Single Molecule Studies of Nuclear Transport

Abstract

The translocation of single biomolecules through nanopores is one of the most fundamental processes of life. For example, the import of histones into the cell nucleus via the nuclear pore complex (NPC) is crucial for DNA condensation (1,2). We aim to gain a deeper understanding of NPC translocation on a single molecule level by combining optical tweezers with patch clamp techniques. This gives us the unique possibility to analyze the interaction forces between a biological macromolecule and the NPC while measuring the relevant time scales by electrophysiological recordings. We present preliminary results on the characterisation of the properties of NPCs concerning mode and duration of macromolecular translocation. Nuclei extracted from the syntactical coconut cocos nucifera are one model system. We demonstrate the selectivity of the NPC using an import signal tagged with GFP. We determine the conductance of a single nuclear pore to be approximately 490pS. Our results allow for estimation of the nuclear pore density to be between one and four per μm2. From observations of the ionic current across the pores, we are able to observe gating and to derive the translocation time of cargo across single NPCs . As a technical development towards nuclear pore force spectroscopy we introduce optical fiber illumination for real-time tracking of optically trapped particles. We demonstrate video-based fluctuation analysis of a single colloid at 10,000 Hz in order to study the dynamics of an attached biological macromolecule with microsecond time resolution (3-5).