Single-Molecule Imaging to Characterize the Transport Mechanism of the Nuclear Pore Complex
In the eukaryotic cell, a large macromolecular channel, known as the Nuclear Pore Complex (NPC), mediates all molecular transport between the nucleus and cytoplasm. In recent years, single-molecule fluorescence (SMF) imaging has emerged as a powerful tool to study the molecular mechanism of transport through the NPC. More recently, techniques such as single-molecule localization microscopy (SMLM) have enabled the spatial and temporal distribution of cargos, transport receptors and even structural components of the NPC to be determined with nanometre accuracy. In this protocol, we describe a method to study the position and/or motion of individual molecules transiting through the NPC with high spatial and temporal precision.
Key wordsNucleus Nuclear pore complex Single-molecule tracking Super-resolution microscopy
Grace Jeremy is supported by a Wellcome Trust studentship. We thank Anthony Roberts for criticial reading of the manuscript. We also thank the Hayward, Waksman and Fassati labs for contributions of reagents, equipment, and expertise. The Lowe lab acknowledges support from the Medical Research Council award MR/K015826/1 Super Resolution Imaging for Cell Biology and Neuroscience at UCL.
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