In vivo detection of cytokeratin filament network breakdown in cells treated with the phosphatase inhibitor okadaic acid.
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.
Strnad
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P
Department of Anatomy, Johannes Gutenberg University Mainz, Becherweg 13, 55128 Mainz, Germany.
Windoffer
R
R
Leube
R E
RE
D000255:Adenosine Triphosphate / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D051180:Desmoplakins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000648:ultrastructure; D005260:Female; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D019319:Okadaic Acid / Q000494:pharmacology*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014846:Vulvar Neoplasms
D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; D007633:Keratins; D000255:Adenosine Triphosphate