Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOLGIA CHILE  

Background: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.
Results: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.
Conclusions: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

DOI： 10.1186/s40659-015-0039-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell-like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time-lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time-lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP-actin transfected cells by time-lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei. Copyright (C) 2011 John Wiley & Sons, Ltd.

DOI： 10.1002/cbf.1814

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The tracheal epithelium can be induced to move as a cellular sheet by heterotopic transplantation, which offers the opportunity to observe migrating cells as a group in an in vivo environment. We therefor investigated the ultrastructural characteristics of migrating tracheal epithelial cells with special reference to the moving front using this transplantation. The migrating epithelial cells underwent squamous metaplasia and lost their differentiated characteristics such as cilia or secretory granules. Several unique observations were made concerning the mechanism of mobility: one is that epithelial cells in the front were elongated in a direction perpendicular to the course of movement, different from previous reports in vitro. The second is that lamellipodia, which are regarded as the major locomotive machinery in the adult wound epithelium, did not make up the major part of the front; the major portion of the anterior fringe of the moving front was usually smooth and gently curved, and actin cables parallel to the elongated cells were observed by confocal laser microscopy, indicating that the purse-string mechanism of epithelial wound healing takes place. The third finding is that the cells in the front had irregular bleb-like structures on their antero-basal surface, which were formed even in the portion where the cells did not attach to the matrix. Few organelles were recognized in these structures. From their location, one might propose that these bleb-like structures play a role in the recognition of the substrate and thus the movement of the cell sheet.

DOI： 10.1679/aohc.71.223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

We established a mesenchymal stem cell clone, 5F9A, from rat bone marrow substrate adherent cells by repeated limiting dilutions. The cells have a fibroblastic shape and form intimate contacts with adjacent cells with interdigitations and junctions similar to adherence and tight junctions in a semi-confluent culture. Analysis of the phenotypes of these cells by RT-PCR and FACS demonstrated that they resembled mesenchymal stem cells, and the cells could differentiate into adiopocytes and osteoblasts under appropriate conditions in vitro showing their oligopotency. Furthermore, the cells were induced to become multinuclear cells by TPA (12-o-tetradecanoylphorbol 13-acetate) stimulation.

DOI： 10.1002/ar.20590

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion. (c) 2007 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.ejcb.2007.04.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Delphilin is identified as a Glutamate receptor delta 2 (GluR delta 2) Subunit interacting protein, consisting of a PDZ domain and formin homology (FH) domains 1 and 2, in addition to a C-terminal coiled-coil structure. Delphilin has been shown to be selectively expressed in cerebellar Purkinje cells where it co-localizes with the GlUR delta 2 Subunit at the Purkinje cell-parallel fiber synapses. Although Delphilin specifically interacts with the GluR delta 2 C-terminus via its PDZ domain, the physiological role of the interaction is not yet understood. Here, we report that the Delphilin protein exhibits diversity at its N-terminus by variable usage of the first several exons. Interestingly, the two Delphilin mRNAs which correspond to the first one initially identified (now designated as Delphilin alpha) and the second that contains a newly identified first exon (designated as Delphilin beta), show different chronological expression profiles. Delphilin beta mRNA was not decreased throughout the cerebellar development in vivo and in vitro, while in vivo Delphilin a mRNA gradually decreases following the first postnatal week. Delphilins alpha and beta also revealed different subcellular distribution with sonic overlap. Specifically, the cerebellar synaptosomal membrane fraction contained the Delphilin beta protein. Both Delphilin alpha and beta localized at the dendritic spines with GluR delta 2; however, dendritic shafts in cultured Purkinje cells also included Delphilin beta. In MDCK cells upon becoming confluent, Delphilin a moved to the cell-cell junction area, whereas Delphilin maintained a diffuse distribution pattern throughout the cytoplasm. Taken as a whole, these two different Delphilins seemed to play functionally different roles in developing and matured cerebellar Purkinje cells. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.molbrainres.2005.08.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Mouse Prrp (mPrrp)/DAZAP1 is a mouse ortholog of Xenopus Prrp, which is involved in vegetal pole localization of Vg1 mRNA in oocytes and is highly expressed in the testis. The mouse protein has been reported to be a shuttling protein which localizes in the nucleus of pre-meiotic spermatogenic cells and round spermatids, and shifts its location into the cytoplasm in elongating spermatids, suggesting that mPrrp may be involved in mRNA transport as well as that of the Xenopus ortholog. We reexamined immunohistochemical analyses of mPrrp/DAZAP1 during spermatogenesis utilizing a newly established monoclonal antibody and reconfirmed it to be a shuttling protein. We also carried out new observations that included remarkable intranuclear movement during spermatogenesis. In addition, we found that a long amino acid stretch which spanned over the C-terminal half of the protein was required for the nuclear import. These observations demonstrated dynamic changes in subnuclear and subcellular localization which might reflect specific functions during spermatogenesis.

DOI： 10.1679/aohc.67.325

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steel(dickie) (SI/SId) mutant mice, which lack expression of membrane bound form SCIF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.

DOI： 10.1095/biolreprod.102.013276

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During student dissecting practice, a rare developmental anomaly showing persistent sciatic artery (PSA) was found in the bilateral lower limbs of a 74-year-old Japanese male cadaver. The PSA was a continuation of the internal iliac artery on both sides, did not anastomose with the perforating arteries and ended by anastomosing with the popliteal artery on both sides. The course and distribution of the PSA were relatively consistent with previous reports. In the present case, however, the PSA and inferior gluteal arteries existed simultaneously on both sides, despite the general assumption that the inferior gluteal artery is a remnant of sciatic artery regression.

DOI： 10.1046/j.0022-7722.2002.00003.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Histochemical Society Inc.  

Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading
fluorescent intensity at the first scan (EM1)
and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EMn = EM1 · A (n-1). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.

DOI： 10.1177/002215540104900304

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The infrared sensory membranes of pit organs of pit vipers have an extremely rich capillary vasculature that forms many vascular loops, each serving a small number of infrared nerve terminals. We clarified the ultrastructure of capillary pericytes in the pit membranes by scanning and transmission electron microscopy, and examined the immunoreactivity in their cytoplasm to two contractile proteins: smooth muscle alpha -actin (SM alpha -actin) and desmin. The capillary pericytes had two major cytoplasmic processes: thickened primary processes that radiate to embrace the endothelial tube and flattened secondary processes that are distributed widely on the endothelium. Coexpression of SM alpha -actin and desmin was observed in the pericytes of entire capillary segments, and SM alpha -actin was characterized by prominent filament bundles directed mainly at right angles to the capillary long axis. This expression pattern was different from that of capillary pericytes of the scales, where SM alpha -actin was expressed diffusely in the cytoplasm. In a series of electron microscopic sections, we often observed the pericyte processes depressing the endothelial wall. We also observed a close relationship of the pericytes with inter-endothelial cell junctions, and pericyte processes connected with the endothelial cells via gap junctions.
From these findings, we surmised that capillary pericytes in the pit membrane have a close functional relationship with the endothelium, and through their contractile and relaxing activity regulate capillary bloodflow to stabilize production of infrared nerve impulses. Anat Rec 260:299-307, 2000. (C) 2000 Wiley-Liss, Inc.

DOI： 10.1002/1097-0185(20001101)260:3<299::AID-AR67>3.0.CO;2-V

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Lipopolysaccharide (LPS) is one of the potent activators of macrophages. LPS was shown to induce cell spreading and large vacuoles in the cytoplasm of a macrophage-like cell line, JY3. These vacuoles were negative for acid phosphatase histochemistry and did not take up Lucifer yellow added to the medium. Latex beads were incorporated into cytoplasmic vesicles distinct from the vacuoles. These results indicated that the vacuoles are neither phagosomes nor lysosomes. DiIC18(3), a specific marker of endoplasmic reticulum (ER), stained the vacuoles intensely, and DiOC6(3) stained the vacuoles at a density similar to nuclear envelope, suggesting ER origin of their membrane. Glucose-6-phosphatase, however, was not detected histochemically. Vacuoles were also stained with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or WGA-biotin, suggesting that the vacuoles originated from plasma membrane-endosome-trans Golgi network-secretory granule pathway. Golgi markers, TPPase or BODIPY-ceramide were not localized to the vacuolar membrane. These results indicate that the vacuoles may have dual origins
ER and plasma membrane-derived organelles.

DOI： 10.1179/096805199101531633

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

An application of double-immunolabelling in combination with a multiple dye filter system demonstrated new findings regarding the distribution pattern of peptidergic fibers in the carotid labyrinth in addition to our previous findings shown by the individual filter system. In high magnification images of about 10% of the yellowish fibers which represent the coexistence of two neuropeptides, there was a definite difference in localization between the fluorescence originating from rhodamine (substance P fibers) and from FITC (vasoactive intestinal polypeptide and neuropeptide Y fibers), but it was clear that they are intertwined within a single nerve bundle. This; combination method was able to discriminate two different peptidergic fibers which run side by side. The coexistence suggested previously by the individual filter system may actually be due to the phenomenon described above. This means that it is necessary to apply the multiple dye filter system for reliable evidence of coexistence of different two substances in a single nerve fiber.

DOI： 10.1016/0006-8993(96)00788-3

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Immunoreactivity of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and galanin is localized in nerve fibres distributed in the fungiform and filiform papillae of the tongue of the bullfrog, Rana catesbeiana. A combination of indirect double immunofluorescence labelling and a multiple dye filter system clearly demonstrated that all substance P fibres in the connective tissue core of the fungiform and filiform papillae, and within the rim of ciliated cells located on the top of the fungiform papillae showed coexistence with calcitonin gene-related peptide. A few fibres in the epithelial discs, which are located in the centre of the top of the fungiform papillae, showed the immunoreactivity of calcitonin gene-related peptide alone. There were no substance P fibres which showed coexistence with vasoactive intestinal polypeptide, galanin, and neuropeptide Y. In high magnification images, substance P and vasoactive intestinal polypeptide, and substance P and galanin fibres were recognized as two intertwined fibres within the same thin nerve bundle. No immunoreactivity of leucine- and methionine-enkephalins can be detected. These findings suggest that the chemoreceptor function of the bullfrog gustatory organ may be under the control of complicated peptidergic innervation.

DOI： 10.1007/BF02409017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Immunoreactivity of galanin (GAL) was detected in the nerve fibers distributed within the intervascular stroma of the bullfrog carotid labyrinth. GAL-immunoreactive fibers are numerous, and some are close to the sinusoidal plexus. Most GAL fibers appear as thin processes with some varicosities. A combination of indirect double immunofluorescence labelling and image processing clearly demonstrated that the distribution pattern of GAL fibers is different from that of SP fibers. This indicates that GAL and SP do not coexist in the same nerve fibers. The role of GAL fibers may be different from that of previously reported neuropeptides (substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and others) as a neuromodulator in controlling vascular tone of the labyrinth.

DOI： 10.1007/BF00307958

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Language：Japanese   Publisher：(公財)金原一郎記念医学医療振興財団  

DOI： 10.11477/mf.2425900583

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DOI： 10.11477/mf.2425900583

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Language：English   Publisher：Bioimaging Society  

In this paper we describe an application of the confocal laser microscope (CLM) to interference reflection (IR) microscopy for analysis of the cell-to-substrate adhesion of cultured cells. The CLM in IR mode provided sharp and high-contrast images of focal contacts of cells grown on glass surfaces without interference from out-of-focus reflections. For simultaneous imaging of fluorescence and IR signals, the 488 nm line of an argon ion laser was used for illumination and a dichroic mirror was placed in front of two photomultipliers to split the light returning from the specimen into reflected (488 nm) and fluorescence (green) signals. Thus we could correlate the IR and fluorescence images of cells stained with fluorescent labels for vinculin or F-actin. To confirm the cell contact, we also examined cultured living cells perfused with a medium containing Lucifer Yellow. Fluorescent dye-excluded spots in the plane of cell adhesion corresponded well to dark spots in IR images. Because of these advantages over the conventional IR microscope, we can corroborate the usefulness of the CLM for IR analysis.

DOI： 10.11169/bioimages.1.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EDITIONS SCIENTIFIQUES ELSEVIER  

When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the 'zinc method' to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.

DOI： 10.1016/S0248-4900(05)80191-1

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Language：English   Publisher：(一社)日本解剖学会  

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Language：Japanese   Publisher：(NPO)日本法医学会  

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Language：Japanese   Publisher：日本顕微鏡学会  

CiNii Books

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：English   Publisher：Oxford University Press  

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Language：English   Publisher：(一社)日本細胞生物学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE SOC ELECTRON MICRO BUS CENTER ACAD SOC JAPAN  

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Presentation type：Poster presentation  

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