Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network.
Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.
Kartasova
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Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
Roop
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Holbrook
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Yuspa
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D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002459:Cell Fusion; D004247:DNA / Q000235:genetics; D004817:Epidermis / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D066298:In Vitro Techniques; D007382:Intermediate Filaments / Q000648:ultrastructure*; D015603:Keratinocytes / Q000648:ultrastructure*; D007633:Keratins / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008854:Microscopy, Electron; D014162:Transfection
D007633:Keratins; D004247:DNA