Abstract. Keratin 5 and keratin 14 have been touted as the hallmarks of the basal keratin networks of all stratified squamous epithelia. Absence of K14 gives rise to epidermolysis bullosa simplex, a human blistering skin disorder involving cytolysis in the basal layer of epidermis. To address the puzzling question of why this disease is primarily manifested in skin rather than other stratified squamous epithelia, we ablated the K14 gene in mice and examined various tissues expressing this gene. We show that a key factor is the presence of another keratin, K15, which was hitherto unappreciated as a basal cell component. We show that the levels of K15 relative to K14 vary dramatically among stratified squamous epithelial tissues, and with neonatal development. In the absence of K14, K15 makes a bona fide, but ultrastructurally distinct, keratin filament network with K5. In the epidermis of neonatal mutant mice, K15 levels are low and do not compensate for the loss of K14. In contrast, the esophagus is unaffected in the neonatal mutant mice, but does appear to be fragile in the adult. Parallel to this phenomenon is that esophageal K14 is expressed at extremely low levels in the neonate, but rises in postnatal development. Finally, despite previous conclusions that the formation of suprabasal keratin filaments might depend upon K5/K14, we find that a wide variety of suprabasal networks composed of different keratins can form in the absence of K14 in the basal layer.

Abstract. The impact of the human/3-and 7-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human/~-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human 3,-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The/3-actin transfectants displayed well-defined filamentous organization of actin; whereas the ,y-actin transfectants displayed a more diffuse organization of the actin cables.

Abstract. We have examined the role of feedbackregulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human ~-and ~-actin genes. In ~-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human T-actin expression resulted in a progressive down-regulation of mouse/~-and ~/-actin mRNAs. Transfection of the ~-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse/3-actin mRNA. In contrast, transfection of a/~-actin gene carrying a single-point mutation ~sm) produced a feedback-regulatory response similar to that of the 7-actin gene. Expression of a /~-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the downregulation of mouse ~,-actin in both the ~-and/3sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse/3-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse 3,-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of B-actin may be distinct from that governing 7-actin. We conclude that actin feedbackregulation provides a biochemical assay for differences between the two nonmuscle actin genes.

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