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First published online 19 February 2003
doi: 10.1242/jcs.00363
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Research Article |
1 Division of Cell and Developmental Biology, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, UK
2 Medical Centre for Molecular Biology, Medical Faculty, University of
Ljubljana, SI-1000, Slovenia
3 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ,
UK
* Author for correspondence (e-mail: e.b.lane{at}dundee.ac.uk)
Accepted 9 January 2003
Keratin intermediate filament networks were observed in living cultured epithelial cells using the incorporation of fluorescently tagged keratin from a transfected enhanced green fluorescent protein (EGFP) construct. In steady-state conditions EGFP-keratin exists not only as readily detectable intermediate filaments, but also as small particles, of which there are two types: a less mobile population (slow or static S particles) and a highly dynamic one (fast or F particles). The dynamic F particles move around the cell very fast and in a non-random way. Their movement is composed of a series of steps, giving an overall characteristic zig-zag trajectory. The keratin particles are found all over the cell and their movement is aligned with microtubules; treatment of cells with nocodazole has an inhibitory effect on keratin particle movement, suggesting the involvement of microtubule motor proteins. Double-transfection experiments to visualize tubulin and keratin together suggest that the movement of keratin particles can be bidirectional, as particles are seen moving both towards and away from the centrosome area. Using field emission scanning and transmission electron microscopy combined with immunogold labelling, we also detected particulate keratin structures in untransfected epithelial cells, suggesting that keratin particles may be a natural component of keratin filament dynamics in living cells.
Key words: Intermediate filaments, Keratins, Microtubules, Epidermolysis bullosa simplex, Microscopy
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