Incubation of cultured cells under specific conditions induces a dramatic change in the actin organization: induction of intranuclear and/or cytoplasmic actin rods (actin paracrystal-like intracellular structures). We have found that cofilin, a 21-kDa actin-binding protein, is a component of these rods. Antibodies directed against cofilin labeled intranuclear actin rods induced in cells treated with dimethyl sulfoxide or exposed to heat shock and also labeled cytoplasmic actin rods induced in cells incubated in specific salt buffers. Moreover, we found that these actin rods are not stained with fluorescent phalloidin derivatives at all and appear to be right-handed helices, different from straight bundles of F-actin such as stress fibers. In vitro experiments revealed that cofilin and phalloidin compete with each other for binding to F-actin. Since cofilin and phalloidin have the ability to stoichiometrically bind actin molecule in the filament in vitro, the above results seem to suggest that cofilin directly binds to actin molecule in nearly an equimolar ratio in these rods. We call these rods "actin/cofilin rods." Cultured fibroblastic cells contain stress fibers consisting of a large number of parallel aligned actin filaments. A variety of incubating conditions are known to disorganize stress fibers and instead induce another prominent actin-containing structure, actin paracrystal-like intracellular structures called actin rods. Dimethyl sulfoxide (Me2SO) and heat shock induce intranuclear actin rods (1-6), whereas incubation in salt buffers induces cytoplasmic rods (7). Certain cytochalasins (8, 9), trifluorperazine (10), A23187 (11), forskolin (12), and non-ionic detergents (7) also induce intranuclear

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The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3n (PZP3n), that can be purified after the removal of sialylated and/or sulfated N-acetylpoly(1actosamine) by digestion with endo-P-galactosidase. In this study, we examined whether N-linked or 0-linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N-linked carbohydrate chains from endo-a-galactosidase-digested PZP3a by digestion with N-glycanase markedly reduced its inhibitory effect on sperm-egg binding in an in vitro competition assay, whereas the elimination of 0-linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N-linked carbohydrate chains of PZP3a play a major role in mediating the sperm binding of zona pellucida in pig.Keywords: zona pellucida; N-linked carbohydrate chains; N-glycanase; sperm-egg binding.The zona pellucida, which surrounds mammalian oocytes, consists of 3-4 glycoproteins and plays an important role in species-specific gamete recognition and the blocking of polyspermy after fertilization [I, 21. Pig zona pellucida consists of three glycoproteins: pig zona protein 1 (PZPI ; 90-kDa glycoprotein composed of disulfide-bonded 65-kDa and 25-kDa polypeptides 13, 41); pig zona protein 3n (PZP3a; 55 kDa); pig zona protein 3P (PZP3p; 55 kDa). PZP3a and PZP3p copurify in the pig zona protein 3 (PZP3) fraction during gel-filtration HPLC [5, 61. The separation of PZP3a and PZP3p is only successful after the removal of sialylated and/or sulfated N-acetylpoly(1ac-tosamine) by digestion with endo-j-galactosidase 16, 71. We refer to the purified PZP3a and PZP3p as endo-p-galactosidasedigested PZP3a (EPG-PZP3a; 46 kDa) and endo-p-galactosidase-digested PZP3P (EpG-PZP3B; 42 kDa), respectively. The major component, PZP3, inhibited sperm-egg binding in an in vitro competition assay and E,b'G-PZP3a retained the inhibitory activity [8]. EpG-PZP3P showed no inhibition in this assay [ S ] . It has been reported that the minor component PZPl has also a small but significant inhibitory effect on sperm-egg binding in an in vitro competition assay 191. Using the fluorescein-labelled components of pig zona pellucida, the binding of PZP3 and EPG-PZP3a to boar sperm has been observed, but the binding of PZPl to boar sperm has not been detected [lo]. PZPl did not effectively inhibit the binding of biotinylated PZP3 to sperm membrane [ l 11. Thus, PZP3a may play a major role in sperm- egg binding and we have focused on the involvement of EPGPZP3a in sperm-egg binding in this study.Since the chemical deglycosylation of PZP3 with trifluoromethane sulfonate abolished its sperm receptor activity 18, 91, it is suggested that the sperm receptor activity depends upon the carbohydrate component of PZP3. The involvement of carbohydrates of zona pellucida in sperm-egg recognition has been well examined in mouse. It has been reported that the sperm receptor activity of mouse ZP3 (83 kDa...

Cofilin is a 21,000-Mr actin-binding protein that widely exists in mammalian tissues. (1) A new purification procedure for porcine brain cofilin has been developed that involves (NH4)2SO4 fractionation and sequential chromatographies on Toyo Pearl and butyl-Toyo Pearl hydrophobic columns, hydroxyapatite, phosphocellulose and Sephadex G-75 gel-filtration columns. The purified cofilin bound to F-actin and increased the amount of G-actin to a limited extent, as previously reported [Nishida, Maekawa & Sakai (1984) Biochemistry 23, 5307-5313]. (2) The binding of cofilin to F-actin was scarcely affected by Mg2+, Ca2+ or by calmodulin. However, the binding was diminished by increasing concentrations of KCl, but was only slightly affected by temperature. (3) Cofilin and either alpha-actinin or filamin could bind to F-actin simultaneously with some competition, but the binding of caldesmon to F-actin was markedly inhibited by cofilin. Phalloidin inhibited the binding of cofilin to F-actin, and protected F-actin from depolymerization by cofilin.