Marilyn C. Hart

Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an alpha/beta heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two beta isoforms. beta1 is at the Z-line; beta2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice. Mice expressing beta2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The beta2 did not localize to the Z-line. Therefore, beta1 has a distinct function that includes interactions at the Z-line. Mice expressing beta1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the beta2-expressing mice. The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a beta1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted. Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform. CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.

Capping protein (CP), a ubiquitous actin binding protein composed of an alpha and a beta subunit, is important for actin assembly and cell motility. Lower organisms have one gene and one isoform of each subunit. Chickens have two very similar alpha-subunit isoforms. To determine if vertebrates in general contain multiple alpha isoforms and if those alpha isoforms have conserved sequences, we isolated and analyzed alpha subunit cDNA's in mice and humans. Both mice and humans also have two alpha isoforms. Phylogenetic analysis of the alpha isoform sequences reveals that vertebrates have two highly conserved subfamilies, alpha1 and alpha2. The alpha1 and alpha2 subfamilies are very similar to each other but can be defined and distinguished from each other by a small number of key amino acid residues. In addition, 3' untranslated cDNA sequences are conserved within the isoform subfamilies. To investigate the function of the alpha isoforms, we examined their expression in mouse cells and tissues. Endothelial cells contain only the alpha2 isoform, and erythrocytes contain almost exclusively the alpha1 isoform. Most tissues have both alpha1 and alpha2 isoforms but the ratio of alpha1:alpha2 varies widely. Together, these findings support the hypothesis that the CP alpha isoforms have conserved, unique and essential roles in vertebrates.

Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC-Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca2+-dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca2+ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-epsilon after PHE or ET exposure of WT preparations. In contrast, myofilament-associated PKC-epsilon was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilament-associated PKC-beta were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca2+ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-beta.

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C2H2 zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl