragments were microinjected. The microinjection again interfered with synaptic vesicle recycling and shallow coated pits accumulated at the plasma membrane. The apparent slowing or blockade of the invagination process after perturbation of the BAR domain suggest two possibilities; (1) that Fab fragments interfere directly with the membrane bending properties of endophilins BAR domain or (2) that they disable the recruitment of endophilin and thereby its binding partners to the plasma membrane.

To further test the role of the BAR domain we injected the N-BAR domain of endophilin which promotes membrane curvature and tubulation in vitro and in vivo (Takei et al., 1999; Masuda et al., 2006). Injection of the N-BAR domain of endophilin resulted in a similar effect with regard to loss of synaptic vesicles and accumulation of shallow coated pits. In addition the N-BAR domain of endophilin also induced membranous tubules on internal membrane structures, indicating that membrane tubulation can occur in synapses in vivo. The estimated concentration of the injected BAR domain in the reticulospinal axon (0.5 µM) did not exceed the concentration used in in vitro tubulation assays (10-40 µM) (Gallop et al., 2006). One explanation for the effect on coated pits could be that the injected N-BAR domain construct bind to the plasma membrane and thereby prevents binding of endogenous endophilin along with its binding partners. This possibility is supported by the fact that injection of a F-BAR domain (of syndapin) caused a similar effect with regard to accumulation of early-stage coated intermediates (F.A. and L.B unpublished results).

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Figure 5. Schematic picture illustrating the effect of different reagents perturbing interaction with the BAR domain of endophilin. IgG and Fab fragments against the BAR domain of endophilin, as well as the N-BAR domain of endophilin reduced the number of synaptic vesicles and increased the number of coated pits. The majority of the coated pits were trapped at a shallow stage.

A major binding partner for endophilin is synaptojanin. In a previous study it was suggested that the phosphatase activity of synaptojanin is important for coated pit invagination (Mani et al., 2007). Studies in Drosophila and C. elegans has suggested that endophilin is important for synaptojanin recruitment (Schuske et al., 2003;

Verstreken et al., 2003). For these reasons IgG were generated against the 5- phosphatase domain of synaptojanin. The antibodies recognized a single band on Western blot of 145 kD and, after microinjection into the reticulospinal axon, it colocalized with IgG against synaptotagmin. The morphology of synapses in microinjected and stimulated axons resembled that of synapses seen after endophilin BAR Fab injection with regard to the accumulation of shallow coated pits and loss of synaptic vesicles in the synaptic vesicle cluster. In addition, filamentous actin matrix (Bloom et al., 2003) was accumulated at the plasma membrane around synapses injected with synaptojanin-5-phosphatase IgG (Figure 6). This effect has also been observed after microinjection of synaptojanin antibodies directed against the proline- rich domain (Gad et al., 2000) and is consistent with the actin regulating effects of PIP2.

Figure 6. Schematic picture illustrating the effect of microinjection of IgG against the phosphatase domain of synaptojanin. Similar effects as for perturbation of endophilin was seen, with the addition of filamentous matrix enriched pockets.

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Although microinjection of both endophilin IgG and synaptojanin IgG results in accumulation of shallow coated pits, it can not be ruled out that dynamin, another binding partner of endophilins SH3 domain, also plays a role in the invagination process. In this study we did not observe any accumulation of deeply invaginated coated pits, which were seen when the SH3 domain of endophilin or a synaptojanin peptide was microinjected (Gad et al., 2000). However, dynamin has also been found to be present on shallow coated pits (Damke et al., 1994; Evergren et al., 2007) and perturbation of dynamin using the membrane-permeable inhibitor dynasore results in accumulation of bucket shaped intermediates (Macia et al., 2006).

The fact that N-BAR and F-BAR domain injections produced similar effects on coated pits (see above) argues against a direct membrane-bending role of the endogenous N- BAR domain in coated pit invagination. Also arguing against such a direct membrane- bending function of the N-BAR domain is the fact that endophilin lacks clathrin/AP2 binding motifs, and that immunogold studies have failed to detect significant levels of endophilin in the clathrin coat (Ringstad et al., 1999). Moreover, live cell imaging studies (in non-neuronal cells) have shown that the recruitment of endophilin primarily occurs at a late stage just preceding fission (Perera et al., 2006). Hence it is plausible that the N-BAR domain does not act directly by driving invagination. This contrasts with the ENTH domain of epsin, which appears to be actively involved in membrane- bending (see above; paper I and Ford et al., 2002).

In summary, this study identifies the N-BAR domain of endophilin as a critical component in the progression from early to late stages of synaptic vesicle endocytosis.

We suggest that its role at this stage is indirect and linked with effects on binding partners of endophilin.

SYNDAPIN IN SYNAPTIC VESICLE RECYCLING (PAPER III)

To analyze the role of syndapin during synaptic vesicle recycling using the lamprey model system we first isolated a lamprey orthologue of syndapin. Lamprey syndapin showed 78% similarity to human syndapin 1, which is the isoform predominantly expressed in CNS. Like mammalian syndapin 1, the lamprey orthologe also contains 2 NPF motifs. Coomassie staining of pull-downs from lamprey CNS with full-length syndapin showed that the major binding partner for lamprey syndapin is dynamin

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(identified on Western blot). Lamprey syndapin also bound to N-WASP as detected using Western blot. Antibodies against full-length syndapin were generated in rabbit.

These antibodies recognized a single protein of about 50 kDa on Western blot. The antibodies co-localized with VAMP IgG at release sites following injection into the reticulospinal axon. While previous studies have suggested a synaptic localization of syndapin (Qualmann et al., 1999), these experiments demonstrate its specific presynaptic localization.

To investigate if syndapin is important for synaptic transmission syndapin IgG were microinjected into reticulospinal axons and the EPSP was recorded in postsynaptic target neurons. During low levels of stimulation (0.2 Hz) the amplitude of EPSPs was not significantly affected by syndapin IgG. Morphological analysis of synapses in axons microinjected with syndapin IgG and stimulated at 0.2 Hz did not reveal any obvious difference from control injected synapses, consistent with the electrophysiology data. Thus, syndapin does not appear to be important for synaptic vesicle recycling during low levels of stimulations. A reduction in the number of synaptic vesicles and accumulation of invaginated coated pits has been observed at low level of stimulation (0.2 Hz) after other perturbations (Shupliakov et al., 1997).

In electrophysiological experiments the recovery of the EPSP amplitude after intense stimulation (50 Hz for 10 min) was found to be altered following microinjection of syndapin IgG. Morphological analysis of syndapin IgG-injected axons after physiological stimulation (30 min at 5 Hz) revealed a loss of synaptic vesicles at release sites. The number of coated intermediates at the plasma membrane was, however unchanged, as compared to synapses in control-injected axons. To follow the recovery of the changes in synapse morphology additional experiments were performed in which axons were analyzed after a recovery period of 15 min following 30 min of 5 Hz stimulation. In control and syndapin IgG injected axons, synapses fixed at the end of a 5 Hz 30 min stimulation period showed a higher number of coated pits, compared to resting synapses. After the recovery period the number of coated pits at the plasma membrane in synapses located in control as well as in syndapin IgG injected axons had returned to the resting level. This observation indicate that perturbation of syndapin did not delay the budding of coated pits.

The decrease in the number of synaptic vesicles corresponded with an accumulation of

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membrane cisternae around release sites (Figure 7). The cisternae remained after a recovery period of 15 min. The cisternae were shown to contain synaptic vesicle membrane by immunogold labeling with IgG against the synaptic vesicle protein VAMP. Coated pits occurred on the cisternae. The cisternae could be connected to each other with narrow tubular structures, and a few connections to the plasma membrane were observed, however occurrence of free cisternae could not be verified even after serial section analysis.

Figure 7. Schematic picture illustrating the effect of microinjection of IgG against syndapin.

Perturbation of syndapin results in stimulus-dependent loss of synaptic vesicles and accumulation of large membranous cisternae in the periactive zone, persisting after 15 min of recovery. No differences were seen with regard to clathrin coated pits at the plasma membrane proper in comparison with control injected axons.

To test the role of syndapin SH3 domain interactions in synaptic vesicle recycling, SH3 domain-directed Fab fragments were generated and purified. In an in vitro pulldown assay, the Fab fragments inhibited the SH3 interaction with N-WASP and dynamin. In microinjection experiments this selective impairment of the SH3 domain of syndapin was sufficient to induce loss of synaptic vesicles and accumulation of membranous cisternae. As in the experiments with syndapin IgG, no change in the number of coated intermediates at the plasma membrane was seen.

The accumulation of membranous cisternae may reflect an impaired bulk endocytosis or reflect compensatory invagination of membrane as it expands by exocytosis when clathrin-mediated endocytosis is inhibited. To study the effect of perturbation of syndapin on a single round of endocytosis, exocytosis and endocytosis were separated (see Methods) and syndapin IgG was injected after intense stimulation and cooling, before the synaptic vesicle cluster had recovered. In control synapses exposed to the same protocol, the number of synaptic vesicles in the synaptic vesicle cluster was reduced, but recovered after the temperature was returned to normal levels. However, when syndapin IgG was microinjected in the interval between exo- and endocytosis the

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synaptic vesicle cluster failed to recover and membranous cisternae accumulated in the synaptic area. This experiment indicates that membranous cisternae trapped by syndapin IgG form by an active process, rather than reflecting a passive invagination of the plasma membrane secondary to a general block in endocytosis.

Both actin and dynamin has previously been proposed to be involved in generation of larger membranous cisternae in the synapse (Itoh et al., 2005). One possible role of syndapin could be to recruit dynamin and actin regulators (N-WASP and cobl) to take part in break down of cisternae. The present data are consistent with the suggested function for an involvement of syndapin in a bulk endocytic pathway (Clayton et al., 2007).

Recently neuromuscular synapses in a Drosophila syndapin mutant were investigated (Kumar et al., 2008). In this study no defect in synaptic vesicle recycling could be detected. This is similar to amphipysin (Razzaq et al., 2001) and epsin (Bao et al., 2008) Drosophila mutants which neither exhibited any detectable defects in synaptic vesicle recycling. The importance of these proteins in the lamprey reticulospinal axon (Evergren et al., 2004) (paper I) and in mice (Di Paolo et al., 2002) suggests differences in the composition of the molecular machinery of synaptic vesicle recycling between vertebrates and invertebrates. Amphiphysin and syndapin have been shown to be involved in functions at the postsynaptic side in the Dropsophila neuromuscular junction (Razzaq et al., 2001; Kumar et al., 2009).

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CONCLUSIONS

Three different proteins containing membrane binding domains known to induce membrane curvature in vitro were investigated. Epsin was shown to be involved in clathrin mediated endocytosis of synaptic vesicles, in agreement with an in vitro-based model. The N-terminal membrane-binding domain of epsin is likely to recruit coat components to the plasma membrane and generate curvature while the clathrin- and adaptor-binding region appears to be important for coordination of coat assembly.

The BAR domain of endophilin was shown to be important for progression of shallow coated pits to invaginated stages. The role of endophilin in coat formation does not seem to be direct, but is most likely due to secondary effects on binding-partners of endophilin.

Syndapin was shown to participate in synaptic vesicle recycling in the lamprey reticulospinal axon. However, no evidence for participation in clathrin-mediated endocytosis from the plasma membrane was obtained. In contrast, when high frequency stimulation was applied after perturbation of syndapin, large endosome-like structures accumulated, indicating a role of syndapin in bulk endocytosis and/or stabilization of the plasma membrane.

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ACKNOWLEDGEMENTS

Many people have helped and supported me during my years at Karolinska Institutet, so there are a couple of people that I especially would like to thank:

Lennart Brodin, my supervisor, for your encouragement and infectious enthusiasm about science. I learned a lot during these years. Thank you for accepting me as PhD student.

Peter Löw, my co supervisor, for teaching me lots of things, from how to grind wooden floors to biochemical methods. I have been lucky to work with you.

My co-authors; Oleg Shupliakov for scientific input and help with EM techniques, Helge Gad for introducing me to the techniques in the lab and Joel Jakobsson for being a great bollplank and a really good friend.

Past and present members in the Brodins and Shupliakovs groups for creating an excellent environment to work in, and helping me with various bothers: Britt, Jenny, Emma, Nikolay, Victoria, Anna, Fransisco, Malin, Kristin, Wei, Cynthia, Arndt, Olga and Frauke.

I am really grateful for working with people at the department of neuroscience and CMB. Thank you for help and friendship especially Johan and Micke for over 1000 km on the road and help with statistics, Zoltan, and Paul for unforgettable morning runs around the lake.

Labgrupp 3, it is impossible to find better people to attend Stockholms universitet with:

Max, Cissi, Karin, Malin, Stina, Ylva, Benita och Lotta.

Killarna with girlfriends and Henke & Zara for being the best of friends.

The Luhr families for recreation outside Stockholm.

Ulla, Torsten, Shinae, Inga, min gode vän Harry, Fredrik and Anna for care, support and being proud of me. It is nice to know that you love me.

Gia for being you and making me happy ♥

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REFERENCES

Ahuja R, Pinyol R, Reichenbach N, Custer L, Klingensmith J, Kessels MM, Qualmann B (2007) Cordon-bleu is an actin nucleation factor and controls neuronal morphology. Cell 131:337-350.

Anggono V, Smillie KJ, Graham ME, Valova VA, Cousin MA, Robinson PJ (2006) Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis. Nat Neurosci 9:752-760.

Bao H, Reist NE, Zhang B (2008) The Drosophila Epsin 1 Is Required for Ubiquitin- dependent Synaptic Growth and Function, But Not for Synaptic Vesicle Recycling. Traffic.

Beck KA, Keen JH (1991) Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem 266:4442-4447.

Bloom O, Evergren E, Tomilin N, Kjaerulff O, Löw P, Brodin L, Pieribone VA, Greengard P, Shupliakov O (2003) Colocalization of synapsin and actin during synaptic vesicle recycling. J Cell Biol 161:737-747.

Brodin L, Shupliakov O (2006) Giant reticulospinal synapse in lamprey: molecular links between active and periactive zones. Cell Tissue Res 326:301-310.

Chen H, De Camilli P (2005) The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin.

Proc Natl Acad Sci U S A 102:2766-2771.

Chen H, Polo S, Di Fiore PP, De Camilli PV (2003) Rapid Ca2+-dependent decrease of protein ubiquitination at synapses. Proc Natl Acad Sci U S A 100:14908-14913.

Chen H, Fre S, Slepnev VI, Capua MR, Takei K, Butler MH, Di Fiore PP, De Camilli P (1998) Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature 394:793-797.

Clayton EL, Evans GJ, Cousin MA (2007) Activity-dependent control of bulk endocytosis by protein dephosphorylation in central nerve terminals. J Physiol.

Clayton EL, Evans GJ, Cousin MA (2008) Bulk synaptic vesicle endocytosis is rapidly triggered during strong stimulation. J Neurosci 28:6627-6632.

Collins BM, McCoy AJ, Kent HM, Evans PR, Owen DJ (2002) Molecular architecture and functional model of the endocytic AP2 complex. Cell 109:523-535.

Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, McCormick DA, De Camilli P (1999) Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99:179-188.

Da Costa SR, Sou E, Xie J, Yarber FA, Okamoto CT, Pidgeon M, Kessels MM, Mircheff AK, Schechter JE, Qualmann B, Hamm-Alvarez SF (2003) Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells. Mol Biol Cell 14:4397-4413.

Damke H, Baba T, Warnock DE, Schmid SL (1994) Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. Journal of Cell Biology 127:915-934.

de Lange RP, de Roos AD, Borst JG (2003) Two modes of vesicle recycling in the rat calyx of Held. J Neurosci 23:10164-10173.

Di Paolo G, Moskowitz HS, Gipson K, Wenk MR, Voronov S, Obayashi M, Flavell R, Fitzsimonds RM, Ryan TA, De Camilli P (2004) Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking.

Nature 431:415-422.

Di Paolo G, Sankaranarayanan S, Wenk M, Daniell L, Perucco E, Caldarone B, Flavell R, Picciotto M, Ryan T, Cremona O, De Camilli P (2002) Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice. Neuron 33:789-804.

Dickman DK, Horne JA, Meinertzhagen IA, Schwarz TL (2005) A slowed classical pathway rather than kiss-and-run mediates endocytosis at synapses lacking synaptojanin and endophilin. Cell 123:521-533.

(34)

Evans GJ, Cousin MA (2007) Activity-dependent control of slow synaptic vesicle endocytosis by cyclin-dependent kinase 5. J Neurosci 27:401-411.

Evergren E, Gad H, Walther K, Sundborger A, Tomilin N, Shupliakov O (2007) Intersectin is a negative regulator of dynamin recruitment to the synaptic endocytic zone in the central synapse. J Neurosci 27:379-390.

Evergren E, Marcucci M, Tomilin N, Löw P, Slepnev V, Andersson F, Gad H, Brodin L, De Camilli P, Shupliakov O (2004) Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse.

Traffic 5:514-528.

Fabian-Fine R, Verstreken P, Hiesinger PR, Horne JA, Kostyleva R, Zhou Y, Bellen HJ, Meinertzhagen IA (2003) Endophilin promotes a late step in endocytosis at glial invaginations in Drosophila photoreceptor terminals. J Neurosci 23:10732- 10744.

Farsad K, Ringstad N, Takei K, Floyd SR, Rose K, De Camilli P (2001) Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J Cell Biol 155:193-200.

Ferguson KM, Lemmon MA, Schlessinger J, Sigler PB (1994) Crystal structure at 2.2 A resolution of the pleckstrin homology domain from human dynamin. Cell 79:199-209.

Ferguson SM, Brasnjo G, Hayashi M, Wolfel M, Collesi C, Giovedi S, Raimondi A, Gong LW, Ariel P, Paradise S, O'Toole E, Flavell R, Cremona O, Miesenbock G, Ryan TA, De Camilli P (2007) A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Science 316:570-574.

Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT (2002) Curvature of clathrin-coated pits driven by epsin. Nature 419:361-366.

Ford MG, Pearse BM, Higgins MK, Vallis Y, Owen DJ, Gibson A, Hopkins CR, Evans PR, McMahon HT (2001) Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291:1051-1055.

Frost A, Perera R, Roux A, Spasov K, Destaing O, Egelman EH, De Camilli P, Unger VM (2008) Structural basis of membrane invagination by F-BAR domains. Cell 132:807-817.

Gad H, Low P, Zotova E, Brodin L, Shupliakov O (1998) Dissociation between Ca2+- triggered synaptic vesicle exocytosis and clathrin-mediated endocytosis at a central synapse. Neuron 21:607-616.

Gad H, Ringstad N, Löw P, Kjaerulff O, Gustafsson J, Wenk M, Di Paolo G, Nemoto Y, Crun J, Ellisman MH, De Camilli P, Shupliakov O, Brodin L (2000) Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 27:301-312.

Gaidarov I, Keen JH (1999) Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits. J Cell Biol 146:755-764.

Gallop JL, Jao CC, Kent HM, Butler PJ, Evans PR, Langen R, McMahon HT (2006) Mechanism of endophilin N-BAR domain-mediated membrane curvature.

Embo J 25:2898-2910.

Gonzalez-Gaitan M, Jäckle H (1997) Role of Drosophila alpha-adaptin in presynaptic vesicle recycling. Cell 88:767-776.

Goodman OB, Jr., Keen JH (1995) The alpha chain of the AP-2 adaptor is a clathrin binding subunit. J Biol Chem 270:23768-23773.

Gu M, Schuske K, Watanabe S, Liu Q, Baum P, Garriga G, Jorgensen EM (2008) Mu2 adaptin facilitates but is not essential for synaptic vesicle recycling in Caenorhabditis elegans. J Cell Biol 183:881-892.

Guichet A, Wucherpfennig T, Dudu V, Etter S, Wilsch-Brauniger M, Hellwig A, Gonzalez-Gaitan M, Huttner WB, Schmidt AA (2002) Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction. Embo J 21:1661-1672.

Harris TW, Hartwieg E, Horvitz HR, Jorgensen EM (2000) Mutations in Synaptojanin Disrupt Synaptic Vesicle Recycling. J Cell Biol 150:589-600.

Hayashi M, Raimondi A, O'Toole E, Paradise S, Collesi C, Cremona O, Ferguson SM, De Camilli P (2008) Cell- and stimulus-dependent heterogeneity of synaptic

(35)

vesicle endocytic recycling mechanisms revealed by studies of dynamin 1-null neurons. Proc Natl Acad Sci U S A 105:2175-2180.

Henne WM, Kent HM, Ford MG, Hegde BG, Daumke O, Butler PJ, Mittal R, Langen R, Evans PR, McMahon HT (2007) Structure and Analysis of FCHo2 F-BAR Domain: A Dimerizing and Membrane Recruitment Module that Effects Membrane Curvature. Structure 15:839-852.

Heuser JE, Reese TS (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. Journal of Cell Biology 57:315-344.

Holt M, Cooke A, Wu MM, Lagnado L (2003) Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells. J Neurosci 23:1329-1339.

Itoh T, Koshiba S, Kigawa T, Kikuchi A, Yokoyama S, Takenawa T (2001) Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 291:1047-1051.

Itoh T, Erdmann KS, Roux A, Habermann B, Werner H, De Camilli P (2005) Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev Cell 9:791-804.

Jost M, Simpson F, Kavran JM, Lemmon MA, Schmid SL (1998) Phosphatidylinositol- 4,5-bisphosphate is required for endocytic coated vesicle formation. Current Biology 8:1399-1402.

Jung N, Haucke V (2007) Clathrin-mediated endocytosis at synapses. Traffic 8:1129- 1136.

Kessels MM, Qualmann B (2004) The syndapin protein family: linking membrane trafficking with the cytoskeleton. J Cell Sci 117:3077-3086.

Kessels MM, Qualmann B (2006) Syndapin oligomers interconnect the machineries for endocytic vesicle formation and actin polymerization. J Biol Chem.

Kim WT, Chang S, Daniell L, Cremona O, Di Paolo G, De Camilli P (2002) Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice. Proc Natl Acad Sci U S A 99:17143-17148.

Kirchhausen T, Harrison SC (1981) Protein organization in clathrin trimers. Cell 23:755-761.

Koenig JH, Ikeda K (1989) Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. Journal of Neuroscience 9:3844-3860.

Koenig JH, Ikeda K (1996) Synaptic vesicles have two distinct recycling pathways.

Journal of Cell Biology 135:797-808.

Kumar V, Alla SR, Krishnan KS, Ramaswami M (2008) Syndapin is dispensable for synaptic vesicle endocytosis at the Drosophila larval neuromuscular junction.

Mol Cell Neurosci.

Kumar V, Fricke R, Bhar D, Reddy-Alla S, Krishnan KS, Bogdan S, Ramaswami M (2009) Syndapin Promotes Formation of a Postsynaptic Membrane System in Drosophila. Mol Biol Cell.

Leenders AG, Scholten G, de Lange RP, Lopes da Silva FH, Ghijsen WE (2002) Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

Neuroscience 109:195-206.

Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10:839-850.

Mani M, Lee SY, Lucast L, Cremona O, Di Paolo G, De Camilli P, Ryan TA (2007) The dual phosphatase activity of synaptojanin1 is required for both efficient synaptic vesicle endocytosis and reavailability at nerve terminals. Neuron 56:1004-1018.

Masuda M, Takeda S, Sone M, Ohki T, Mori H, Kamioka Y, Mochizuki N (2006) Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms. Embo J 25:2889-2897.

McPherson PS, Garcia EP, Slepnev VI, David C, Zhang X, Grabs D, Sossin WS, Bauerfeind R, Nemoto Y, De Camilli P (1996) A presynaptic inositol-5- phosphatase. Nature 379:353-357.

(36)

Micheva KD, Kay BK, McPherson PS (1997) Synaptojanin forms two separate complexes in the nerve terminal. Interactions with endophilin and amphiphysin.

Journal of Biological Chemistry 272:27239-27245.

Miki H, Takenawa T (2003) Regulation of actin dynamics by WASP family proteins. J Biochem 134:309-313.

Miki H, Miura K, Takenawa T (1996) N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. Embo J 15:5326-5335.

Modregger J, DiProspero NA, Charles V, Tagle DA, Plomann M (2002) PACSIN 1 interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's disease brains. Hum Mol Genet 11:2547-2558.

Morgan JR, Prasad K, Jin S, Augustine GJ, Lafer EM (2001) Uncoating of clathrin- coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin. Neuron 32:289-300.

Morgan JR, Zhao X, Womack M, Prasad K, Augustine GJ, Lafer EM (1999) A role for the clathrin assembly domain of AP180 in synaptic vesicle endocytosis. J Neurosci 19:10201-10212.

Motley A, Bright NA, Seaman MN, Robinson MS (2003) Clathrin-mediated endocytosis in AP-2-depleted cells. J Cell Biol 162:909-918.

Nonet ML, Holgado AM, Brewer F, Serpe CJ, Norbeck BA, Holleran J, Wei L, Hartwieg E, Jorgensen EM, Alfonso A (1999) UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol Biol Cell 10:2343-2360.

Pearse BM (1976) Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci U S A 73:1255-1259.

Perera RM, Zoncu R, Lucast L, De Camilli P, Toomre D (2006) Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages. Proc Natl Acad Sci U S A 103:19332-19337.

Perez-Otano I, Lujan R, Tavalin SJ, Plomann M, Modregger J, Liu XB, Jones EG, Heinemann SF, Lo DC, Ehlers MD (2006) Endocytosis and synaptic removal of NR3A-containing NMDA receptors by PACSIN1/syndapin1. Nat Neurosci 9:611-621.

Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon HT (2004) BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303:495-499.

Pieribone VA, Shupliakov O, Brodin L, Hilfiker-Rothenfluh S, Czernik AJ, Greengard P (1995) Distinct pools of synaptic vesicles in neurotransmitter release. Nature 375:493-497.

Plomann M, Lange R, Vopper G, Cremer H, Heinlein UA, Scheff S, Baldwin SA, Leitges M, Cramer M, Paulsson M, Barthels D (1998) PACSIN, a brain protein that is upregulated upon differentiation into neuronal cells. Eur J Biochem 256:201-211.

Polo S, Sigismund S, Faretta M, Guidi M, Capua MR, Bossi G, Chen H, De Camilli P, Di Fiore PP (2002) A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Nature 416:451-455.

Praefcke GJ, McMahon HT (2004) The dynamin superfamily: universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol 5:133-147.

Pucadyil TJ, Schmid SL (2008) Real-time visualization of dynamin-catalyzed membrane fission and vesicle release. Cell 135:1263-1275.

Qualmann B, Kelly RB (2000) Syndapin isoforms participate in receptor-mediated endocytosis and actin organization. Journal of Cell Biology 148:1047-1062.

Qualmann B, Roos J, DiGregorio PJ, Kelly RB (1999) Syndapin I, a synaptic dynamin- binding protein that associates with the neural Wiskott-Aldrich syndrome protein. Molecular Biology of the Cell 10:501-513.

Razzaq A, Robinson IM, McMahon HT, Skepper JN, Su Y, Zelhof AC, Jackson AP, Gay NJ, O'Kane CJ (2001) Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila. Genes Dev 15:2967-2979.

Richards DA, Rizzoli SO, Betz WJ (2004) Effects of wortmannin and latrunculin A on slow endocytosis at the frog neuromuscular junction. J Physiol 557:77-91.

Studies of membrane-binding proteins involved in synaptic vesicle recycling