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Issei Mabuchi

Publications and source records attributed to Issei Mabuchi.

At least 19 recordsLinked to original sources
PubMed2006

Actin-depolymerizing protein Adf1 is required for formation and maintenance of the contractile ring during cytokinesis in fission yeast.

The role of the actin-depolymerizing factor (ADF)/cofilin-family protein Adf1 in cytokinesis of fission yeast cells was studied. Adf1 was required for accumulation of actin at the division site by depolymerizing actin at the cell ends, assembly of the contractile ring through severing actin filaments, and maintenance of the contractile ring once formed. Genetic and cytological analyses suggested that it collaborates with profilin and capping protein in the mitotic reorganization of the actin cytoskeleton. Furthermore, it was unexpectedly found that Adf1 and myosin-II also collaborate in assembling the contractile ring. Tropomyosin was shown to antagonize the function of Adf1 in the contractile ring. We propose that formation and maintenance of the contractile ring are achieved by a balanced collaboration of these proteins.

Actin Depolymerizing Factors↗
PubMed2006

Actin-capping protein is involved in controlling organization of actin cytoskeleton together with ADF/cofilin, profilin and F-actin crosslinking proteins in fission yeast.

Actin-capping protein (CP) is a heterodimeric protein which is expressed in various eukaryotic cells. CP binds to the barbed end of the actin filaments in vitro and inhibits both the association and dissociation of actin monomers at this end. However, the cellular role of CP has not been uncovered. Here we investigated the function of CP in fission yeast cells. The fission yeast CP is composed of Acp1 and Acp2. It was found that Acp2 accumulated as cortical dots at the cell ends during interphase and the mid-region of mitotic cells, which disappeared in the absence of Acp1 or F-actin. Acp1 and Acp2, when co-over-expressed, decreased F-actin structures in cells, and cytokinesis was often interrupted in these cells. On the other hand, disruption of one of the CP genes affected the distribution of F-actin patches at cell ends and decreased the rate of actin depolymerization in vivo. Moreover, genetic analysis showed that CP controls actin dynamics together with ADF/cofilin and profilin. In addition, CP is likely involved in assembling the F-actin contractile ring and F-actin patch with F-actin-crosslinking proteins.

Actin Capping Proteins↗
PubMed2005

Small GTPase Rho5 is a functional homologue of Rho1, which controls cell shape and septation in fission yeast.

The small GTPase Rho1 plays an essential role in controlling the organization of the actin cytoskeleton and synthesis of the cell wall in the fission yeast Schizosaccharomyces pombe. Here we studied the role of Rho5 whose primary structure is very similar to that of Rho1. It was found that elevated expression of Rho5 was able to compensate for the lethality of cells lacking Rho1. Rho5 was localized to the ends of interphase cells and the mid-region of mitotic cells. Overexpression of Rho5 caused depolarization of F-actin patches and abnormal formation of the cell wall, as did Rho1. Although rho5(+) was not essential for maintaining the cell shape, rho1 rho5-double null cells showed more severe defects in cell viability than rho1-null cells. Thus, it is likely that Rho5 has an overlapping function with Rho1 in controlling cell growth and division in S. pombe.

Amino Acid Sequence↗
PubMed2005

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

Actins↗
PubMed2005

Directionality of F-actin cables changes during the fission yeast cell cycle.

Longitudinal F-actin cables are thought to be important for transporting materials for polarized cell growth in fission yeast. We show that most F-actin in the cables is oriented such that the barbed end faces the nearest cell tip during interphase; however, this directionality is reversed during mitosis. These orientations of F-actin ensure proper transport of materials to growing sites during these cell-cycle stages.

Actin Cytoskeleton↗
PubMed2005

Rho1-GEFs Rgf1 and Rgf2 are involved in formation of cell wall and septum, while Rgf3 is involved in cytokinesis in fission yeast.

The Rho GTPase acts as a binary molecular switch by converting between a GDP-bound inactive and a GTP-bound active conformational state. The guanine nucleotide exchange factors (GEFs) are critical activators of Rho. Rho1 has been shown to regulate actin cytoskeleton and cell wall synthesis in the fission yeast Schizosaccharomyces pombe. Here we studied function of fission yeast RhoGEFs, Rgf1, Rgf2, and Rgf3. It was shown that these proteins have similar molecular structures, and function as GEFs for Rho1. Disruption of either rgf1 or rgf2 did not show a serious effect on the cell. On the other hand, disruption of rgf3 caused severe defects in contractile ring formation, F-actin patch localization, and septation during cytokinesis. Rgf1 and Rgf2 were localized to the cell ends during interphase and the septum. Rgf3 formed a ring at the division site, which was located outside the contractile ring and inside the septum where Rho1 was accumulated. In summary, Rgf1 and Rgf2 show functional redundancy, and roles of these RhoGEFs are likely to be different from that of Rgf3. Rho1 is likely to be activated by Rgf3 at the division site, and involved in contractile ring formation and/or maintenance and septation.

Cell Division↗
PubMed2004

Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast.

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.

Actin Cytoskeleton↗
PubMed2003

A novel actin-bundling kinesin-related protein from Dictyostelium discoideum.

Actin filaments and microtubules are two major cytoskeletal systems involved in wide cellular processes, and the organizations of their filamentous networks are regulated by a large number of associated proteins. Recently, evidence has accumulated for the functional cooperation between the two filament systems via associated proteins. However, little is known about the interactions of the kinesin superfamily proteins, a class of microtubule-based motor proteins, with actin filaments. Here, we describe the identification and characterization of a novel kinesin-related protein named DdKin5 from Dictyostelium. DdKin5 consists of an N-terminal conserved motor domain, a central stalk region, and a C-terminal tail domain. The motor domain showed binding to microtubules in an ATP-dependent manner that is characteristic of kinesin-related proteins. We found that the C-terminal tail domain directly interacts with actin filaments and bundles them in vitro. Immunofluorescence studies showed that DdKin5 is specifically enriched at the actin-rich surface protrusions in cells. Overexpression of the DdKin5 protein affected the organization of actin filaments in cells. We propose that a kinesin-related protein, DdKin5, is a novel actin-bundling protein and a potential cross-linker of actin filaments and microtubules associated with specific actin-based structures in Dictyostelium.

Actins↗
PubMed2003

Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis.

Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells.

Actin Cytoskeleton↗
PubMed2003

An IQGAP-like protein is involved in actin assembly together with Cdc42 in the sea urchin egg.

We isolated a gene homologous to human cdc42 (ucdc42) from a sea urchin cDNA library. The GTPgammaS-bound UCdc42 induced actin assembly in sea urchin egg extract. Proteins that are involved in this actin assembly system were searched using UCdc42-bound agarose beads. A 180-kDa protein (p180), which showed a homology to human IQGAPs, bound to the GTPgammaS-UCdc42 beads. Immunodepletion of p180 from the sea urchin egg extract abolished this actin assembly on the UCdc42 beads. Immunofluorescent localization of p180 was similar to that of the actin cytoskeleton in the egg cortex and it was concentrated in the cleavage furrow during cytokinesis. A possible role of p180 in actin assembly is discussed.

Actins↗
PubMed2003

The small GTPase Rho4 is involved in controlling cell morphology and septation in fission yeast.

BACKGROUND: Rho family small GTPases have been shown to be involved in various cellular activities, including the organization of actin cytoskeleton in eukaryotic cells. There are six rho genes in the fission yeast Schizosaccharomyces pombe. Cdc42 is known to control the polarity of the cell. Rho1, Rho2 and Rho3 play important roles in controlling cell shape and septation. On the other hand, Rho4 and Rho5 have not yet been characterized. Here we report the function of rho4+ in fission yeast. RESULTS: Gene disruption revealed that rho4+ is not essential for cell growth. However, rho4-null cells were abnormally elongated and had multiple septa of irregular shape at 37 degrees C. In these cells, F-actin patches were randomly localized all over the cell periphery, and cytoplasmic microtubules (MTs) were misoriented. On the other hand, the exogenous expression of a constitutively active Rho4-G23V or Rho4-Q74L in wild-type cells induced depolarization of F-actin patches and cytoplasmic MTs. Rho4 was localized to the cell periphery during interphase and septum during mitosis. Both the binding of GTP and isoprenylation of its C-terminus were necessary for the localization. Furthermore, the localization of Rho4 was likely to be controlled by Rho GAP and Rho GDI. CONCLUSION: Rho4 may control cell morphogenesis and septation by regulating both the actin cytoskeleton and cytoplasmic MTs.

Actins↗
PubMed2002

The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast.

We identified a novel Rho gene rho3(+) and studied its interaction with diaphanous/formin for3(+) in the fission yeast Schizosaccharomyces pombe. Both rho3 null cells and for3 null cells showed defects in organization of not only actin cytoskeleton but also cytoplasmic microtubules (MTs). rho3 for3 double null cells had defects that were more severe than each single null cell: polarized growth was deficient in the double null cells. Function of For3 needed the highly conserved FH1 and FH2 domains, an N-terminal region containing a Rho-binding domain, and the C-terminal region. For3 bound to active forms of both Rho3 and Cdc42 but not to that of Rho1. For3 was localized as dots to the ends of interphase cells and to the mid-region in dividing cells. This localization was probably dependent on its interaction with Rho proteins. Overexpression of For3 produced huge swollen cells containing depolarized F-actin patches and thick cytoplasmic MT bundles. In addition, overexpression of a constitutively active Rho3Q71L induced a strong defect in cytokinesis. In conclusion, we propose that the Rho3-For3 signaling system functions in the polarized cell growth of fission yeast by controlling both actin cytoskeleton and MTs.

Cell Cycle Proteins↗
PubMed2002

Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast.

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1-3]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4-5]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division.

Actomyosin↗
PubMed2002

F-actin ring formation and the role of F-actin cables in the fission yeast Schizosaccharomyces pombe.

Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell. We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy. In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase. The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase. During anaphase, the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring. These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation. In the nda3 mutant, which has a mutation in ss-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature. Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring. This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase. We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants. As a result, Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase. In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.

Actins↗
PubMed2002

Localized calcium signals along the cleavage furrow of the Xenopus egg are not involved in cytokinesis.

It has been proposed that a localized calcium (Ca) signal at the growing end of the cleavage furrow triggers cleavage furrow formation in large eggs. We have examined the possible role of a Ca signal in cleavage furrow formation in the Xenopus laevis egg during the first cleavage. We were able to detect two kinds of Ca waves along the cleavage furrow. However, the Ca waves appeared after cleavage furrow formation in late stages of the first cleavage. In addition, cleavage was not affected by injection of dibromoBAPTA or EGTA into the eggs at a concentration sufficient to suppress the Ca waves. Furthermore, even smaller classes of Ca release such as Ca puffs and Ca blips do not occur at the growing end of the cleavage furrow. These observations demonstrate that localized Ca signals in the cleavage furrow are not involved in cytokinesis. The two Ca waves have unique characteristics. The first wave propagates only in the region of newly inserted membrane along the cleavage furrow. On the other hand, the second wave propagates along the border of new and old membranes, suggesting that this wave might be involved in adhesion between two blastomeres.

Animals↗