Biomedical subjects

I Mabuchi

Publications and source records attributed to I Mabuchi.

At least 37 records · Page 2Linked to original sources

PubMed1997

Purealin blocks the sliding movement of sea urchin flagellar axonemes by selective inhibition of half the ATPase activity of axonemal dyneins.

Ciliary and flagellar movements are explained by active sliding between the outer doublet microtubules of an axoneme via their inner and outer dynein arms. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, blocked the motility of Triton-demembranated sea urchin sperm flagella within 5 min at concentrations above 20 microM. In a similar concentration range, purealin blocked the sliding movement of the flagellar axonemes in vitro within a few minutes judging from the turbidity measurements. The ATPase activity of axonemes was partially inhibited by purealin in a concentration-dependent manner. The maximum inhibition reached approximately 50% at concentrations above 20 microM, indicating that half the axonemal ATPase activity is sensitive to purealin. Similar results were observed on the ATPase activity of outer-arm-depleted axonemes and that of a mixture of 21S dynein and salt-extracted axonemes. On the other hand, ATPase activity of isolated 21S dynein was not inhibited by purealin. The inhibitory action of purealin on the axonemal ATPases was reversed by dilution of purealin. The effect of purealin on the double-reciprocal plot of the ATPase activity as a function of ATP concentrations showed that the inhibition was not a competitive type. In accord with this finding, purealin did not affect the vanadate-mediated UV photocleavage of axonemal dyneins. These results suggest that purealin binds reversibly to a site other than the catalytic ATP-binding site and inhibits half the ATPase activity of axonemes. Taken together, our results suggest that purealin-sensitive ATPase activity of the dynein arms plays an essential role in generating the sliding movement of flagellar axonemes.

Adenosine Triphosphatases↗

PubMed1997

Identification of p34cdc2 kinase from sea urchin Hemicentrotus pulcherrimus and its involvement in the phosphorylation of myosin II regulatory light chain in the metaphase extract.

We present here the nucleotide sequence for a cDNA clone encoding p34cdc2 from sea urchin, Hemicentrotus pulcherrimus. The obtained cDNA comprised 301 amino acid residues that contained the PSTAIRE domain to be important for binding to cyclins. Amino acid sequence similarity between this clone and other eukaryotic cdc2 sequences averaged approximately 72%. Using p13suc1-conjugated Sepharose 4B and a selective inhibitor of p34cdc2 kinase, butyrolactone I, it was first suggested that p34cdc2 kinase is involved in the phosphorylation of MRLC at both MLCK site and two PKC sites.

Amino Acid Sequence↗

PubMed1997

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe.

BACKGROUND: The small GTP-binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+, from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1. RESULTS: We found that Rho1 is essential for cell viability and cell polarity using gene disruption and by exogenous expression of botulinum C3 ADP-ribosyltransferase. In cells expressing either a constitutively active Rho1 or a dominant-negative Rho1, actin patches were delocalized. Both the cell wall and secondary septum were thick and stratified in cells expressing the constitutively active Rho1, while the cell wall of cells expressing the dominant-negative Rho1 seemed to be loosely organized. Furthermore, inactivation of Rho1 is apparently required for the separation of daughter cells. Cell fractionation studies suggested that Rho1 is predominantly membrane-bound. Moreover, we observed that Rho1 is localized to the cell periphery and to the septum. CONCLUSIONS: Rho1 is involved in actin patch localization, the control of cell polarity, the regulation of septation, and cell wall synthesis.

ADP Ribose Transferases↗

PubMed1997

Identification of actin-binding proteins from sea urchin eggs by F-actin affinity column chromatography.

Novel F-actin binding proteins of sea urchin eggs were searched for in order to study regulation of the actin cytoskeleton during fertilization and cell division. An extract of unfertilized eggs was analyzed by F-actin column chromatography. Several previously characterized F-actin-modulating proteins such as spectrin, myosin, and fascin bound to the column. The eluates from the column also contained proteins having apparent molecular weights of 225K, 150K, 70K, 60K, 45K, 40K, 38K, 36K, 34K, 20K, and 15K, which were thought to be novel cytoskeletal proteins judging from their molecular weights and non-reactivity to antibodies against previously characterized F-actin-modulating proteins. Most of the proteins in the F-actin column eluates co-sedimented with F-actin. Partial amino acid sequences of the peptides derived from the 45K and 40K proteins showed that these proteins are homologous to Arp3 and Arp2 subfamilies of actin-related proteins, respectively. The 150K protein seemed to be an unconventional myosin, that belongs to myosin VI subfamily. Amino acid sequences of two fragments from the 60K protein showed homology to that of coronin. The 150K protein was localized by immunofluorescence microscopy to the cleavage furrows in both whole cell sample and isolated cortex of dividing eggs. The 70K protein was uniformly localized in the cortical layer in the whole egg, but weak staining of the cleavage furrow region with the antiserum was observed in the isolated cortex. The 60K protein was localized to both the bulk cortical layer and the cleavage furrow, but the modes of localization were different.

Actins↗

PubMed1997

Isolation of cleavage furrows from eggs of regular sea urchins and identification of furrow-specific proteins.

We have developed a method for the isolation of cleavage furrows from dividing sea urchin eggs, which is applicable to various sea urchin species. The new method differs from that used for isolating cleavage furrows from sand dollar Clypeaster japonicus eggs [Yonemura, S., Mabuchi, I., and Tsukita, S. (1991) J. Cell Sci. 100, 73-84] in the type and concentration of detergent included in the isolation medium, the temperature during the treatment of dividing eggs with the isolation medium, and the centrifugation conditions. The contractile ring was included in the isolated cleavage furrows, as seen on rhodamine-phalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and beta-glycerophosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus. When the isolation was carried out in the absence of both NaF and beta-glycerophosphate, cleavage furrows without the mitotic apparatus were obtained. The development of a method of isolation of cleavage furrows from regular sea urchin eggs enabled us to compare protein constituents among furrows from different sea urchin and sand dollar species. We found that 32, 36, and 51 kDa proteins were concentrated in common in the cleavage furrows isolated from eggs of the sand dollars, C. japonicus and Scaphechinus mirabilis, and the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus nudus, on two-dimensional gel electrophoreses.

Animals↗

PubMed1997

Interaction of flagellar inner arm dynein isolated from sea urchin sperm with microtubules in the presence of ATP.

We have isolated C/A dynein, which is considered to be a component of inner arms, from flagellar axonemes of sea urchin sperm (E. Yokota, I. Mabuchi, J. Cell Sci. 107, 345-351 (1994). C/A dynein binds to and bundles the microtubules in the absence of ATP. In contrast to outer arm 21S dynein, C/A dynein is not released from the microtubules in the presence of ATP (E. Yokota, I. Mabuchi, J. Cell Sci. 107, 353-361 (1994)). We further investigated the interaction of C/A dynein with microtubules in the presence of ATP. The turbidity at 350 nm of a mixture of C/A dynein and microtubules increased by the addition of ATP. Both the initial rate and final extent of the turbidity increase were dependent on C/A dynein or ATP concentration and were inhibited by vanadate. ATP hydrolysis by C/A dynein was linear during the time course of the turbidity increase. Negative staining electron microscopy revealed that microtubular bundles which formed in the presence of C/A dynein became thicker and longer after addition of ATP. Furthermore, sliding movements of microtubule(s) in the individual bundles were observed in the presence of ATP. This mode of interaction of C/A dynein with microtubules was distinct from that of flagellar or ciliary dyneins reported so far. These results suggest that C/A dynein, as a component of inner arms, may play a distinct role in the flagellar movement of sea urchin sperm.

Adenosine Triphosphate↗

PubMed1996

A single Drosophila melanogaster myosin light chain kinase gene produces multiple isoforms whose activities are differently regulated.

BACKGROUND: Myosin light chain kinase (MLCK) specifically phosphorylates the myosin regulatory light chain in a calcium/calmodulin (Ca2+/CaM)-dependent manner in animal cells. The roles of MLCK are not fully understood, particularly in nonmuscle cells. Therefore, we cloned and characterized a Drosophila MLCK gene as the first step in a genetic analysis of this process. RESULTS: Four transcripts are produced from this gene. These transcripts encode at least three isoforms (isoform-I, -II and -III), which share a kinase domain, a fibronectin type III motif and an immunoglobulin C2 motif. However, regulatory regions differ between isoform-I/II and -III due to the alternative splicing of the exon encoding a CaM-binding domain. As a result, isoform-I and -II are Ca2+/CaM-dependent forms, whereas isoform-III is a Ca2+/CaM-independent form. Northern blotting and in situ hybridization showed that the expressions of these isoforms are distinctly regulated in stage- and tissue-dependent manners. Isoform-I seems to be expressed ubiquitously, while isoform-III is expressed predominantly in muscle tissues. In contrast to these isoforms, isoform-II is specific to late pupa and adult. CONCLUSION: In Drosophila, a single MLCK gene produces multiple isoforms whose regulatory regions and expression patterns are different. These differences suggest various cellular functions of MLCK in Drosophila.

Amino Acid Sequence↗

PubMed1996

Cell cycle-dependent phosphorylation of smooth muscle myosin light chain in sea urchin egg extracts.

We studied enzymatic activities in sea urchin egg extracts that phosphorylate myosin regulatory light chain (MRLC) from chicken gizzard smooth muscle. The activity in the presence of EGTA showed cell cycle-dependent changes similar to that of histone H1 kinase, namely, it peaked shortly before cleavage, while that in the presence of Ca2+ ions did not show significant change during division cycle. Phosphopeptide mapping revealed that both the sites phosphorylatable by smooth muscle myosin light chain kinase (MLCK sites) and the sites phosphorylatable by protein kinase C (PKC sites) were phosphorylated in the presence or absence of Ca2+ ions. By analyses using an inhibitor of cdc2 kinase, butyrolactone-I, and ion exchange column chromatography, at least three kinases were detected as kinases that phosphorylate MRLC in vitro. These kinases phosphorylated distinct sites on MRLC. The first one, which phosphorylated the PKC sites, was identified as cdc2 kinase. The second one phosphorylated the MLCK sites in the absence of Ca2+ ions. The third one phosphorylated unknown sites. Possible implication of these activities in regulation of cytokinesis is discussed.

4-Butyrolactone↗

PubMed1995

Isolation and sequencing of two cDNA clones encoding Rho proteins from the fission yeast Schizosaccharomyces pombe.

The rho genes encode a group of low-molecular-weight GTP-binding proteins that show about 30% identity in the amino-acid sequence to the ras gene product. Two cDNA clones, both of which are similar to the rho genes, were isolated from a cDNA library of the fission yeast Schizosaccharomyces pombe, using the human rhoA cDNA as a probe. These genes were called rho1+ and rho2+. The encoded Rho1 protein showed 72.7% identity to the budding yeast RHO1 and 66.8% to human RhoA, and the encoded Rho2 protein showed 53.2% identity to the budding yeast RHO2 and RhoA.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes.

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22-23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of 'A' heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.

Animals↗

PubMed1994

Cleavage furrow: timing of emergence of contractile ring actin filaments and establishment of the contractile ring by filament bundling in sea urchin eggs.

Cleavage furrow formation at the first cell division of sea urchin and sand dollar eggs was investigated in detail by fluorescence staining of actin filaments with rhodamine-phalloidin of either whole eggs or isolated egg cortices. Cortical actin filaments were clustered at anaphase and then the clusters became fibrillar at the end of anaphase. The timing when the contractile ring actin filaments appear was precisely determined in the course of mitosis: accumulation of the contractile ring actin filaments at the equatorial cell cortex is first noticed at the beginning of telophase (shortly before furrow formation), when the chromosomal vesicles are fusing with each other. The accumulated actin filaments were not well organized at the early stage but were organized into parallel bundles as the furrowing progressed. The bundles were finally fused into a tightly packed filament belt. Wheat germ agglutinin (WGA)-binding sites were distributed on the surface of the egg in a manner similar to the actin filaments after anaphase. The WGA-binding sites became accumulated in the contractile ring together with the contractile ring actin filaments, indicating an intimate relationship between these sites and actin filament-anchoring sites on the plasma membrane. Myosin also appeared in the contractile ring together with the actin filaments. The 'cleavage stimulus', a signal hypothesized by Rappaport (reviewed by R. Rappaport (1986) Int. Rev. Cytol. 105, 245-281) was suggested to induce aggregation or bundling of the actin filaments in the cortical layer.

Actin Cytoskeleton↗

PubMed1994

C/A dynein isolated from sea urchin sperm flagellar axonemes. Enzymatic properties and interaction with microtubules.

C/A dynein is a novel dynein isolated from sea urchin sperm flagellar axonemes. It is composed of C and A heavy chains and some additional lower molecular mass polypeptide chains. The characterization of ATPase activity and the interaction of this dynein with microtubules polymerized from calf brain tubulin were investigated in this study. The ATPase activity of C/A dynein (0.3-0.4 mumol Pi/min per mg) was about one half that of outer arm 21 S dynein (0.6-0.8 mumol Pi/min per mg) at 25 degrees C. Vanadate inhibited the ATPase activity with a half-maximal inhibition at 1 microM. C/A dynein absorbed to the glass surface was able to translocate the microtubules towards its plus end. The velocity of the microtubule movement in the presence of 1 mM ATP was 4.0 to 4.5 microns/s at 22 degrees C. C/A dynein binds to and bundles the microtubules even in the presence of ATP. Cross-bridges were found between adjacent microtubules in the bundle with an axial periodicity of about 24 nm. The ATPase activity of C/A dynein was enhanced up to several-fold by the microtubules at concentration as low as 1 mg/ml. On the other hand, 21 S dynein bound to the microtubules with 24 nm axial periodicity only in the absence of ATP. Its ATPase activity was not activated by the microtubules. From these results, it is concluded that the manner of interaction with microtubules of C/A dynein is different from that of the outer arm dynein.

Animals↗

PubMed1993

A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs.

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.

ADP Ribose Transferases↗

PubMed1993

45K actin filament-severing protein from sea urchin eggs: interaction with phosphatidylinositol-4,5-bisphosphate.

An actin filament-severing activity of 45K protein isolated from sea urchin eggs was abolished when this protein was incubated with phosphatidylinositol-4,5-bisphosphate (PIP2). This effect was specific to PIP2 since phosphatidylinositol, phosphatidylinositol-4-monophosphate, inositol-1,4,5-trisphosphate, and phosphatidylserine did not show such an effect at the same concentration. Digestion of PIP2 with phospholipase C eliminated the effect. On the other hand, PIP2 did not affect either the formation of 45K protein-actin complex or actin filament-capping activity of the complex. Possible implication of the binding of PIP2 to 45K protein in cytoskeleton formation after fertilization of sea urchin eggs is discussed.

Actins↗

PubMed1992

Calyculin A induces contractile ring-like apparatus formation and condensation of chromosomes in unfertilized sea urchin eggs.

Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone H1 kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.

Actin Cytoskeleton↗

PubMed1990

Primary structure of profilins from two species of Echinoidea and Physarum polycephalum.

Profilin is a small G-actin-binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N-termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465-476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N-terminus was acylated, and the calculated molecular mass was 13132 Da. The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those form other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins. The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N-terminal region, which was suggested to be a common actin-binding region. The C-terminal region was also conserved, although to a lesser extent than the N-terminal region.

Amino Acid Sequence↗

PubMed1990

Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection.

In attempting to produce a mutant mouse with embryonic stem cells, the critical step is the efficient isolation of homologous recombinants; the frequency of the homologous recombination is usually low and the potency of the cells to differentiate into germ cells is unstable in culture. Here, we report an efficacious method for such isolation in which the diphtheria toxin A-fragment gene is used to negatively select nonhomologous recombinants. In contrast to the use of the herpes simplex virus thymidine kinase gene, the selection can be made singly by the neomycin analog G418 without using a drug such as ganciclovir, a nucleoside analog. At the c-fyn locus, the diphtheria-toxin negative selection enriched the recombinants about 10-fold, and half of the cells integrating with the neomycin phosphotransferase gene were homologous recombinants.

Animals↗

PubMed1990

The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes.

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.

Animals↗