Funktionelle Charakterisierung der Adaptorproteine Stonin 1 und gamma-BAR beim intrazellulären Membrantransport
Zusammenfassung der Projektergebnisse
Membrane traffic within the endocytic system is based on the recognition of membrane cargo by adaptor proteins and their associated coats. How membrane traffic regulates signalling and dynamic cell behavior remains incompletely understood. Here we have tested the hypothesis that specific adaptors modulate dynamic cell behavior by connecting key components of the cytoskeleton to membrane-associated cargo. Specifically, we have demonstrated that Gadkin (previously termed γ-BAR), a palmitoylated membrane protein that partitions between the TGN and endosomal vesicles, directly associates with and regulates the actin polymerizing ARP2/3 complex via a W-containing acidic cluster motif similar to those found in NPFs. Furthermore, we demonstrated that complex formation between Gadkin and ARP2/3 negatively regulates cell spreading and motility. These data thus identify Gadkin as the first known ARP2/3 inhibitor present on intracellular membranes. In additional work conducted within this project we have demonstrated that deletion of the endocytic adaptor Stonin 2 in mice compromises the fidelity of synaptic vesicle protein sorting, whereas the apparent speed of synaptic vesicle retrieval was increased. Loss of Stonin 2 led to selective missorting of Synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated synaptic vesicle protein endocytosis, a phenotype mimicked by overexpression of endocytosis- defective variants of Synaptotagmin 1. These results are consistent with a model whereby Stonin 2 is required to preserve synaptic vesicle protein composition but is dispensable for maintaining the speed of synaptic vesicle recycling. The role of Stonin 2 is distinct from that of its close paralog Stonin 1, an endocytic protein expressed in the lung, reproductive organs, and in mouse embryonic fibroblasts (MEFs). We have shown that Stonin 1 associates with components of focal adhesions and with the endocytic machinery. Loss of Stonin 1 in MEFs alters the size, number, and dynamics of focal adhesions, thereby regulating cell motility. Further preliminary experiments indicate that these changes at least in part are due to the surface accumulation and clustering of the proteoglycan NG2, an important regulator of PDGF signaling, cell migration, and differentiation. Current efforts are directed to dissect the underlying molecular mechanisms in further detail and to analyze the importance of Stonin 1-mediated NG2 sorting in tumorigenesis in vivo.
Projektbezogene Publikationen (Auswahl)
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(2009) Regulation of endosomal membrane traffic by a Gadkin-kinesin KIF5 complex. Proc. Natl. Acad. Sci. USA, 106, 15344-15349
Schmidt, M.R., Maritzen, T., Kukhtina, V., Higman, V.A., Doglio, L., Barak, N.N., Strauss, H.M., Volkmer, R., Gensler, S., Oschkinat, H., Dotti, C.G., Haucke, V.
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(2010) Stonins - specialized adaptors for synaptic vesicle recycling and beyond? Traffic, 11, 8-15
Maritzen, T., Podufall, J., Haucke, V.
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(2010) A novel subtype of AP-1 binding motif within the palmitoylated TGN/ endosomal accessory protein Gadkin/ γ-BAR. J. Biol. Chem., 285, 4074-4086
Maritzen, T., Schmidt, M.R., Kukhtina, V., Higman, V.A., Strauss, H.M., Volkmer, R., Gensler, S., Oschkinat, H., Dotti, C.G., Haucke, V.
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(2011) Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes. Mol. Biol. Cell, 22, 2068- 2082
Laulagnier, L., Maritzen, T., Haucke, V., Parton, R.G., Gruenberg, J.
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(2012) Gadkin negatively regulates cell spreading and motility via sequestration of the actin-nucleating ARP2/3 complex. Proc. Natl. Acad. Sci. USA, 109, 10382-10387
Maritzen, T., Zech, T., Schmidt, M.R., Krause, E., Machesky, L.M., Haucke, V.
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(2013) Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2. Proc Natl Acad Sci USA, 110, E526-E535
Kononenko, N., Diril, M.K., Puchkov, D., Kintscher, M., Koo, S.J., Pfuhl, G, Winter, Y., Wienisch, M., Klingauf, J., Breustedt, J., Schmitz, D., Maritzen, T., Haucke, V.
