Development of a super-resolution light microscopy approach to investigate the architecture of yeast nuclear pore complex

• Applicant: Dr. Robert Kasper
• Subject Area: Biophysics
• Term: from 2011 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 201610929

Biological systems on the molecular level often rely on complex interaction of several proteins. It is important to resolve the structural arrangement to understand the function of protein assemblies. The aim of this project is to develop a new approach based on super-resolution fluorescence microscopy to dissect the structure of macromolecular assemblies in situ. My approach is to label individual protein units within the complex, determine their relative position, and then reconstruct the full architecture. On the method side, I will further improve the STORM technique (Stochastic Optical Reconstruction Microscopy) so that it can achieve a spatial resolution at the scale of a protein molecule, which is approximately a factor of 4 better over current achievements. I plan to reach this goal by optimizing the microscope mechanical stability, dye photophysics, and fluorescent probe size. On the biology side, I will choose the yeast nuclear pore complex (NPC) as the model system to demonstrate this new approach. The large size of the NPC makes it extremely challenging for current structural biology method to obtain detailed structural understandings. In this proposal, I will focus on the Nup84 subcomplex, which known to be essential for the overall scaffold and is so far the best characterized component of the NPC. I will use the method that I have developed to investigate how the Nup84 subcomplexes are arranged to form the NPC ring, which is still heavily debated nowadays. The new super-resolution microscopy approach for macromolecular complex architecture will not be limited to the NPC. I expect it to become a widely applicable structural biology method that is complimentary to crystallography, NMR and EM because of its unique visualization ability in a cellular environment. Further developments using computer modelling to combine its outcome with other methods will undoubtedly bring us much more understandings about molecular processes in cells.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Bo Huang, Ph.D.