Nuclear Protein Import: Methods

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Nuclear Protein Import: Methods

Kylie M Wagstaff, Monash University, Clayton, Australia
David A Jans, Monash University, Clayton, Australia

Published online: June 2014

DOI: 10.1002/9780470015902.a0002616.pub2

Full Article on Wiley Online Library

Abstract
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Abstract

Protein synthesis takes place predominantly in the cytoplasm, meaning that proteins that are needed in the nuclear compartment, such as those that control gene transcription, have to be transported from the cytoplasm to the nucleus. Analysis of the regulation of nuclear import, which is central to cell responses to signalling pathways, and stress responses such as viral infection, requires dynamic experimental systems able to provide quantitative kinetic information. This article describes an in vitro reconstituted system as well as quantitative live cell imaging approaches, including the technique of fluorescence recovery after photobleaching, that enable the rate and extent of nuclear import to be quantitatively determined, and assist mechanistic studies with respect to the nuclear transporters and targeting signals involved. This is critical to a full understanding of the importance of nuclear trafficking in biological systems.

Key Concepts:

The regulation of nuclear transport is critical to transcriptional control in response to cellular and developmental signals, viral infection, etc.
Using quantitative confocal laser scanning microscopy (CLSM) can enable small changes in nuclear localisation to be identified.
Systems to reconstitute nuclear transport in vitro can enable the delineation of nuclear transport mechanisms.
The technique of fluorescence recovery after photobleaching can be used to determine nuclear transport kinetics in the living cell.
Quantitative techniques to examine nuclear transport allow for sequences and mechanisms underpinning regulated nucleocytoplasmic protein transport to be dissected.

Keywords: nuclear transport; nuclear envelope; transcription factors; in vitro reconstituted system; fluorescence recovery after photobleaching

Article Title:	Nuclear Protein Import: Methods
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	Figure 1. Steady state analysis of nuclear protein import using transfection. Using chemical (e.g. liposome‐mediated) or physical (e.g. electroporation) methods, cells can be induced to take up plasmid DNA that encodes the fluorescent protein to be analysed, which will subsequently translocate from the cytoplasm to the nucleus. After 1–2 days the subcellular localisation of the protein of interest is determined using live cell confocal laser scanning microscopy (CLSM, cells can also be fixed and stained using immunofluorescence). Results (bottom) are shown for cells transfected in parallel expressing either green fluorescent protein (GFP, left) or a GFP‐tagged‐NLS‐containing protein (right). Digitised images can be analysed (using software packages such as ImageJ: NIH) for their nuclear to cytoplasmic fluorescence ratio (Fn/c), according to the formula: Fn/c=(Fn−Fb)/(Fc−Fb), where Fn=nuclear fluorescence, Fc=cytoplasmic fluorescence and Fb=background fluorescence. An Fn/c>1 indicates nuclear accumulation and <1 indicates nuclear exclusion.
	Figure 2. Reconstitution of signal‐dependent nuclear protein import in vitro. Nuclear protein import of fluorescently labelled protein can be reconstituted in vitro using mechanically perforated rat hepatoma cells (Hearps and Jans, ). Mechanical perforation removes the plasma membrane of cells, but leaves the nuclear envelope intact, the perforated cells then being inverted onto a microscope slide over a chamber of artificial ‘cytoplasm’ containing cytosolic factors, an adenosine triphosphate (ATP) regenerating system, fluorescently labelled transport substrate and a 70 kDa Texas red‐labelled dextran. The movement of the fluorescent protein is monitored over time by CLSM in the green channel as it translocates from the cytoplasm to the nucleus. Nuclear integrity is monitored simultaneously in the red channel using the 70 kDa Texas red‐labelled dextran. The dextran is too large to enter the nuclear pore by simple diffusion and is thus excluded from intact nuclei. Conversely, nuclear accumulation of the dextran indicates over perforation. Cells are scanned every 1–2 min and digitised images such as those shown (bottom left) are analysed to determine the Fn/c of the NLS‐containing protein over time (bottom right).
	Figure 3. Kinetic analysis of the regulation of NLS‐dependent nuclear protein import using FRAP. The kinetics of protein nucleocytoplasmic transport can be analysed by FRAP. Cells expressing a fluorescently tagged protein of interest are initially scanned by CLSM to determine the protein's subcellular localisation (prebleach). A region of interest, such as the nucleus, is then subjected to high laser power, permanently bleaching the fluorescent protein within that region. The cell is then monitored over time (scans taken every 20 s) for the return of fluorescent protein to the bleached area. Digitised images can be analysed for the Fn‐b and are presented as fractional recovery (bottom graph), from which kinetic measurements such as the t_1/2 (time taken to reach half maximal binding) and the maximum recovery (max. recovery) can be determined.