Keiko Kanda

α-Actinins, Calspectin (Brain Spectrin or Fodrin), and Actin Participate in Adhesion and Movement of Growth Cones

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.

Actin polymerization induced by calspectin, a calmodulin-binding spectrinlike protein

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 M r 240 000 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.

Primary structure and functional expression of h-caldesmon complementary DNA.

Recently, the two Mr forms of caldesmon (Mrs in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence. This sequence predicts a protein of 771 amino acids with a Mr of 88,743. The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids. Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized. Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol. wt. with 150kDa as measured by SDS-PAGE. This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions. A recombinant protein produced in E. coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon.

Caldesmon, a calmodulin-binding, F actin-interacting protein, is present in aorta, uterus and platelets

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) Ca2+‐dependent calmodulin‐binding; (b) binding to F actin in such way that the binding was broken on Ca2+‐dependent binding of calmodulin; (c) cross‐reactivity in immune blotting procedures with affinity‐purified antibody against gizzard caldesmon; (d) similar subunit M r‐values on SDS‐gel to those of gizzard caldesmon. Like gizzard caldesmon, platelet caldesmon was composed of two polypeptide bands of M r 150000 and 147000, but caldesmon in aorta and uterus gave a single band of M r 150000. A polypeptide of M r 165000 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.

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

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 M r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca2+ and calmodulin. Peak II revealed a basal activity in the absence of Ca2+ and calmodulin, which was stimulated 2‐fold by addition of Ca2+. Calmodulin had no effect on the peak II activity.

Reconstitution of Ca2+-sensitive gelation of actin filaments with filamin, caldesmon and calmodulin

The discovery of Ca2+-activatable cyclic nucleotide phosphodiesterase [1 ] and the subsequent demonstration of a protein factor which confers Ca2÷-sensitivity upon this enzyme [2,3] coincided with the discovery of a protein activator of brain phosphodiesterase [4]. The identity of the 2 proteins as a Ca2÷-binding protein (calmodulin) was established subsequently [5]. The structural similarity of calmodulin and troponin Cs [6,7] suggests that both proteins may have stemmed from a common ancestral protein and may perform analogous functions as 4-domain calcium receptive proteins. However, surprisingly few studies have been done with regard to the interaction of calmodulin with components of the contractile or cytoskeletal system except for myosin light chain kinase. Calmodulin associates with skeletal muscle troponin components to form a soluble hybrid complex and neutralizes the inhibitory action of troponin I on the actomyosin ATPase activity [8]. A similar observation was made in [9]. We have purified, from erythrocytes [10,11 ], brain [12] and chicken gizzard smooth muscle [13,14], actin-related proteins that bind to calmodulin in the presence of Ca 2÷. The calmodulinbinding protein from chicken gizzard, named caldesmon, interacted with calmodulin and F actin in the presence or absence, respectively, of Ca 2÷ and forma. tions of the 2 species of protein complexes is regulated by [Ca 2+] in a flip-flop fashion [14]. Here, this mechanism is extended to the control of the filamininduced gelation of actin filaments: calmodulincaldesmon system conferred Ca2+-sensitivity upon the

Localization of pp60c-src in growth cone of PC12 cell.

By immunocytochemical and biochemical techniques, we observed the localization and expression of pp60c-src in nerve growth factor (NGF)-treated PC12 cells. Immunostaining of pp60c-src is detected in the neuronal soma and the tips of neurites (growth cones). Immunofluorescence in the neurites is less significant. High-resolution microscopy reveals that the location of pp60c-src in growth cone is in good agreement with the adhesive site of growth cone to the substratum. The pp60c-src kinase activity and the pp60c-src protein level increase 3.1- to 3.5-fold and 2.0-fold during differentiation of PC12 cells, respectively. The pp60c-src levels in the neurite fraction are also higher than those in the neuronal soma fraction. These results support the immunocytochemical finding that pp60c-src is localized in growth cones of differentiated PC12 cells. Furthermore, we discuss the possible role of pp60c-src in growth cone.

Crosslinking of actin filaments is caused by caldesmon aggregates, but not by its dimers

Caldesmon Actin‐associated protein Calmodulin‐binding protein Actin‐crosslinker Chicken gizzard

Calspectin (fodrin or nonerythroid spectrin)-actin interaction: A possible involvement of 4.1-related protein

The calspectin/actin complex extracted from the bovine brain membrane crosslinks F-actin, resulting in the increasing viscosity of F-actin determined by low-shear viscometry. We demonstrated the presence of a protein factor in this complex, which regulated the calspectin-F-actin interaction in a Ca2+- and calmodulin-dependent manner. Erythrocyte protein 4.1, but not synapsin I, mimics the function of this brain factor using a reconstitution system including purified calspectin, calmodulin and F-actin. In the brain complex, the Mr 120,000 and the Mr 80,000/77,000 polypeptides were detected to crossreact with anti-protein 4.1 antibody.

Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats, with special regard to the relationship between actin filaments and secretory granules*

SummaryAs previously reported, in anterior pituitary cells of the rat, secretory granules are linked with adjacent granules, cytoorganelles, microtubules, and plasma membrane by thin filaments, 4–10 nm in diameter. The quick-freeze, deep-etching method revealed that some of the filaments linking adjacent secretory granules show 5 nm-spaced striations on their surface which are known to be characteristic of actin. Immunocytochemistry showed that actin is localized in the cytoplasm beneath the plasma membrane, and around or between secretory granules. The heavy meromyosin decoration method demonstrated that actin filaments are mainly located in the cytoplasm beneath the plasma membrane, while some actin filaments are connected with the limiting membrane of the secretory granules. The actin filaments associated with the secretory granules are considered to be involved in the intracellular transport of the granules, while those localized in the peripheral cytoplasmic matrix might control the approach of the secretory granules to the plasma membrane and their release.