A01 - Structural and functional organization of endocytic scaffolds within the periactive zone

Principal Investigators:

Prof. Dr. Volker Haucke, Freie Universität Berlin

Dr. Tanja Maritzen, Freie Universität Berlin

Communication within the nervous system involves the exocytic fusion of neurotransmitter-filled synaptic vesicles (SVs), neurosecre­tory organelles that comprise distinct sets of proteins and lipids, at active zone membranes. Following exocytic fusion SVs are recycled locally (1) (Figure 1) largely via clathrin-mediated endocytosis (CME) of SV proteins within the surrounding periactive zone. Clathrin-based SV recycling in addition to clathrin (2) involves endocytic adaptors such as stonin 2 (3) and AP180 (4) as well as accessory proteins that contribute to SV cargo sorting, membrane deformation, remodelling, and fission. Although sub­stantial progress has been made towards unravelling the mechanics of CME, the spatio-temporal dynamics of endocytic proteins within the endocytic or periactive zone and the underlying molecular mechanisms have remained elusive. Such spatio-temporal control of the SV cycle within the nerve terminal likely involves membrane-associated multidomain scaffolds including intersectin (5) (Figure 2) and Eps15.

Within project A01 we investigate the role of Eps15/intersectin as a molecular scaffold linking SV sorting adaptors such as stonin 2/stoned B to periactive zone organization. We make combined use of cell biologi­cal, biochemical, and genetic approaches, paired with electron and super-resolution light micro­scopy (SRLM) techniques to unravel the structural and functional organization of the Eps15/ inter­sectin-based endocytic scaffold at synapses. On the long run we expect to gain new insights into the role of endocytic scaffolds in presynaptic organization and exo-endocytic coupling.

Exo-endocytic cycle of SVs

Fig. 1: Schematic depiction of the exo-endocytic cycle of SVs. The AZ is a specialized matrix for exocytosis, whereas SV endocyto­sis preferentially occurs at the sur­rounding periactive zone. SV pro­teins other than synaptobrevin are summarized as "cargo proteins" (taken from Haucke et al., Nat Rev Neurosci 2011).

Domain structure and protein interactions of intersectin 1L (ITSN1-L) (taken from Pechstein et al., Biochem. Soc. Trans. 2010)

Fig. 2: Domain structure and protein interactions of intersectin 1L (ITSN1-L) (taken from Pechstein et al., Biochem. Soc. Trans. 2010).

References:

1. Haucke, V., Neher, E., Sigrist, S.J. (2011) Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat Rev Neurosci. 12, 125-136

2. von Kleist, L. et al (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell, 146, 471-484

3. Diril, M. K., Wienisch, M., Jung, N., Klingauf, J., and Haucke, V. (2006) Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization. Dev. Cell 10, 233-244

4. Koo, S.Y., Markovic, S., Puchkov, D., Mahrenholz, C., Beceren-Braun, F., Maritzen, T., Dernedde, J., Volkmer, R., Oschkinat, O., Haucke, V. (2011) SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors CALM and AP180 at synapses. Proc. Natl. Acad. Sci. USA, 108, [advance online publication]

5. Pechstein, A., Bacetic, J., Vahedi-Faridi, A., Gromova, K., Sundborger, A., Tomlin, N., Krainer, N., Vorontsova, O., Schäfer, J.G., Owe, S.G., Cousin, M.A., Saenger, W., Shupliakov, O., and Haucke, V. (2010) Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2. Proc. Natl. Acad. Sci. USA, 107, 4206-4211

2nd International Symposium Membranes and Modules