In vivo detection of cytokeratin filament network breakdown in cells treated with the phosphatase
Strnad P, Windoffer R, Leube RE, 2001
We have previously described vulva carcinoma-derived A-431 subclone AK13-1, which stably expresses fluorescently labeled cytokeratin filaments (CKFs). Time-lapse fluorescence microscopy of these cells permits the continuous monitoring of the dynamics of the CKF cytoskeleton in vivo. To study mechanisms and principles of CKF disassembly as it occurs, e.g., during mitosis and liver disease, we have treated cells with the phosphatase inhibitor okadaic acid (OA), which induces complete CKF network breakdown within 3-5 h without significantly affecting the organization of the actin- and tubulin-based cytofilaments. In time-lapse movies, we find that the network breakdown starts at the cell periphery and proceeds toward the cell center, where residual filaments become compacted into a prominent perinuclear ring.
The progressing disassembly is paralleled by an increase of diffuse fluorescence throughout the cytoplasm and the appearance of non-filamentous spheroidal aggregates. They are formed in the filament-free cell periphery from non-filamentous precursors and can sometimes be detected in the proximity of desmosomes. Other aggregates are either found in close apposition to CKFs or are generated directly from the compacted perinuclear material. Primary granules later fuse, thereby producing structures of considerable size. We show that CKF network breakdown and granule formation rely on metabolic energy and that the continued presence of OA is needed for its completion. We conclude that phosphorylation/dephosphorylation is an important mechanism regulating CKF network dynamics in vivo with far-reaching implications for the understanding of epithelial plasticity and pathology.
Time-lapse fluorescence microscopy of AK13-1 cells; detection of changes in the distribution of human CK 13-EGFP chimera HK13-EGFP in the presence of OA (0.025 µg/ml). The image series was recorded at 1.5 min intervals. The typical dynamics of the spread-out network prior to addition of OA are seen from -36 min to 0 min. In the presence of OA, the centripetal movement of peripheral fluorescence continues, but filaments disappear progressively starting at the periphery. Granules are formed that subsequently fuse into larger aggregates (arrows). Concurrently, a dense perinuclear ring is formed that fragments into granules toward the end of the sequence.
Time-lapse fluorescence microscopy of AK13-1 cells detecting altering dynamics of human CK 13-EGFP chimera HK13-EGFP in response to ATP depletion (addition of 50 mM 2-deoxy-D-glucose and 0.05% sodium azide). The movie shows first the characteristic interphase dynamics of the CKF network that are abruptly inhibited upon addition of the drugs.
The movie depicts the onset of OA-induced (0.05µg/ml) CKF network reorganization during the first 90 min when ATP is depleted by addition of 50 mM 2-deoxy-D-glucose and 0.05% sodium azide. Note that granule motility, granule formation, and granule fusion do not proceed significantly after ATP depletion and that residual filaments remain mostly unaltered.
Time-lapse fluorescence microscopy of AK13-1 cells detecting HK13-EGFP during and after a short pulse of OA treatment. Cells were treated for 30 min with OA at 0.1 µg/ml (only last 18 min of OA treatment are shown) followed by incubation in OA-free medium. Partial collapse of the CK 13 filament network and concentration of filament bundles in the perinuclear region still occur after removal of the drug. Note, however, that complete CKF network breakdown does not take place but that, instead, compacted and aggregated material remains stationary, and no cytoplasmic aggregates are formed. In addition, a fine filamentous network is seen in the cell periphery in the last few frames.
Fluorescence microscopy of living AK13-1 cells detecting HK13-EGFP during and after a short pulse of OA treatment. Treatment of cells was as in movie 4 (30 min OA at 0.1 µg/ml and subsequent incubation in OA-free medium). Note the formation of granules in the cell periphery which disappear after removal of the drug.