• Immunogold and electron tomography protocols were developed, making it possible to localize synaptic proteins in Drosophila NMJs and to study the structural organization of this synapse at high resolution.
• Fas II delineates the active zone area in the Drosophila NMJ under resting conditions and immunolabeling for this protein can be reliably used to evaluate changes in the size of the active zone region.
• The presynaptic dense projection of the active zone consists of three distinct components: the central core, legs connecting the central core to the plasma membrane, and cytoplasmic extensions emerging from the central core.
• The SV pool in the Drosophila synapse is structurally composed of the proximal pool attached to the dense projection by thin filaments and distal pool constituted by interconnected vesicles. Vesicles tethered to the presynaptic membrane are able to release immediately in response to stimulation.
• The scaffolding molecules Dap160 and Eps15 and the GTPase dynamin reside in the distal pool of SV at rest and relocate to the periactive zone during synaptic activity.
• Dap160 and Eps15 directly interact and form a complex, which is required to maintain synaptic transmission in the Drosophila NMJ at high activity rates.
This complex is essential to concentrate dynamin at the periactive zone during synaptic activity.
ACKNOWLEDGEMENTS
First of all I would like to thank my supervisor Oleg Shupliakov for taking me as a PhD student and introducing me to the field of neuroscience. Thanks for giving me the guidance, encouragement and telling me to be strong to face any problem.
Big thanks go to former and present labmates: Nikolay, for all the EM teaching;
Peter, for all the care and nice tea time; Fredrik and Joel, for helping me with moving and being great lunch companies; Cynthia, for all the fun together and keeping in touch from so long distance, and also your proofreading; Lennart, for all the knowledge I have learned from you; Kristin, for the comfort and company the first couple of years here; Frauke, for the support and believe in my graduation; Sevi, for the nice time in the office and meeting in Greece; Anna and Arndt, for all the funny stories during lunch time; Åsa, for helping me with fly work during the busy thesis writing time; Olga, for the help with biochemistry; Elena, for continuing my EM work and warm greetings;
Emma, Felicia, Malin, Joshua, Sara and Ronia, for creating a friendly working environment; All members from Neurovetenskap, CMB and CEDB/DBRM for being enthusiastic and helpful.
Thanks to my collaborators: Professor Hugo J. Bellen, Tong-Wey and Karen, for the work of generating all the mutant flies. Sergej and Oscar, for the help with tomography and reconstruction.
I also want to thank Professor Christos Samakovlis, for kindness and support for my fly work.
所有在瑞典相识的朋友，这个寒冷的国度，半年的白雪，一季的阳光，因为有你
们，才显得温暖。
戴琦，汪神俅，谢谢你们对刚来瑞典的我的照顾，为了打升级而手制的纸牌依旧 是我美好的回忆。
巧琳，谢谢你姐姐一样的关怀，血拼的时候也总是不忘记帮我注意好东西。金 哲， 你的好手艺让我很饱口服啊。
绍波，曾几何时，来去路上都有你做伴，现在住的远了，一个人等公车的时候还 是会想到你。赵建，很想偷师你的川菜手艺啊。（好吧，我承认我是吃货。）
隶宁，还记得两人在Gotland东走走，西看看的悠闲，谢谢你时不时来自旧金山的 问候。
28
赵斌，尤记得那些互相宽慰的茶余饭后时光，你需要新的茶友和饭友啦。王柠 莎，我喜欢你湘妹子的个性，够直爽够呛，就象那火红的辣椒。
我在Lappis认识的朋友：丽如，我们做了两年的室友，一起走过大半个欧洲，虽然 现在你远在台湾，还是希望有再见的时候。崔威和李静，你们是最好客的男女主人，
你们家的客厅曾经容纳了多少我们的欢声笑语。简亮，曾经任劳任怨的司机，希望你 心想事成。老朱，武汉和长沙不远，有机会探望探望。姜岩，谢谢你的好脾气和包 容。
我的青梅——糖粒子（谭丽），永远记得分别八年后异国的重逢，希望在纽约的 你幸福安好。
亲爱的小猪（朱莹），酒逢知己千杯少，我虽然不会喝酒，也愿意为了你饮下几 杯伏特加。
洁欣，羡慕你的勤劳和贤惠，谢谢你总是做好吃的点心给我打牙祭（再次声明，
我是吃货）。玉兰，谢谢狮子座之间的理解和寄自非洲的明信片，满足我收集的热 衷。施耀，你的光辉形象总是和美餐联系在一起的（吃货……）。新蕊，谢谢在我暗 无天日论文时期的关心和照顾。立群，谢谢你不辞辛苦的帮我订果蝇食物。还有所有 我认识的朋友：燕铃，王迪，李昱，万敏，薛远，颜沁子，王恒，新松，新明，嘉 琪，宋辞，畅征，宋直励，陆明，晓伟，晓峰，李芳，钱禹，睿雪，Sofia，相逢既是 有缘。
一路行来，除了朋友给予的温暖，最让我感动与牵挂的是我的父母。来瑞典念书 其实是我任性的决定。当时年纪小，不懂父母心，现在长大了，尤感父母恩。这么多 年，因为有你们，艰难困苦才可以走过，我只想说：爸爸妈妈，我爱你们！
REFERENCES
Anderson, M. S., M. E. Halpern, et al. (1988). "Identification of the neuropeptide transmitter proctolin in Drosophila larvae: characterization of muscle fiber-specific neuromuscular endings." J Neurosci 8(1): 242-255.
Ashley, J., M. Packard, et al. (2005). "Fasciclin II signals new synapse formation through amyloid precursor protein and the scaffolding protein dX11/Mint." J Neurosci 25(25): 5943-5955.
Atwood, H. L., C. K. Govind, et al. (1993). "Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae." J Neurobiol 24(8): 1008-1024.
Benmerah, A., M. Bayrou, et al. (1999). "Inhibition of clathrin-coated pit assembly by an Eps15 mutant." J Cell Sci 112 ( Pt 9): 1303-1311.
Benmerah, A., J. Gagnon, et al. (1995). "The tyrosine kinase substrate eps15 is
constitutively associated with the plasma membrane adaptor AP-2." J Cell Biol 131(6 Pt 2): 1831-1838.
Betz, W. J. and J. K. Angleson (1998). "The synaptic vesicle cycle." Annu Rev Physiol 60: 347-363.
Bloom, F. E. and G. K. Aghajanian (1968). "Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid." J Ultrastruct Res 22(5): 361-375.
Brand, A. H. and N. Perrimon (1993). "Targeted gene expression as a means of altering cell fates and generating dominant phenotypes." Development 118(2): 401-415.
Broadie, K. (2004). "Synapse scaffolding: intersection of endocytosis and growth."
Curr Biol 14(19): R853-855.
Brodin, L., P. Low, et al. (1997). "Sustained neurotransmitter release: new molecular clues." Eur J Neurosci 9(12): 2503-2511.
Ceccarelli, B., W. P. Hurlbut, et al. (1973). "Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction." J Cell Biol 57(2): 499-524.
Cochilla, A. J., J. K. Angleson, et al. (1999). "Monitoring secretory membrane with FM1-43 fluorescence." Annu Rev Neurosci 22: 1-10.
Couteaux, R. and M. Pecot-Dechavassine (1970). "[Synaptic vesicles and pouches at the level of "active zones" of the neuromuscular junction]." C R Acad Sci Hebd Seances Acad Sci D 271(25): 2346-2349.
Farsad, K., V. Slepnev, et al. (2003). "A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis."
Neuropharmacology 45(6): 787-796.
Feeney, C. J., S. Karunanithi, et al. (1998). "Motor nerve terminals on abdominal muscles in larval flesh flies, Sarcophaga bullata: comparisons with Drosophila."
J Comp Neurol 402(2): 197-209.
Fouquet, W., D. Owald, et al. (2009). "Maturation of active zone assembly by Drosophila Bruchpilot." J Cell Biol 186(1): 129-145.
Gaidarov, I. and J. H. Keen (1999). "Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits." J Cell Biol 146(4): 755-764.
Gerlitz, O., D. Nellen, et al. (2002). "A screen for genes expressed in Drosophila imaginal discs." Int J Dev Biol 46(1): 173-176.
Gonzalez-Gaitan, M. and H. Jackle (1997). "Role of Drosophila alpha-adaptin in presynaptic vesicle recycling." Cell 88(6): 767-776.
Gramates, L. S. and V. Budnik (1999). "Assembly and maturation of the Drosophila larval neuromuscular junction." Int Rev Neurobiol 43: 93-117.
Grant, D., S. Unadkat, et al. (1998). "Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster." Genetics 149(2): 1019-1030.
Guichet, A., T. Wucherpfennig, et al. (2002). "Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction." EMBO J 21(7): 1661-1672.
30
Hallermann, S., R. J. Kittel, et al. (2010). "Naked dense bodies provoke depression." J Neurosci 30(43): 14340-14345.
Hao, W., Z. Luo, et al. (1999). "AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin." J Biol Chem 274(32): 22785-22794.
Harlow, M. L., D. Ress, et al. (2001). "The architecture of active zone material at the frog's neuromuscular junction." Nature 409(6819): 479-484.
Heidelberger, R., C. Heinemann, et al. (1994). "Calcium dependence of the rate of exocytosis in a synaptic terminal." Nature 371(6497): 513-515.
Henne, W. M., E. Boucrot, et al. (2010). "FCHo proteins are nucleators of clathrin-mediated endocytosis." Science 328(5983): 1281-1284.
Heuser, J. E. and T. S. Reese (1973). "Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction." J Cell Biol 57(2): 315-344.
Hinshaw, J. E. and S. L. Schmid (1995). "Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding." Nature 374(6518): 190-192.
Hirst, J. and M. S. Robinson (1998). "Clathrin and adaptors." Biochim Biophys Acta 1404(1-2): 173-193.
Ikeda, K., S. Ozawa, et al. (1976). "Synaptic transmission reversibly conditioned by single-gene mutation in Drosophila melanogaster." Nature 259(5543): 489-491.
Johansen, J., M. E. Halpern, et al. (1989). "Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae." J Neurosci 9(2): 710-725.
Johansen, J., M. E. Halpern, et al. (1989). "Axonal guidance and the development of muscle fiber-specific innervation in Drosophila embryos." J Neurosci 9(12):
4318-4332.
Kittel, R. J., C. Wichmann, et al. (2006). "Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release." Science 312(5776): 1051-1054.
Klingauf, J., E. T. Kavalali, et al. (1998). "Kinetics and regulation of fast endocytosis at hippocampal synapses." Nature 394(6693): 581-585.
Koenig, J. H. and K. Ikeda (1989). "Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval." J Neurosci 9(11): 3844-3860.
Koh, T. W., P. Verstreken, et al. (2004). "Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis." Neuron 43(2): 193-205.
Kristiansen, L. V. and M. Hortsch (2008). "Fasciclin II: The NCAM Ortholog in Drosophila melanogaster." Neurochem Res.
Kuromi, H. and Y. Kidokoro (2003). "Two synaptic vesicle pools, vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction." J Neurocytol 32(5-8): 551-565.
Landis, D. M., A. K. Hall, et al. (1988). "The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse." Neuron 1(3):
201-209.
Loewen, C. A., J. M. Mackler, et al. (2001). "Drosophila synaptotagmin I null mutants survive to early adulthood." Genesis 31(1): 30-36.
Majumdar, A., S. Ramagiri, et al. (2006). "Drosophila homologue of Eps15 is essential for synaptic vesicle recycling." Exp Cell Res 312(12): 2288-2298.
Mao, Y. and M. Freeman (2009). "Fasciclin 2, the Drosophila orthologue of neural cell-adhesion molecule, inhibits EGF receptor signalling." Development 136(3):
473-481.
Marie, B., S. T. Sweeney, et al. (2004). "Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth." Neuron 43(2): 207-219.
Martina, J. A., C. J. Bonangelino, et al. (2001). "Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery." J Cell Biol 153(5):
1111-1120.
Masuda, M., S. Takeda, et al. (2006). "Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms." EMBO J 25(12): 2889-2897.
McDonald, K. L. and M. Auer (2006). "High-pressure freezing, cellular tomography, and structural cell biology." Biotechniques 41(2): 137, 139, 141 passim.
McNiven, M. A., H. Cao, et al. (2000). "The dynamin family of mechanoenzymes:
pinching in new places." Trends Biochem Sci 25(3): 115-120.
Mears, J. A., P. Ray, et al. (2007). "A corkscrew model for dynamin constriction."
Structure 15(10): 1190-1202.
Miele, A. E., P. J. Watson, et al. (2004). "Two distinct interaction motifs in
amphiphysin bind two independent sites on the clathrin terminal domain beta-propeller." Nat Struct Mol Biol 11(3): 242-248.
Monastirioti, M., M. Gorczyca, et al. (1995). "Octopamine immunoreactivity in the fruit fly Drosophila melanogaster." J Comp Neurol 356(2): 275-287.
Muresan, V., A. Lyass, et al. (1999). "The kinesin motor KIF3A is a component of the presynaptic ribbon in vertebrate photoreceptors." J Neurosci 19(3): 1027-1037.
Okamoto, P. M., B. Tripet, et al. (1999). "Multiple distinct coiled-coils are involved in dynamin self-assembly." J Biol Chem 274(15): 10277-10286.
Parks, A. L., K. R. Cook, et al. (2004). "Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome." Nat Genet 36(3):
288-292.
Peter, B. J., H. M. Kent, et al. (2004). "BAR domains as sensors of membrane curvature: the amphiphysin BAR structure." Science 303(5657): 495-499.
Phillips, G. R., J. K. Huang, et al. (2001). "The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution." Neuron 32(1): 63-77.
Pyle, J. L., E. T. Kavalali, et al. (2000). "Rapid reuse of readily releasable pool vesicles at hippocampal synapses." Neuron 28(1): 221-231.
Ramaswami, M., K. S. Krishnan, et al. (1994). "Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions."
Neuron 13(2): 363-375.
Richards, D. A., C. Guatimosim, et al. (2000). "Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals." Neuron 27(3):
551-559.
Rizzoli, S. O. and W. J. Betz (2005). "Synaptic vesicle pools." Nat Rev Neurosci 6(1):
57-69.
Rizzoli, S. O., D. A. Richards, et al. (2003). "Monitoring synaptic vesicle recycling in frog motor nerve terminals with FM dyes." J Neurocytol 32(5-8): 539-549.
Robinson, M. S. (1994). "The role of clathrin, adaptors and dynamin in endocytosis."
Curr Opin Cell Biol 6(4): 538-544.
Roos, J. and R. B. Kelly (1998). "Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin." J Biol Chem 273(30): 19108-19119.
Rorth, P. (1996). "A modular misexpression screen in Drosophila detecting tissue-specific phenotypes." Proc Natl Acad Sci U S A 93(22): 12418-12422.
Roux, A., G. Koster, et al. (2010). "Membrane curvature controls dynamin polymerization." Proc Natl Acad Sci U S A 107(9): 4141-4146.
Royle, S. J. and L. Lagnado (2003). "Endocytosis at the synaptic terminal." J Physiol 553(Pt 2): 345-355.
Rubin, G. M. and A. C. Spradling (1982). "Genetic transformation of Drosophila with transposable element vectors." Science 218(4570): 348-353.
Salim, K., M. J. Bottomley, et al. (1996). "Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase." EMBO J 15(22): 6241-6250.
Sara, Y., M. G. Mozhayeva, et al. (2002). "Fast vesicle recycling supports
neurotransmission during sustained stimulation at hippocampal synapses." J Neurosci 22(5): 1608-1617.
Schmid, S. L. (1997). "Clathrin-coated vesicle formation and protein sorting: an integrated process." Annu Rev Biochem 66: 511-548.
Schmid, S. L., M. A. McNiven, et al. (1998). "Dynamin and its partners: a progress report." Curr Opin Cell Biol 10(4): 504-512.
Schmidt, A., M. Wolde, et al. (1999). "Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid." Nature 401(6749): 133-141.
32
Schneggenburger, R., A. C. Meyer, et al. (1999). "Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse." Neuron 23(2): 399-409.
Sengar, A. S., W. Wang, et al. (1999). "The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15." EMBO J 18(5): 1159-1171.
Shupliakov, O., P. Low, et al. (1997). "Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions." Science 276(5310): 259-263.
Simpson, F., N. K. Hussain, et al. (1999). "SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation." Nat Cell Biol 1(2): 119-124.
Skoglund, U., L.-G. Öfverstedt, et al. (1998). "Procedures for three-dimensional reconstruction from thin sections with electron tomography. In: Schenkel, J.(Ed.) " RNP Particles, Splicing and Autoimmune Diseases. Springer Verlag;
Berlin, Heidelberg, New York: pp. 72–94.
Slepnev, V. I. and P. De Camilli (2000). "Accessory factors in clathrin-dependent synaptic vesicle endocytosis." Nat Rev Neurosci 1(3): 161-172.
Smirnova, E., D. L. Shurland, et al. (1999). "A model for dynamin self-assembly based on binding between three different protein domains." J Biol Chem 274(21):
14942-14947.
Smith, C. J. and B. M. Pearse (1999). "Clathrin: anatomy of a coat protein." Trends Cell Biol 9(9): 335-338.
Steyer, J. A. and W. Almers (2001). "A real-time view of life within 100 nm of the plasma membrane." Nat Rev Mol Cell Biol 2(4): 268-275.
Sudhof, T. C. (1995). "The synaptic vesicle cycle: a cascade of protein-protein interactions." Nature 375(6533): 645-653.
Sundborger, A., C. Soderblom, et al. (2011). "An endophilin-dynamin complex
promotes budding of clathrin-coated vesicles during synaptic vesicle recycling."
J Cell Sci 124(Pt 1): 133-143.
Sweitzer, S. M. and J. E. Hinshaw (1998). "Dynamin undergoes a GTP-dependent conformational change causing vesiculation." Cell 93(6): 1021-1029.
Takei, K., P. S. McPherson, et al. (1995). "Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals." Nature 374(6518): 186-190.
Traub, L. M., M. A. Downs, et al. (1999). "Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly." Proc Natl Acad Sci U S A 96(16): 8907-8912.
van der Bliek, A. M. and E. M. Meyerowitz (1991). "Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic." Nature
351(6325): 411-414.
Venken, K. J. and H. J. Bellen (2005). "Emerging technologies for gene manipulation in Drosophila melanogaster." Nat Rev Genet 6(3): 167-178.
Verstreken, P., O. Kjaerulff, et al. (2002). "Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release." Cell 109(1): 101-112.
Verstreken, P., T. W. Koh, et al. (2003). "Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating." Neuron 40(4): 733-748.
von Gersdorff, H. and G. Matthews (1994). "Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals." Nature 367(6465): 735-739.
von Gersdorff, H. and G. Matthews (1999). "Electrophysiology of synaptic vesicle cycling." Annu Rev Physiol 61: 725-752.
Wagh, D. A., T. M. Rasse, et al. (2006). "Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila." Neuron 49(6): 833-844.
Wigge, P., Y. Vallis, et al. (1997). "Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain." Curr Biol 7(8): 554-560.
Yamabhai, M., N. G. Hoffman, et al. (1998). "Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains." J Biol Chem 273(47):
31401-31407.
Yang, P., S. A. Shaver, et al. (2000). "Abnormal turning behavior in Drosophila larvae.
Identification and molecular analysis of scribbler (sbb)." Genetics 155(3): 1161-1174.