Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments.
We have deleted cDNA sequences encoding portions of the amino- and carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have defined the limits of K14 sequence necessary to incorporate into a keratin filament network in vivo without disrupting its architecture. We have also uncovered major differences in the behavior of carboxy- and amino-terminal alpha-helical mutants which do perturb the cytoskeletal network of IFs: whereas carboxy terminal mutants give rise to aggregates of keratin in the cytoplasm, amino-terminal mutants tend to produce aggregates of keratins which seem to localize at the nuclear surface. An examination of the phenotypes generated by the carboxy and amino-terminal mutants and the behavior of cells at late times after transfection suggests a model whereby initiation of filament assembly occurs at discrete sites on the nuclear envelope and filaments grow from the nucleus toward the cytoplasm.
Albers
K
K
Howard Hughes Medical Institute, University of Chicago, Illinois 60637.
Fuchs
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003062:Codon; D003599:Cytoskeleton / Q000648:ultrastructure*; D004247:DNA / Q000235:genetics*; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005796:Genes; D007382:Intermediate Filaments / Q000378:metabolism / Q000648:ultrastructure*; D007633:Keratins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014162:Transfection
D003062:Codon; D007633:Keratins; D004247:DNA