Abstract. From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 Ixm) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope.Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 ° to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length.Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction.We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmillingtype mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.T o move over a natural or synthetic substrate, metazoan cells protrude a thin layer of cytoplasm, a lamellipodium, that establishes new anterior contacts required for forward locomotion (see review by Heath and Holifield, 1991). The lamellipodium is thus the primary locomotory organelle (Abercrombie et al., 1970), but its gross moveme...

Calponin is a basic smooth-muscle-specific protein capable of binding to F-actin, tropomyosin and calmodulin in vitro. Using two-dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a PI identical to the most basic adult a variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees-Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985 -59911, which has sequence motifs related to calponin, displays a similar isoform pattern during development; one isoform (a) is present in the embryo and three in the adult.In living smooth-muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32P04, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two-dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. In vitro translation of porcine and chicken smooth-muscle mRNA produced only a single (a) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the a isoform and a second more basic spot, not observed in tissue samples.Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down-regulated like calponin, metavinculin and heavy-caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the a SM 22 isoform during smooth-muscle phenotype modulation in vitro.Smooth muscle differentiation is characterized by the expression of a family of cytoskeletal and contractile proteins, among them specific actin and myosin isoforms [l -31, desmin [4], metavinculin [5, 61 and the higher-molecular-mass variant of caldesmon [A. Two basic proteins, conspicious only in twodimensional gels, are also strongly and specifically expressed in smooth muscle tissue, namely calponin [8] and SM 22 [9]. Calponin has recently received much attention since current data suggest that this protein may reside on the actin thin filaments and serve as a modulator of actomyosin ATPase [9a; reviewed in 12 and 131. The mechanism of actin-linked regulation via calponin is, however, by no means clear, either with regard to the possible role of phosphorylation [9a, 101 or with respect to the reported additional involvement of caldesmon [14]. Since SM 22 could not be shown to bind to any contractile proteins with significant affinity [9, 151, not even a tentative role could be ascribed to this protein. It was noteworthy, however, that independent sequence analysis of Correspondence to M. Gimona. Institute of Molecular Biology, Austrian Acadcmy of Sciences,...

Using a synthetic peptide mimicking the NH2-terminus of beta-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the beta isoform, by its exclusive recognition of the synthetic beta-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the beta-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function.

Abstract. Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, a,-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and ~-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged •4.5 #m. Myosin filaments in the supercontracted dimers averaged 1.6 #m in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.

Calpomn is a smooth muscle specific, actin-, tropomyosmand calmodulin-bindmg protein thought to be involved in some way m the regulation or modulatton of contraction.Here we describe the cloning and bacterral expression of two calponin species from murine and porcine smooth muscle tissues. Primary and secondary structural analyses of the deduced amino actd sequences revealed a high degree of homology to avian calponm with the exception of a short and variable C-terminal segment. The sequence data demonstrate that the two mammalian calponin variants do not arise via alternative splicing but are encoded by dtfferent genes Smooth muscle: Calponin: Actm-binding protein: Bacterial expression