Related Experiment Videos
The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines
Abstract:
We have deleted cDNA sequences encoding portions of the 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 identified a collection of mutants that have a wide range of morphological effects on the endogenous keratin filament networks of transfected cells. Mutants that are missing most of the nonhelical carboxy-terminal domain of K14 incorporate into the endogenous keratin filaments without any visible perturbations on the network. In contrast, mutants that are missing as few as 10 of the 310 amino acids of the central alpha-helical domain of the polypeptide cause gross alterations in the keratin network. In some cases, the entire cytoskeletal network of keratins was disrupted, leaving no evidence of 8-nm filaments. These results reveal the existence of a dynamic exchange between newly synthesized subunits and preexisting keratin filaments.
Insights
Altering human keratin K14 protein structure impacts keratin filament networks. Even small changes in the alpha-helical domain significantly disrupt the cytoskeleton, revealing dynamic subunit exchange.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Keratins are intermediate filament proteins crucial for cellular structure and tissue integrity.
- Type I epidermal keratin K14 is a key component of the keratin cytoskeleton in epithelial cells.
Purpose of the Study:
- To investigate the molecular consequences of deleting carboxy-terminal portions of human keratin K14.
- To determine how keratin K14 mutants affect endogenous keratin filament networks in transfected cells.
Main Methods:
- Deletion mutagenesis of human keratin K14 cDNA.
- Expression of K14 mutants in simple epithelial and squamous cell carcinoma lines.
- Immunohistochemistry using an antibody against substance P to track mutant K14 expression.
Main Results:
- Mutants lacking most of the nonhelical carboxy-terminal domain incorporated into keratin filaments without apparent disruption.
- Mutants with deletions in the central alpha-helical domain caused significant alterations to the keratin network.
- Complete disruption of the keratin cytoskeleton was observed with certain alpha-helical domain mutants.
Conclusions:
- The central alpha-helical domain of K14 is critical for maintaining keratin filament network integrity.
- Newly synthesized keratin subunits dynamically exchange with preexisting keratin filaments.