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We have purified from a membrane fraction of bovine brain a calmodulin‐binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with Mr240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F‐actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G‐actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.

Caldesmon, a protein originally found in chicken gizzard, was concluded also to be present in bovine aorta, uterus, and human platelets by demonstration of a protein with the following properties: (a) Ca 2+-dependent calmodulin-binding; (b) binding to F actin in such way that the binding was broken on Ca 2+-dependent binding of calmodulin; (c) cross-reactivity in immune blotting procedures with affinity-purified antibody against gizzard caldesmon; (d) similar subunit Mr-values on SDS-gel to those of gizzard caldesmon. Like gizzard caldesmon, platelet caldesmon was composed of two polypeptide bands of Mr150000 and 147000, but caldesmon in aorta and uterus gave a single band of Mr150000. A polypeptide of Mr165000 that was immunologically distinct from caldesmon but, like caldesmon, bound to calmodulin and F actin in a flip-flop fashion, was also demonstrated in aorta and uterus.

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca 2-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 × 10 6M −1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca 2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated Mrof 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca 2+and calmodulin. Peak II revealed a basal activity in the absence of Ca 2+and calmodulin, which was stimulated 2-fold by addition of Ca 2+. Calmodulin had no effect on the peak II activity.

CaldesmonActin‐associated proteinCalmodulin‐binding proteinActin‐crosslinkerChicken gizzard

The addition of large amounts of myosin light chain kinase to the reconstituted gizzard actomyosin shows diphosphorylation of 20 kDa myosin light chain. Accompanying diphosphorylation, the actin-activated myosin ATPase activity was also enhanced. The extent of diphosphorylation and the myosin ATPase activity were clearly demonstrated to be in a linear relationship. From the time course experiment, the conversion of monophosphorylated light chain into one which was diphosphorylated seemed to be a sequential process. Moreover, analyzing phospho-amino acid by using a two-dimensional electrophoresis technique revealed that monophosphorylated light chain contained phosphoserine and diphosphorylated one contained phosphothreonine in addition to phosphoserine.

Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluo-rescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions. The peripheral adhesions were observed as a darker region than podosomes by inter-ference reflection microscopy. They were more easily destroyed by incubating the cells with RGD-containing peptide than were the focal adhesions. In contrast to focal adhesions and podosomes, actin bundles were not detected within the peripheral adhesions, where pp60v-src and tyrosine-phosphorylated proteins accumu-lated. Expression of the integrin was determined by the substratum composition when BY1 cells were cultured in serum-free culture medium. Under such conditions, BY1 cells expressed the peripheral adhesions within 3 hours on adhesion molecule-coated glass. On the other hand, in serum-containing medium, they first developed focal adhesions transiently at their early stage of adhe-sion, and then the peripheral adhesions were predomi-nantly expressed within 12 hours. Podosomes were formed in a time course similar to that of the periph-eral adhesions. These findings suggest that the periph-eral adhesion is a class of stable adhesive structure dis-tinct from the focal adhesion or podosome of BY1 cells. Similar close contact-type peripheral adhesions with the integrin were also observed in a variety of cultured cells such as normal fibroblasts at their logarithmic growth phase, phorbol ester-treated fibroblasts, and several malignant tumor cells, with poorly organized focal adhe-sions and stress fibers. These findings further suggest that the peripheral adhesions may be widely involved in the adhesion of cells that inadequately develop stress fibers and focal adhesions.

The expression and intracellular distribution of four contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal fibroblasts and their transformed counterparts by Rous or avian sarcoma virus were compared. By analyzing the isoformal expression of actin, caldesmon and tropomyosin using two-dimensional gel electrophoresis, only tropomyosin showed significant alteration in its isoformal expression accompanied by transformation. Morphological study revealed that in normal cells, myosin, caldesmon and tropomyosin were distributed periodically along stress fibers, but were excluded from focal adhesions (adhesion plaques), at which stress fibers terminate. By contrast, the contractile proteins were concentrated within the protrusions of the ventral cell surface of transformed cells, which are cell-adhesive structures with high motility (podosomes). Regional analysis indicated that the contractile proteins do not show diffuse distribution within podosomes. Myosin, some caldesmon and tropomyosin in association with F-actin were localized in the region surrounding the core domains of podosomes. A major part of the caldesmon was, however, located in the core domain with short F-actin bundles. In order to compare the stability and the molecular organization of stress fibers with that of the short F-actin bundles within podosomes, the dorsal plasma membranes of the cells were removed by lysis and squirting. Then, the ruptured cells were treated with various buffers containing high salt, ATP or Ca2 /calmodulin. Myosin, caldesmon and tropomyosin were strongly associated with stress fibers of the ruptured normal fibroblasts even in a buffer containing high salt or Ca2 /calmodulin. On the other hand, myosin and tropomyosin within podosomes were easily extracted by lysis and squirting. And, the remaining caldesmon in podosomes was separated from the short F-actin bundles with high salt or Ca2 /calmodulin buffer. The present findings suggest that the high motility of podosomes from transformed cells is based on the actomyosin system, and that the stable adherence of focal adhesions of normal cells is due to a lack of this system. The accumulation of contractile proteins and their dynamic association within podosomes might be the cause of the short half-life of the structures. In relation to its localization in the core domain of podosomes without myosin and tropomyosin, the function of caldesmon has been discussed.