Cytoskeleton in motion: The Dynamics of Keratin Intermediate Filaments in Epithelia
Windoffer R, Beil M, Magin TM, Leube RE, 2011
The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells.
The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation.
These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.
Cytoskeleton in motion: The case of keratin intermediate filaments. The video presents aspects of the keratin cycle of assembly and disassembly that are also depicted in Fig. 2 and summarized schematically in Fig. 1. Time-lapse fluorescence recordings (between 0:00 min and 1:05 min and between 1:45 min and 3:30 min of the video) were prepared from two hepatocellular carcinoma-derived PLC cells of clone PK18-5 producing human K18-YFP fusion proteins (details in Kölsch et al., 2010). One half of one of the cells was bleached to observe fluorescence recovery. Image stacks (11 focal planes) were recorded every 5 min. Global cellular movement was compensated for by an image-intensity-based method. The projected images are shown in inverse at a display rate of 12 frames/s. The fluorescence images (also inverse presentation) presented between 1:05 and 1:45 min of the video were taken from a time-lapse epifluorescence recording (30 s recording interval) of K18-YFP in a peripheral region of an adrenal cortex carcinoma-derived SW13 cell of clone SK8/18-2 producing fluorescent keratin fusions K8-CFP and K18-YFP (details in Wöll et al., 2005) highlighting details of juxtamembranous keratin filament formation.
The high magnification images (corresponding areas are demarcated in the accompanying overviews) first depict stable filaments that are presumably anchored to the nucleus (12 frames/s). Next, inward-moving keratin filament particles are shown which nucleate in the cell periphery (marked by orange ellipsoids in a PK18-5 cell (3 frames/s) and by arrows in a SK8/18-5 cell (2 frames/s)). Newly-formed keratin particles elongate and/or fuse prior to integration into the peripheral keratin network. Inward-moving and bundling filaments are then tracked by bars in the following high resolution sequences (2.5 frames/s). The ensuing disassembly of filament bundles (first shown at 5 frames/s, then at 2 frames/s) is highlighted by arrows. The last high magnification sequence shows, how new keratin filaments appear in the cell periphery and move toward the cell interior in the bleached area (2.5 frames/s). The front of the newly-built keratin filaments is delineated by a red line.