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F-actin serves as a template for cytokeratin organization in cell free extracts

¹ Department of Zoology, University of Wisconsin, Madison, Madison, WI 53706, USA
² Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Madison, WI 53706, USA

View larger version (96K): [in a new window]   Fig. 3. Cytoskeleton interactions in rapidly frozen, fixed samples. Triple label confocal micrographs showing interactions between microtubules (red), F-actin (green) and cytokeratin filaments (blue). (A) Most of the cytokeratin is associated with F-actin cables (arrows). Frequently, the distal ends of microtubules were bent at points of contact with cables of F-actin and cytokeratin, and appeared to track along them (arrowheads). (B) En face view of cytoskeletal interactions and the same view at a tilt of 70°. The interaction of F-actin with cytokeratin (arrowheads) is apparent in both views. (C) The interaction of cytokeratin with F-actin was further examined by quantifying overlap (in pixel number) with and without rotating the channel representing cytokeratin 90° clockwise. The error bar for colocalization with rotation was too small to appear on this graph (P<0.05).

View larger version (54K): [in a new window]   Fig. 1. Cytoskeleton interactions in unfixed samples. Triple-label confocal micrograph showing interactions of microtubules (red), F-actin (green), and cytokeratin (blue) in an unfixed aster. All three systems could be found in close proximity to each other (arrows). Microtubule—F-actin associations could be observed in the absence of cytokeratin (arrowheads), and microtubule-cytokeratin interactions in the absence of F-actin (chevrons).

View larger version (61K): [in a new window]   Fig. 2. Perturbation of any single cytoskeletal system in unfixed samples does not prevent interaction of the remaining two. Triple label confocal micrographs showing that inhibition of F-actin (green) fails to prevent cytokeratin (blue) association with aster microtubules (red) (latrunculin, arrows); inhibition of cytokeratin polymerization does not inhibit F-actin association with aster microtubules (C11, arrows); inhibition of microtubule polymerization does not prevent interactions between F-actin and cytokeratin (nocodazole, arrows).

View larger version (45K): [in a new window]   Fig. 4. Perturbation of any single cytoskeletal system in rapidly frozen, fixed samples does not prevent interaction of the remaining two. Triple label confocal micrographs showing that inhibition of microtubule polymerization (nocodazole) does not prevent association of cytokeratin (blue) with F-actin (green, arrowheads). Inhibition of F-actin polymerization (latrunculin) fails to prevent cytokeratin (blue) from associating with aster microtubules (red, arrowheads). However, most of the cytokeratin is found in large aggregates on the substrate (arrow). Inhibition of cytokeratin polymerization (C11) does not inhibit F-actin association with aster microtubules (arrowheads). However, microtubules were found in small bundles on the substrate rather than as large asters. Inhibition of both microtubule and latrunculin polymerization (noc/lat) resulted in cytokeratin forming large, unorganized aggregates.

View larger version (165K): [in a new window]   Fig. 5. Cytokeratin—F-actin interactions change over time. Double-label confocal micrographs showing F-actin (red) or cytokeratin (green) rapidly frozen at increasing time intervals following warming of extract to room temperature. Immediately after warming (0'), F-actin cables are not present and cytokeratin was found as particulates on the substrate. At 5' and 10', cytokeratin filaments are present, and invariably associated with F-actin cables. By later time points (20'), both F-actin and cytokeratin cables are thicker, and cytokeratin cables are frequently found without associated F-actin (arrowheads).

View larger version (79K): [in a new window]   Fig. 6. -actinin changes the organization of both the F-actin and the cytokeratin network. Low magnification (left panels) and high magnification (right panels) double-label confocal micrographs showing F-actin (red) and cytokeratin (green) in rapidly frozen extract samples prepared after the addition of -actinin. The exogenous -actinin results in fine, highly crosslinked meshworks of F-actin cables in extracts that colocalize with cytokeratin. At high magnification, cytokeratin localization along F-actin cables in these networks appears punctate. The bar graph shows that the addition of exogenous -actinin significantly decreased the mean distance between both adjacent F-actin cables and cytokeratin relative to control samples (P<0.05).

View larger version (111K): [in a new window]   Fig. 7. Cytokeratin can move in concert with and release from dynamic actin cables. 4D analysis kymographs that exhibit colocalization of cytokeratin (green) with F-actin (red) in living extract samples. (A) Cytokeratin is initially associated with F-actin, but releases and remains behind (arrowheads) after the F-actin moves away (arrows). (B) Cytokeratin remains associated with moving F-actin throughout the time of imaging (arroheads). Arrows on either side each represent elapsed time of one minute for their respective kymograph.

View larger version (210K): [in a new window]   Fig. 8. -actinin inhibits zippering and contraction of F-actin cables in extracts. In untreated extracts, networks of F-actin zipper and contract over time (control). However, in extracts containing exogenously provided -actinin, zippering is prevented, resulting in formation of a stable network of fine cables (-actinin).

View larger version (183K): [in a new window]   Fig. 9. F-actin is cleared by astral microtubules in the absence of the cytokeratin network. Images of living extracts showing microtubules (MTs) and F-actin (Actin) with intact (Control) and disassembled (C11) cytokeratin networks. In both the presence and absence of cytokeratin filaments, the microtubules clear F-actin from around asters, indicating that cytokeratin is not required for the microtubule-actin interactions involved in clearing.

View larger version (158K): [in a new window]   Fig. 10. F-actin is required for normal cytokeratin assembly and organization in vivo. Confocal, single label micrographs showing cytokeratin distribution in Xenopus eggs. In the absence of activation, both control (A) and latrunculin-treated (C) eggs have no apparent cytokeratin network, although cytokeratin aggregates not seen in controls were seen in latrunculin-treated, unactivated eggs. Following activation, cytokeratin assembles into a fine network in control eggs (B). Cytokeratin in activated, latrunculin-treated eggs ranges from moderately disordered, forming unusually thick cables and loops (D), to completely disordered, forming extremely large aggregates (E). F is a bar graph displaying for two experiments the difference in width of filaments and/or aggregates in control versus latrunculin-treated, activated eggs. Latrunculin treatment resulted in increased thickness, as well as overall variability. The error bars for the width of filaments in control, activated eggs were too small to appear on this graph (P<0.05).

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