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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Abstract. We have addressed the question of whether two highly conserved noncoding regions of the y-actin gene are of functional importance. Human y-actin gene constructs deleted for either the entire 3' untranslated region (UTR) and 3' flank or intron III sequences were transfected into mouse myoblasts and the resulting clones were analyzed for cell morphology and actin protein expression. Transfectants carrying the y-actin gene deleted for the 3' end (y22) exhibited numerous long pseudopods and increased surface area. In contrast, transfectants expressing the y-actin gene deleted for intron III (~/156) were rounded with blebs over the cell surface and showed decreased surface area. The relative expression of/3-to 3,-actin protein decreased for both transfectant types. The total actin protein levels remained constant for the y22 cells but decreased for the 3,156 cells. The results indicate that alterations to transfectant cell morphology can be influenced by the presence or absence of different noncoding regions in the transfected 3,-actin gene. The mechanisms by which noncoding regions of the y-actin gene influence the impact of the gene are unknown. Nevertheless, these noncoding regions are isoform specific and highly conserved in evolution. We propose that the functional significance of the different actin isoforms may involve the properties of these noncoding regions in addition to the differences in protein sequence.

Abstract. Phenotypically altered C2 myoblast cells, generated by the stable transfection of human nonmuscle actin genes (Schevzov, G., C. Lloyd, and P. Gunning. 1992. J. Cell Biol. 117:775-786), exhibit a differential pattern of tropomyosin cellular organization and isoform gene expression. The/~-actin transfectants displaying a threefold increase in the cell surface area, showed no significant changes in the pattern of organization of the high Mr tropomyosin isoform, Tm 2, or the low Mr tropomyosin isoform, Tm 5. In contrast, the 3'-and Bsm-actin gene transfectants, exhibiting a twofold decrease in the cell surface area, had an altered organization of Tm 2 but not Tm 5. In these actin transfectants, Tm 2 did not preferentially segregate into stress fiber-like structures and the intensity of staining was greatly diminished. Conversely, a welldefined stress fiber-like organization of Tm 5 was observed. The pattern of organization of these tropomyosin isoforms correlated with their expression such that a profound decrease in Tm 2 expression was observed both at the transcript and protein levels, whereas Tm 5 remained relatively unchanged. These results suggest that relative changes in nonmuscle actin gene expression can affect the organization and expression of tropomyosin in an isoform specific manner. Furthermore, this apparent direct link observed between actin and tropomyosin expression suggests that nonpharmacological signals originating in the cytoskeleton can regulate cytoarchitectural gene expression.

Previous studies have demonstrated a strong correlation between the expression of vinculin and the shape and motility of a cell (Rodriguez Fernandez et al., 1992a, b, 1993). This hypothesis was tested by comparing the expression of vinculin and talin with the motility of morphologically altered myoblasts. These mouse C2 myoblasts were previously generated by directly perturbing the cell cytoskeleton via the stable transfection of a mutant-form of the beta-actin gene (beta sm) and three different forms of the gamma-actin gene; gamma, gamma minus 3'UTR (gamma delta'UTR), and gamma minus intron III (gamma delta IVSIII) (Schevzov et al., 1992; Lloyd and Gunning, 1993). In the case of the beta sm and gamma-actin transfectants, a two-fold decrease in the cell surface area was coupled, as predicted, with a decrease in vinculin and talin expression. In contrast, the gamma delta IVSIII transfectants with a seven-fold decrease in the cell surface area showed an unpredicted slight increase in vinculin and talin expression and the gamma delta 3'-UTR transfectants with a slight increase in the cell surface area showed no changes in talin expression and a decrease in vinculin expression. We conclude that changes in actin gene expression alone can impact on the expression of vinculin and talin. Furthermore, we observed that these actin transfectants failed to show a consistent relationship between cell shape, motility, and the expression of vinculin. However, a relationship between talin and cell motility was found to exist, suggesting a role for talin in the establishment of focal contacts necessary for motility.