Keratin incorporation into intermediate filament networks is a rapid process.
The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.
Miller
R K
RK
Northwestern University Medical School, Department of Cell, Molecular, and Structural Biology, Chicago, Illinois 60611.
Vikstrom
K
K
Goldman
R D
RD
D000818:Animals; D001710:Biotin; D002460:Cell Line; D006224:Cricetinae; D004173:Dipodomys; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D015603:Keratinocytes / Q000378:metabolism* / Q000648:ultrastructure; D007633:Keratins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D013997:Time Factors
D007633:Keratins; D001710:Biotin