A06 - Organization of the synaptic active zone scaffold by Bruchpilot and RIM-binding protein
Synaptic vesicles fuse at specialized membrane compartments called active zones (AZs). The intracellular face of the AZ membrane is decorated by a proteinaceous cytomatrix, in which multiple copies of Bruchpilot (BRP) and RIM-binding protein (RBP) act as the main scaffolding proteins (Figure1; Haucke et al., 2011). In project A06 we want to elucidate the architecture of the AZ scaffold organized around BRP and RBP and delineate how this structural organization supports AZ functions. Within the last funding period we found that two BRP isoforms alternate in a circular array of oligomeric clusters to form the central body of the AZ scaffold, likely involving homo-oligomerization via a coiled coil domain in the central region of BRP. We also showed that RBP binds apparently stoichiometrically to the BRP central body and that RBP incorporation depends on its N-terminal region that also comprises a putative CC domain (Siebert et al., 2015). Results from our biochemical/biophyscial and structural investigations of the interactions of two SH3 domains in RBP with various partners support a role of RBP in concentrating effectors for synaptic vesicle release at the base of the AZ scaffold (Matkovic et al., 2013). We also elucidated the crystal structure of a triple fibronectin 3 (FN3)-like domain-containing region of RBP and are presently analyzing whether this domain array acts as an architectural element in RBP or as a protein-interaction platform in the AZ scaffold. We started to investigate the regulation of AZ scaffold assembly and function by reversible protein phosphorylation and small GTPases of the Rab family (Lardong et al., 2015). Finally, by crystallographic analysis we described the molecular interaction between Neurexin (Nrx) and PDZ domains of the early AZ assembly factors Syd-1 and Spinophilin, which contribute to BRP and RBP assembly at the AZ (Karzan et al., 2015). We will focus on fine-mapping of interaction surfaces between the core AZ components BRP and RBP as well as between these proteins and other AZ components by yeast two hybrid. Furthermore, we will continue to explore how BRP phosphorylation and binding of Rab proteins influence AZ assembly and remodeling. Finally, we plan to investigate how PDZ domain protein-Nrx interactions correlate with AZ scaffold assembly and how thereby the number and size of AZ scaffolds are determined. To these ends, we will continue to combine high precision protein localization and morphological analyses of Drosophila neuromuscular junction model synapses via nanoscopic techniques with genetics and structural biochemical approaches.
Figure 1: Molecular model of the core AZ scaffold shaped by BRP/RBP and proposed interactions.
- Petzoldt A, Götz TWB, Driller JHD, Lützkendorf J, Reddy-Alla S, Matkovic-Rachid T, Liu S, Knoche E, Mertel S, Ugorets V, Lehmann M, Ramesh N, Beuschel C, Kuropka B, Freund C, Stelzl U, Loll B, Liu F, Wahl MC, Sigrist SJ. RIM-binding protein couples synaptic vesicle recruitment to release sites. J Cell Biol 219, e201902059 (2020)
- Driller JH, Lützkendorf J, Depner H, Siebert M, Kuropka B, Weise C, Piao C, Petzoldt AG, Lehmann M, Stelzl U, Zahedi R, Sickmann A, Freund C, Sigrist SJ, Wahl MC. Phosphorylation of the Bruchpilot N-terminus unlocks axonal transport of active zone building blocks. J Cell Sci 132, jcs225151 (2019)
- Rademacher N, Kuropka B, Kunde SA, Wahl MC, Freund C, Shoichet SA. Intramolecular domain dynamics regulate synaptic MAGUK protein interactions. eLife 8, e41299 (2019)
- Gerth F, Jaepel M, Sticht J, Schmitt XJ, Kuropka B, Driller J, Loll B, Wahl MC, Pagel K, Haucke V, Freund C. Exon inclusion modulates conformational plasticity and autoinhibition of the intersectin 1 SH3A domain. Structure 27, 977-987 (2019)
Siebert, M., Böhme, M.A., Driller, J.H., Babikir, H., Mampell, M.M., Holton, N., Rey, U., Reddy-Alla, S., Gottfert, F., Kamin, D., Quentin, C., Klinedinst, S. Andlauer, T.F.M., Hell, S.W., Collins, C.A., Wahl, M.C., Loll, B., Sigrist, S.J. A high affine RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones. eLife;4:e06935 (2015)
Lardong, J.A., Driller, J.H., Depner, H., Weise, C., Petzholdt, A., Wahl, M.C., Sigrist, S.J., Loll, B. Crystal structure of Drosophila melanogaster Rab2 and Rab3 bound to GMPPNP. Acta Crystallogr. F Biol. Crystallogr. F71, 34-40 (2015)
Karzan, GH. Muhammad, K.G.H., Reddy-Alla, S., Jan H. Driller, J.H., Schreiner, D., Rey, U., Böhme, M.A., Hollmann, C., Ramesh, N., Depner, H., Lützkendorf, J., Matkovic, T., Bergeron, D.D., Schmoranzer, J., Goettfert, F., Holt, M., Wahl, M.C., Hell, S.W., Scheiffele, P., Walter, A., Loll, B., Sigrist, S.J. Presynaptic Spinophilin tunes Neurexin signaling to control active zone architecture and function. Nat Commun 8, 8362 (2015)
- Matkovic, T., Siebert, M., Knoche, E., Depner, H., Mertel, S., Owald, D., Schmidt, M., Thomas, U., Sickmann, A., Kamin, D., Hell, S.W., Bürger, J., Hollmann, C., Mielke, T., Wichmann, C. & Sigrist, S.J. The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles. J Cell Biol 202, 667-683 (2013)
- Haucke, V., Neher, E., Sigrist, S.J. Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat Rev Neurosci. 12, 125-136 (2011)