Abstract. Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP~/S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L. Schmid, and P. De Camilli.1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K ÷ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin-and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the a, subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis.Strong evidence implicates the GTPase dynamin (1, 2) in the internalization of synaptic vesicle membranes after exocytosis and, more generally, in internalization of clathrin-coated vesicles. Temperature-sensitive mutations of the dynamin gene (shibire) in Drosophila cause a selective arrest of the synaptic vesicle cycle at the stage of invaginated plasmalemmal pits (3)(4)(5)(6), and transfection of dominant negative dynamin mutants in fibroblastic cells blocks clathrin-mediated endocytosis (7,8). Recent studies have shown that dynamin forms rings at the neck of invaginated clathrin-coated vesicles and suggested that a conformational change of the rings which correlates with GTP hydrolysis leads to vesicle fission (9, 10). The identification of dynamin's physiological binding partner will be an important next step toward a full elucidation of endocytotic mechanisms.Dynamin has a proline-rich C-terminal region that binds to a subset of SH3 domains. It was found to bind most effectively to the SH3 domains of Grb2, phospholipase Cy,, and the p85 subunit of phosphatidylinositol 3-kinase (11-14). However, none of these proteins was shown to be concentrated in nerve terminals and the significance of these interactions for synaptic vesicle recycling remains unclear. In this study we have explored the possibility that amphiphysin, a neuronal SH3 domain-containing protein selectively concentrated in axon endings (15-17), may represent a physiological partner for dynamin. Amphiphysin is a hydrophilic, highly acidic protein, which is found in soluble and particulate fractions of brain homogenates including synaptic vesicle membranes but is not enriched in purified synaptic vesicles (15)(16)(17) MATERIALS AND METHODS Antibodies. Polyclonal antibodies (CD5 and CD6) directed against full-length glutathione S-transferase (GST)-amphiphysin were raised in rabbits and affinity purified on polyhistidinetagged amphiphysin (His-amph) fusion proteins. Polyclonal antibodies directed against dynamin were obtained by injecting rabbits with gel slices containing rat brain dynamin purified on a Grb2 column. A polyclonal anti-synapsin antibody (G246) was previously described (20). The T7 tag antibody which recognizes an 11-amino acid (aa) sequence in the pTrcHis constructs was from Novagen. The following antibodies were generous gifts: ...

Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse.

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cellmediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. Mol Cancer Ther; 12(10); 2031-42. Ó2013 AACR.