Probing Nucleocytoplasmic Transport with Fluorescence Fluctuation Spectroscopy and Two-photon Activation of Photoactivable GFP

Abstract

Large proteins and macromolecular complexes have to enter and leave the nucleus in an efficient and selective manner. Macromolecules that are greater than 40 kD are transported actively across the nuclear envelope through nuclear pore complexes using soluble transport factors or carrier molecules that cycle between the cytoplasm and nucleus. The carrier proteins themselves interact with each other in order to transport cargo proteins across the nuclear pore complexes. In this work, we apply dual-color time-integrated fluorescence cumulant analysis (TIFCA), a fluorescence fluctuation spectroscopy technique, to investigate the protein interactions of the carrier proteins directly in cells. In addition, we apply two-photon activation to directly examine the nucleocytoplasmic transport of photoactivable GFP tagged carrier proteins. With these two approaches, we are able to probe the nucleocytoplasmic transport process of NTF2 directly inside cells and under equilibrium conditions. We investigate the oligomerization of NTF2 in cells and its transport properties when crossing the nuclear pore complexes.The experiments identify the presence of NTF2 dimers in the cytoplasm and nucleus of cells, while the measured transport properties across the nuclear pore complex are not consistent with dimeric NTF2. We will discuss the implications of these results for functions of nuclear envelope and models of nucleocytoplasmic transport. This work is supported by the National Science Foundation (PHY-0346782) and NIH grant R01GM064589.