Optical single transporter analysis of the nuclear pore complex

• Applicant: Professor Dr. Reiner Peters
• Subject Area: Biophysics
• Term: from 2000 to 2007
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5239456

Substantial progress has been made in identifying and characterizing the components involved in nucleocytoplasmic transport. However, the mechanisms by which macromolecules are actually translocated across the nuclear pore complex (NPC) are still not very well understood. We recently described a fluorescence microscopic method, Optical Single-Transporter Recording (OSTR), which can provide new access to the topic by permitting to record the transport across single NPCs and to independently manipulate the media on both the cytoplasmic and nuclear side of the NPC. In this project we want to further explore the possibilities of OSTR for the elucidation of nucleocytoplasmic transport mechanisms concentrating on two fundamental aspects: - the cross-talk between individual translocation events, - the energy requirements and reversibility of translocation. To accomplish these aims we want: a) to use defined mixtures of recombinant transport factors instead of cell extracts; b) to generate new recombinant transport substrates which permit to simultaneously study signal-mediated import and export as well as passive transport; c) to employ a flash photolysis method to photochemically generate concentration steps of various nucleotides across individual NPCs; d) to control the temperature during OSTR measurements to determine the activation energy of translocation and thus to identify and possibly 'freeze in' kinetic intermediates.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1050: Funktionelle Architektur des Zellkerns