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Actin–microtubule interactions have been postulated to play an important role in the formation of the preprophase microtubule band, which predicts the future division site in plants. Electron tomography reveals that microfilaments are used as bridging structures to draw widely spaced microtubules together during microtubule band formation.

Effective defense responses involve the entire organism. To maintain body homeostasis after tissue damage, a systemic wound response is induced in which the response of each tissue is tightly orchestrated to avoid incomplete recovery or an excessive, damaging response. Here, we provide evidence that in the systemic response to wounding, an apoptotic caspase pathway is activated downstream of reactive oxygen species in the midgut enterocytes (ECs), cells distant from the wound site, in Drosophila. We show that a caspase-pathway mutant has defects in homeostatic gut cell renewal and that inhibiting caspase activity in fly ECs results in the production of systemic lethal factors after wounding. Our results indicate that wounding remotely controls caspase activity in ECs, which activates the tissue stem cell regeneration pathway in the gut to dampen the dangerous systemic wound reaction.

In this report, we demonstrate that an optic cup structure can form by self-organization in human ESC culture. The human ESC-derived optic cup is much larger than the mouse ESC-derived one, presumably reflecting the species differences. The neural retina in human ESC culture is thick and spontaneously curves in an apically convex manner, which is not seen in mouse ESC culture. In addition, human ESC-derived neural retina grows into multilayered tissue containing both rods and cones, whereas cone differentiation is rare in mouse ESC culture. The accumulation of photoreceptors in human ESC culture can be greatly accelerated by Notch inhibition. In addition, we show that an optimized vitrification method enables en bloc cryopreservation of stratified neural retina of human origin. This storage method at an intermediate step during the time-consuming differentiation process provides a versatile solution for quality control in large-scale preparation of clinical-grade retinal tissues.

Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA–cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB–cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA–talB–cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA–cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB–cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA–talB–cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.

ABSTRACTTalin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideumhas two talin homologues, talAand talB, and earlier studies have characterized the single knockout mutants. talA-cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB-cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talAand talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talAand talB. The talA-talB-cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA-cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB-cells at the loose-mound stage. Moreover, overexpression of either talAor talBcompensated for the loss of the other talin, respectively. The analysis of talA-talB-cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.

Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton,especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2],affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and-independent activation of ERM proteins require a local elevation of PtdIns(4,5)P2 concentration in vivo.

Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Here we report that forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. We believe that this is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells.

Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, we monitored its localization in cell cycle. We have found: (1) Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron-K in the cortex of ana- to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody; (3) inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevented its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, we expressed active Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure was localized at the same plane as actin stress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells.

At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127–142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1–positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1–positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1. J. Cell. Physiol. 179:115–125, 1999. © 1999 Wiley-Liss, Inc.

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

Ezrin/radixin/moesin (ERM) proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand, experiments at the cellular level and in vitrohave suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e.platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level.

Cellular ezrin-radixin-moesin (ERM) family proteins, members of the actin-binding proteins of the band 4.1 superfamily, were detected in the virions of enveloped viruses, such as rabies, vesicular stomatitis, Newcastle disease, and influenza viruses. To elucidate the mechanism of ERM protein incorporation, we investigated possible association of ERM proteins with viral components in rabies virus-infected BHK-21 cells. Double immunofluorescence studies demonstrated that the ERM proteins are concentrated in the microvilli, where the colocalized viral G protein was also seen. Viral G protein expressed in the G cDNA-transfected COS-7 cells also displayed similar distributions to those seen in the virus-infected cells. Both the ERM and viral envelope proteins were coprecipitated by anti-viral G antibody from lysates of the virus-infected cells, while the anti-ERM antibody coprecipitated viral G and ERM proteins. These observations suggest that the ERM proteins are closely associated with viral envelope proteins in the cell, which would be involved in the selective incorporation of cellular actin into the virion.

To develop a mass isolation procedure for the cleavage furrow from synchronized sea urchin eggs, we compared the stability of the cleavage furrow with that of the rest of the cortex (polar-region cortex) and the inner cytoplasm under various conditions using the rhodamine-phalloidin staining method. As a result, to remove the polar-region cortex and leave the cleavage furrow intact, it became clear that the type and concentration of detergent, the pH and Ca concentration of the isolation solution and the temperature were of critical importance, and that 0.04-0.1 % Nonidet P-40, pH 7.0-7.5, low calcium ion concentration and room temperature were optimal conditions. To solubilize the inner cytoplasm to release intact cleavage furrows, two factors, osmotic pressure and sea urchin species, were found to be important: 0.16 M glucose (or sucrose) was optimal, and we found Clypeaster japonicus to be the most appropriate. A shearing force, by gentle pipetting, was also required for furrow isolation. Taking these results into consideration, we have succeeded in developing a mass isolation procedure for cleavage furrow from C. japonicus. A total of 20–50 μg of protein of isolated cleavage furrow was recovered from 1 ml of packed dividing eggs. The structural integrity of the isolated cleavage furrow was well maintained and it was characterized by remnants of plasma membranes, actin filament meshwork including a contractile ring, and cytoplasmic vacuoles. Although the isolated furrow contained myosin II molecules, it showed no capability of in vitro reactivation.

Cadherin has an intimate spatial relationship with actin filaments (AF) in various types of cells, forming the cell-to-cell adherens junction (AJ). We compared the AJ/AF rela-tionship between non-polarized fibroblasts (NRK cells) and polarized epithelial cells (MTD-1A cells). E/P-cadherin, α-catenin, ZO-1 and vinculin were localized with reference to AF in these cells using laser scan microscopy as well as conventional light and electron microscopy. NRK cells adhered to each other at the tips of thin cellular processes, where spot-like AJ were formed, where P-cadherin, α-catenin, ZO-1 and vinculin were concentrated. Some stress-fiber-like AF bundles ran axially in these processes and terminated at spot-like AJ on their tips. At the electron micro-scopic level these spot-like AJ were seen as aggregates of small ‘units’ of AJ, where AF were densely and perpendicularly associated with the plasma membrane. In MTD-1A cells, the AJ/AF relationship was investigated during the cell polarization process after replating or wounding. At the early stage, the AJ/AF relationship was quite similar to that in NRK cells. As polarization proceeded, the spot-like AJs were gradually fused side by side with the concomitant shortening of the associated stress-fiber-like AF bundles. Finally, the belt-like AJ was established, which was lined with circumferential AF bundles. The similarities and differences in the AJ/AF relationship between non-polarized fibroblasts and polarized epithelial cells are discussed.

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a ‘transition zone’. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium–basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.Audio S1Audio S1Respiratory sound of a P50 Camsap3mutant mouse.Movie S1Movie S1Beating multicilia in a wild-type airway epithelial cell. The time-lapse images were acquired at 300 fps and displayed at 31 fps.Movie S2Movie S2Beating multicilia in a Camsap3-mutated airway epithelial cell. The time-lapse images were acquired at 300 fps and displayed at 31 fps.Movie S3Movie S3Beating multicilia in a wild-type airway epithelial cell, which is overlaid with a flow vector field calculated by PIV analysis. The flow vector field was also shown separately at the right. The scale arrow and scale bar are 20 μm/s and 1 m, respectively. Part of Video S1 was used for this analysis.Movie S4Movie S4Beating multicilia in a Camsap3-mutated airway epithelial cell, which is overlaid with a flow vector field calculated by PIV analysis. The flow vector field was also shown separately at the right. The scale arrow and scale bar are 20 μm/s and 1 μm, respectively. Part of Video S2 was used for this analysis.Movie S5Movie S5Animation of sequential optical sections, each of which is 0.2 m thick, of a wild-type multi-ciliated epithelial cell, in which GFP-Centrin (green) and -tubulin (magenta) are visualized by fluorescence signals. The animation view begins around the level where the array of GFP-Centrin is detectable, then shifts toward a more basal view of the cell at a speed of 2 fps. See also Figure 6A.Movie S6Movie S6Animation of sequential optical sections, each of which is 0.2 m thick, of a Camsap3-mutated multi-ciliated epithelial cell, in which GFP-Centrin (green) and -tubulin (magenta) are visualized by fluorescence signals. The animation view begins at the level where the array of GFP-Centrin is detectable, then shifts toward a more basal view of the cell at a speed of 2 fps. See also Figure 6B.