Issei Mabuchi

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 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.

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 p 180 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.

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. (C) 2003 Wiley-Liss, Inc.

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. (C) 2003 Wiley-Liss, Inc.

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 degreesC. 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.

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 degreesC. 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.

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 F112 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 NIT 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.

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 F112 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 NIT 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.

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.

Background: The small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeast Schizosaccharomyces pombe. Activity of Rho1 must be precisely regulated in vivo, since both increases! and decreases in its activity affect cell growth and shape. Thus, it is important to clarify the mechanism by which the activity of Pho1 is regulated in vivo. Results: Seven genes encoding putative GAPs, GTPase-activating proteins, for the function of the Rho-family proteins were isolated from S. pombe. After disruption of these genes, rga1(+) was found to splay important roles in cell growth and morphogenesis. In rga1 null cells, delocalized F-actin patches and extraordinary thickening of the cell wall and the septum were observed. On the other hand, overexpression of Rga1 produced shrunken or dumpy cells. The phenotype of the rga1 null cells or the Rga1-over-expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively. Moreover, direct association of Rga1 with Rho1 was shown. Rga1 was localized to the cell ends and septum where Pho1 is known to function. Conclusions: In S. pombe, Rga1 is involved in the F-actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1.

Background: The small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeast Schizosaccharomyces pombe. Activity of Rho1 must be precisely regulated in vivo, since both increases! and decreases in its activity affect cell growth and shape. Thus, it is important to clarify the mechanism by which the activity of Pho1 is regulated in vivo. Results: Seven genes encoding putative GAPs, GTPase-activating proteins, for the function of the Rho-family proteins were isolated from S. pombe. After disruption of these genes, rga1(+) was found to splay important roles in cell growth and morphogenesis. In rga1 null cells, delocalized F-actin patches and extraordinary thickening of the cell wall and the septum were observed. On the other hand, overexpression of Rga1 produced shrunken or dumpy cells. The phenotype of the rga1 null cells or the Rga1-over-expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively. Moreover, direct association of Rga1 with Rho1 was shown. Rga1 was localized to the cell ends and septum where Pho1 is known to function. Conclusions: In S. pombe, Rga1 is involved in the F-actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1.

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca2+-dependent manner.

Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca2+-dependent manner.

We characterized the novel Schizosaccharomyces pombe genes myo4(+) and myo5(+), both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

We characterized the novel Schizosaccharomyces pombe genes myo4(+) and myo5(+), both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a shea period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immune-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a P-32 in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. (C) 2001 Wiley-Liss, Inc.

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a shea period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immune-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a P-32 in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. (C) 2001 Wiley-Liss, Inc.

Background: Type I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes. In the budding yeast, two myosin I species have been identified and their role in F-actin assembly has been inferred. Results: We cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein. Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F-actin patches. An abnormal deposition of cell wall materials was also seen. Homothallic myo1 Delta cells could mate, but heterothallic myo1 Delta cells were poor in conjugation. Myo1p was necessary for the encapsulation of spores. The tail domain of Myo1p was pivotal for its function. Calmodulin could bind to Myo1p through the IQ domain at the neck. Conclusions: Myo1p appears to control the redistribution of F-actin patches during the cell cycle. Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes.

Background: Type I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes. In the budding yeast, two myosin I species have been identified and their role in F-actin assembly has been inferred. Results: We cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein. Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F-actin patches. An abnormal deposition of cell wall materials was also seen. Homothallic myo1 Delta cells could mate, but heterothallic myo1 Delta cells were poor in conjugation. Myo1p was necessary for the encapsulation of spores. The tail domain of Myo1p was pivotal for its function. Calmodulin could bind to Myo1p through the IQ domain at the neck. Conclusions: Myo1p appears to control the redistribution of F-actin patches during the cell cycle. Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes.

We studied reorganization of actin-myosin cytoskeleton at the growing ends of the cleavage furrow of Xenopus eggs in order to understand how the contractile ring is formed during cytokinesis. Reorganization of F-actin structures during the furrow formation was demonstrated by rhodamine-phalloidin staining of the cleavage furrow and by time-lapse scanning with laser scanning microscopy of F-actin structures in the cleavage furrow of live eggs to which rhodamine-G-actin had been injected. Actin filaments assemble to form small clusters that we call 'F-actin patches' at the growing end of the furrow. In live recordings, we observed emergence and rapid growth of F-actin patches in the furrow region. These patches then align in tandem, elongate and fuse with each other to form short F-actin bundles. The short bundles then form long F-actin bundles that compose the contractile ring. During the furrow formation, a cortical movement towards the division plane occurs at the growing ends of the furrow, as shown by monitoring wheatgerm agglutinin-conjugated fluorescent beads attached to the egg surface. As a result, wheatgerm agglutinin-binding sites accumulate and form 'bleb-like' structures on the surface of the furrow region. The F-actin patch forms and grows underneath this structure. The slope of F-actin accumulation in the interior region of the furrow exceeds that of accumulation of the cortex transported by the cortical movement. In addition, rhodamine-G-actin microinjected at the growing end is immediately incorporated into the F-actin patches. These data, together with the rapid growth of F-actin patches in the live image, suggest that actin polymerization occurs in the contractile ring formation. Distribution of myosin II in the cleavage furrow was also examined by immunofluorescence microscopy, Myosin II assembles as spots at the growing end underneath the bleb-like structure. It was suggested that myosin is transported and accumulates as spots by way of the cortical movement, F-actin accumulates at the position of the myosin spot a little later as the F-actin patches. The myosin spots and the F-actin patches are then simultaneously reorganized to form the contractile ring bundles.

How actin filaments (F-actin) and myosin II (myosin) assemble to form the contractile ring was investigated with fission yeast and Xenopus egg. In fission yeast cells, an aster-like structure composed of F-actin cables is formed at the medial cortex of the cell during prophase to metaphase, and a single F-actin cable(s) extends from this structure, which seems to be a structural basis of the contractile ring. In early mitosis, myosin localizes as dots in the medial cortex independently of F-actin. Then they fuse with each other and are packed into a thin contractile ring. At the growing ends of the cleavage furrow of Xenopus eggs, F-actin at first assembles to form patches. Next they fuse with each other to form short F-actin bundles. The short bundles then form long bundles. Myosin seems to be transported by the cortical movement to the growing end and assembles there as spots earlier than F-actin. Actin polymerization into the patches is likely to occur after accumulation of myosin. The myosin spots and the F-actin patches are simultaneously reorganized to form the contractile ring bundles. The idea that a Ca signal triggers cleavage furrow formation was tested with Xenopus eggs during the first cleavage. We could not detect any Ca signals such as a Ca wave, Ca puffs or even Ca blips at the growing end of the cleavage furrow. Furthermore, cleavages are not affected by Ca-chelators injected into the eggs at concentrations sufficient to suppress the Ca waves. Thus we conclude that formation of the contractile ring is not induced by a Ca signal at the growing end of the cleavage furrow.

We studied reorganization of actin-myosin cytoskeleton at the growing ends of the cleavage furrow of Xenopus eggs in order to understand how the contractile ring is formed during cytokinesis. Reorganization of F-actin structures during the furrow formation was demonstrated by rhodamine-phalloidin staining of the cleavage furrow and by time-lapse scanning with laser scanning microscopy of F-actin structures in the cleavage furrow of live eggs to which rhodamine-G-actin had been injected. Actin filaments assemble to form small clusters that we call 'F-actin patches' at the growing end of the furrow. In live recordings, we observed emergence and rapid growth of F-actin patches in the furrow region. These patches then align in tandem, elongate and fuse with each other to form short F-actin bundles. The short bundles then form long F-actin bundles that compose the contractile ring. During the furrow formation, a cortical movement towards the division plane occurs at the growing ends of the furrow, as shown by monitoring wheatgerm agglutinin-conjugated fluorescent beads attached to the egg surface. As a result, wheatgerm agglutinin-binding sites accumulate and form 'bleb-like' structures on the surface of the furrow region. The F-actin patch forms and grows underneath this structure. The slope of F-actin accumulation in the interior region of the furrow exceeds that of accumulation of the cortex transported by the cortical movement. In addition, rhodamine-G-actin microinjected at the growing end is immediately incorporated into the F-actin patches. These data, together with the rapid growth of F-actin patches in the live image, suggest that actin polymerization occurs in the contractile ring formation. Distribution of myosin II in the cleavage furrow was also examined by immunofluorescence microscopy, Myosin II assembles as spots at the growing end underneath the bleb-like structure. It was suggested that myosin is transported and accumulates as spots by way of the cortical movement, F-actin accumulates at the position of the myosin spot a little later as the F-actin patches. The myosin spots and the F-actin patches are then simultaneously reorganized to form the contractile ring bundles.

In order to identify additional components important for cell division in the fission yeast Schizosaccharomyces pombe we have screened a bank of conditional cold-sensitive mutants for cytokinesis defects. One of these mutants showed a delay in cell cleavage, and strong genetic interactions with other genes implicated in medial ring formation. Cloning of the corresponding gene indicates that it encodes a protein with significant homology to the regulatory light chain of non-muscle myosins, We have named the gene rlc1 (regulatory light chain 1), The gene is not essential for division, but null mutants display a cell cleavage defect and form an aberrant F-actin ring. Two myosin-II heavy chains have been identified in fission yeast: Co-immunoprecipitation experiments indicate that rlc1p associates more strongly with myo3p than myo2p.

Schizosaccharomyces pombe cells divide by virtue of the Factin-based contractile ring (F-actin ring), Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring, Here, me investigated the mechanism of myosin-II, assembly at the division site in S, pombe cells, First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and R;Myo3, Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells, It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis, Next, me investigated the dynamics of myosin-II during mitosis in S, pombe cells, In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex. Myo2 fibers also became visible following the appearance of the dots, The Myo2 dots and fibers then fused,vith each other to form a medial cortical network, Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region, Finally these structures were packed into a thin contractile ring, In mutant cells that cannot form the F-actin ring such as cdc(3ts), cdc(8ts) and cdc(12ts), Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4(ts) cells, Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation, A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4(ts) mutant cells and cdc4 null cells, The Myo2dIQs could assemble into a normal-shaped ring in these cells, Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3.

Background: In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C-related PRKs, is responsible for an interaction with Rho-GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho-dependent protein kinase C-related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2. Results: Rho1 is shown to interact with the conserved N-terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co-expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2-Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located. Conclusions: The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho-GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.

Background: Elongation factor lo (EF1 alpha), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F-actin-binding and -bundling, microtubule-severing, and the activity of making fibroblasts highly susceptible to transformation. However, our understanding of the biological significance of EF1 alpha with respect to these various biochemical or biological activities remains Limited. Here we report the identification of EF1 alpha-encoding genes as genes whose over-expression causes aberrant cell morphology in fission yeast. Results: Overproduction of EF1 alpha caused aberrant cell morphology-elliptic, curved or branched-and growth defects in yeast cells at high temperatures. EF1 alpha-overproducing cells showed a supersensitivity to the actin inhibitor cytochalasin D and to the tubulin inhibitor thiabendazole. Genetic analyses using cdc mutants suggested that excess EF1 alpha disturbed the establishment and the maintenance of growth polarity in the G1 phase by preventing the localization of F-actin to the polarized growing site and the organization of microtubules. Results from DNase I column chromatography indicated that EF1 alpha was bound to G-actin. Indeed, the fission yeast actin was immunoprecipitated along with EF1 alpha. Moreover, the temperature sensitivity caused by the overproduction of EF1 alpha was restored by co-overproduction of actin. Conclusions: Fission yeast EF1 alpha has the ability to alter the cell morphology of yeast by affecting the control of actin and microtubule cytoskeletons.

We isolated the urho1 (urchin rho in English or uni rho in Japanese) gene from the sea urchin cDNA library which encodes a Rho GTPase, Anti-URho1 antibodies specifically recognized a 22 kDa protein in the extracts of echinoderm eggs. URho1 was concentrated in the cortices from both unfertilized and fertilized eggs as judged by immunoblot analysis. URho1 may bind directly to the cell membrane but not be a component of the cortical layer. Immunofluorescence microscopy revealed that URho1 is localized to the cleavage furrow and the midbody during cytokinesis. (C) 1998 Federation of European Biochemical Societies.

To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression, The isolated genes were classified on the basis of morphology conferred, One of the genes causing a rounded morphology was identified as the rho2(+) gene encoding a small GTP-binding protein. The overexpression of rho2(+) resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall. Analyses using cdc mutants suggested that the overexpression of rho2(+) prevents the establishment of growth polarity in G(1). The rho2(+) gene was not essential, but among cells deleted for rho2(+), those with an irregular shape were observed, The disruptant also showed a defect in cell wall integrity, An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment. A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site, The localization of GFP-Rho2 during interphase was partially dependent on sts5(+), These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction, Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.

To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression, The isolated genes were classified on the basis of morphology conferred, One of the genes causing a rounded morphology was identified as the rho2(+) gene encoding a small GTP-binding protein. The overexpression of rho2(+) resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall. Analyses using cdc mutants suggested that the overexpression of rho2(+) prevents the establishment of growth polarity in G(1). The rho2(+) gene was not essential, but among cells deleted for rho2(+), those with an irregular shape were observed, The disruptant also showed a defect in cell wall integrity, An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment. A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site, The localization of GFP-Rho2 during interphase was partially dependent on sts5(+), These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction, Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.

We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain, myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F-actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S. pombe.

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 μM. 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 μM, 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.

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.