Nanodomains in the secretory pathway investigated by super-resolution microscopy

• Antragsteller: Professor Dr. Silvio-Olivier Rizzoli, Ph.D.
• Fachliche Zuordnung: Molekulare Biologie und Physiologie von Nerven- und Gliazellen
• Förderung: Förderung von 2010 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 178963281

Membrane traffic in the secretory pathway requires the budding of carrier vesicles from different compartments, and their subsequent fusion to their intended targets. Both processes are based on the formation of highly organized protein nanodomains (such as those involved in carrier vesicle generation or in the assembly of the docking machinery at the interface of fusing organelles). The organelle nanopatterning, however, has not been trivial to investigate, as many of the organelles themselves are on the order of the diffraction limit of light (200-300 nm). A recently developed light microscopy technique, stimulated emission depletion (STED), which narrows the focal spot far beyond the diffraction limit (Willig, Rizzoli et al., 2006; Westphal, Rizzoli et al., 2008) makes this investigation possible. I propose here to use super-resolution microscopy to investigate nanodomain patterning in two relatively well-known pathways: early endosomal recycling, using transferrin as the marker of choice, and synaptic vesicle trafficking, from the biogenesis of organelles containing vesicle markers in the Golgi apparatus to their recycling in the synapse. The short-term goal of the work is to provide a characterization of the sorting and fusion nanodomains of the early endosome, and equally to provide a thorough analysis of the nanopatterning of the synaptic vesicles, vesicle precursor membranes, and of the fused synaptic vesicles in the plasma membrane. The long-term implication of this study is to provide a better understanding of nanodomain sorting in secretory membranes, comparing two alternative hypotheses: 1) presence of minimal core structures, perhaps formed by self-organization, which persist throughout membrane recycling; 2) molecular mixing upon fusion of different membranes, followed by renewed sorting at each step of the particular pathways. The first hypothesis would indicate that stable “sub-organelles” are the basic unit of the secretory pathway; alternatively, the second hypothesis would indicate that the pathway is extremely fluid, with continuous sorting being a requirement.

• DFG-Verfahren: Sachbeihilfen