Cleavage furrow formation and actin-modulating proteins.
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Actins↗
Biomedical subjects
I Mabuchi
Publications and source records attributed to I Mabuchi.
End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin.
A 1:1 complex of actin and depactin, an actin-depolymerizing protein isolated from starfish oocytes [Mabuchi, I. (1983) J. Cell Biol. 97, 1612-1621], was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to introduce covalent bonds at their contact site. Locations of cross-linking sites were identified along the depactin sequence by the end-label fingerprinting, which employed site-directed antibodies against the N- and C-termini of depactin as end labels. Mappings with these end labels have revealed that the N-terminal segment of depactin (residues 1-20) contains sites in contact with the N- and C-terminal segments of actin, both of which participate in interaction with depactin [Sutoh, K., & Mabuchi, I. (1986) Biochemistry 25, 6186-6192].
Actin Depolymerizing Factors↗
[Mechanism of cytokinesis: isolation and properties of cleavage furrow].
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Animals↗
Amino acid sequence of starfish oocyte depactin.
The amino acid sequence of starfish oocyte depactin was determined by aligning lysyl endopeptidase, Staphylococcus aureus V8 protease, and cyanongen bromide peptides. Starfish oocyte depactin is composed of 150 amino acid residues and the N terminus is free proline. The molecular weight is calculated to be 17,590, in good agreement with the value estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Prediction of the secondary structure shows that depactin contains 64% alpha-helix. Comparison of depactin sequence with those of other proteins shows no significant similarity.
Actin Depolymerizing Factors↗
Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization.
Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM- or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC- or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 microM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.
Actins↗
Cleavage furrow isolated from newt eggs: contraction, organization of the actin filaments, and protein components of the furrow.
The cleavage-furrow region was isolated surgically from newt eggs at the early stage of the first cleavage. The isolated furrow contracted in the presence of ATP at a Ca2+ concentration of 10 or 100 nM, although the speed was less than that of the furrow in vivo. Cytochalasin B, cytochalasin D, phalloidin, p-chloromercuribenzoate, and N-ethyl-maleimide interfered with the contraction, but colchicine did not. The furrow contained bundles of actin filaments of opposite polarities oriented parallel to the long axis of the furrow; these bundles may be the main component of the contractile arc. From electron microscopic observation of thin sections of the furrow, it was suggested that the actin bundles of the contractile arc were organized from preexisting cortical filaments that were connected to the plasma membrane by granular materials at their barbed ends. Contractile-arc actin filaments were revealed to be crosslinked by thin strands by the rapid freezing/deep etching-replication technique. Two-dimensional polyacrylamide gel electrophoresis showed that several proteins found in the furrow cortex are absent from the cortical layer before the cleavage furrow is formed.
Actin Cytoskeleton↗
A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex.
A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.
Actin Depolymerizing Factors↗
The amino acid sequence, immunofluorescence and microinjection studies on the 15 kDa calcium-binding protein from sea urchin egg.
The 15 kDa protein is the most abundant low molecular weight Ca2+-binding protein, different from calmodulin, in eggs of sea urchin, Hemicentrotus pulcherrimus. The data from the amino acid sequence demonstrated that the 15 kDa protein belonged to the troponin C superfamily. Based on immunofluorescent and immunomicroscopic observations, we showed that the 15 kDa protein localized in the nuclei of fertilized eggs and mitotic apparatus of dividing eggs. Microinjection of the antibody against 15 kDa protein into sea urchin blastomeres resulted in the arresting of cell division. These results suggest that the 15 kDa protein plays an important role in mitosis of sea urchin egg.
Amino Acid Sequence↗
Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg.
We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".
Actins↗
Wave of cortical actin polymerization in the sea urchin egg.
The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.
Actins↗
Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes.
When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.
Actin Cytoskeleton↗
A 250K-molecular-weight actin-binding protein from actin-based gels formed in sea urchin egg cytoplasmic extract.
The actin-based gel formed at 35 degrees C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the presence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170 nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20 mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R.E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.
Animals↗
Improved method for mapping the binding site of an actin-binding protein in the actin sequence. Use of a site-directed antibody against the N-terminal region of actin as a probe of its N-terminus.
An antibody was raised against the N-terminal 18 residues of rabbit skeletal muscle actin. By the use of this antibody as the N-terminal probe of actin and the fluorescent label at Cys-374 as its C-terminal probe, binding sites of depactin (an actin-depolymerizing protein from starfish oocytes) were identified in the actin sequence according to the method of Sutoh [Sutoh, K. (1982) Biochemistry 21, 3654-3661]. Cross-linking of the one-to-one complex of actin and depactin with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) generated two types of cross-linked products with slightly different apparent molecular weights, denoted as 60KU and 60KL. By the use of the N-terminal probe, it was unequivocally revealed that the C-terminal actin segment of residues 357-375 participated in cross-linking with depactin to form 60KL. On the other hand, by the use of the C-terminal probe it was revealed that the N-terminal actin segment of residues 1-12 participated in cross-linking with depactin to form 60KU. Since EDC cross-links Lys residue with Asp or Glu residue only when they are in direct contact, the result indicates that some of the N-terminal residues 1-12 and the C-terminal residues 357-375 of actin participate in binding depactin. The introduction of the N-terminal probe (the antibody recognizing the actin N-terminus) has increased the flexibility of the mapping method for locating binding sites of actin-binding proteins in the actin sequence.
Actin Depolymerizing Factors↗
A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus.
A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.
Amino Acids↗
An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg.
An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.
Actin Depolymerizing Factors↗
Biochemical aspects of cytokinesis.
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Actin Cytoskeleton↗
Acrosomal actin bundles of Asian and American horseshoe crab sperm differ in protein composition.
Acrosomal actin bundles were isolated from the sperm of horseshoe crabs from four different sources, three from Asia and one from North America, and their protein constituents and structures were compared. The bundle from the American Limulus polyphemus sperm was composed of actin and two associated proteins of MW 95,000 and MW 52,000, as reported previously (Tilney, L. G. (1975) J. Cell Biol. 64, 289-310). However, those from the three Asian species (Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda) were composed only of actin and the protein of MW 95,000. Electron microscopic and optical diffraction studies indicated that both the helical structures and the interfilament spacing of the actin filaments composing the acrosomal bundle were indistinguishable among the four species. These results suggest that the MW 95,000 protein crosslinks actin filaments in the bundle. Moreover, they support the idea that Limulus and the three Asian species have evolved independently from a common ancestor.
Acrosome↗
The 45K molecular weight actin-modulating protein from sea urchin eggs forms a complex with actin in the presence of calcium ions.
The 45K protein from sea urchin eggs forms a complex with actin in the presence of Ca2+. The fraction was not readily dissociated on depletion of Ca2+ by gel filtration, but was dissociated on dialysis against an EGTA-containing solution. The free 45K protein and the 45K protein-actin complex affect F-actin in different manners in the presence of Ca2+. The former severs F-actin in the presence of Ca2+ but not in its absence, while the latter caps the barbed end of F-actin in a Ca2+-independent manner thereby lowering the viscosity of F-actin slowly.
Actins↗