Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.

Physarum actinin previously isolated [Hatano, S., & Owaribe, K. (1976) in Cell Motility (Goldman, R., Pollard, T., & Rosenbaum, J., Eds.) Vol. 3, Book B, p 499, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] was found to be a 1:1 complex of actin and fragmin which is a regulatory factor in the formation of actin filaments. Since fragmin did not contain a cysteine residue, it was purified from the complex by the selective cleavage of actin with 2-nitro-5-thiocyanobenzoic acid, followed by column chromatography. Fragmin had nearly the same molecular weight as actin, but had a quite different amino acid composition. When added to G-actin before polymerization, fragmin accelerated the initial viscosity increase of actin solutions induced by salts, but kept the final viscosity much lower than normal F-actin. When added to F-actin after polymerization, fragmin drastically reduced the viscosity of actin solutions. In both cases, the final products of reaction of fragmin with actin were short F-actin filaments. The number average length of the filaments decreased with the increasing molar ratio of fragmin to actin, and the length distribution was always exponential. Fragmin required for its activity a concentration of free Ca2+ higher than 10(-6) M. When the concentration of free Ca2+ was lower than 10(-7) M, fragmin affected neither actin polymerization nor F-actin. The regulation by Ca2+ was reversible.

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region . This process can be generated in less than 30 s . Within this process is a bundle of microfilaments . Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP . Within this extract is a protein with the same molecular weight as muscle actin . It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract . The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin . The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by : (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps . Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin . This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state .

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A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.

Abstract. Fragmin is a Ca2÷-sensitive F-actin-severing protein purified from a slime mold, Physarum polycephalum (Hasegawa, T., S. Takahashi, H. Hayashi, and S. Hatano. 1980. Biochemistry. 19:2677-2683. It binds to G-actin to form a 1:1 fragmin/actin complex in the presence of micromolar free Ca 2+. The complex nucleates actin polymerization and caps the barbed end of the short F-actin (Sugino, H., and S. Hatano. 1982. Cell Motil. 2:457-470). Subsequent removal of Ca 2÷, however, hardly dissociates the complex. This complex nucleates actin polymerization and caps the F-actin regardless of Ca 2+ concentration. Here we report that this activity of fragmin-actin complex can be abolished by phosphorylation of actin of the complex.When crude extract from Physarum plasmodium was incubated with 5 mM ATP and 1 mM EGTA, the activities of the complex decreased to a great extent. The inactivation of the complex in the crude extract was not observed in the presence of Ca 2+. In addition, the activities of the complex inactivated in the crude extract were restored under conditions suitable for phosphatase reactions. We purified factors that inactivated fragmin-actin complex from the crude extract. These factors phosphorylated actin of the complex, and the activities of the complex decreased with an increased level of phosphorylation of the complex. These factors, termed actin kinase, also inactivated the complex that capped the barbed end of short F-actin, leading to elongation of the short F-actin to long F-actin. Thus the length of F-actin can be controlled by phosphorylation of fragmin-actin complex by actin kinase. IT is considered that actin-binding or actin regulatory proteins play key roles in dynamic behaviors of actin cytoskeletons in nonmuscle cells (9,14,22,29). Actin organizes into bundles of F-actin in plasmodium of a slime mold, Physarum polycephalum. These bundles could be observed as birefringent fibers under a sensitive polarizing microscope. They appear and disappear in accordance with the contraction-relaxation cycle of plasmodium (12). Fragmin (6, 8, 27), profilin (21), and a high molecular weight actinbinding protein (25) , fragmin severs F-actin to produce shorter filaments in the presence of Ca 2÷ of more than 10-6 M (6, 8, 24). It binds to G-actin to form fragmin-actin complex. The fragmin-actin complex becomes a nucleus for actin polymerization and caps the barbed (or fast-growing) end of the short F-actin, so that annealing of the short F-actin is inhibited (24). Because these activities of fragmin are regulated by the physiological concentration of Ca 2÷, fragmin may be responsible for regulating the actin organization. However, reversal of these fragmin activities is not achieved by only removal of Ca 2+. Fragmin bound to actin in the presence of Ca 2÷ hardly dissociated even when Ca 2÷ was removed so as to be <10 -6 M. A prolonged incubation (on the order of days) of fragmin-actin complex with EGTA is required for dissociation of the complex.It has been recently reported that actin or actin re...

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