Masanobu Obara

Site-directed mutagenesis of the cell-binding domain of human fibronectin: Separable, synergistic sites mediate adhesive function

Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell‐binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively‐spliced type III homology‐connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

Role of Fibronectin in Adhesion, Migration, and Metastasis

AbstractAdhesive macromolecules of the extracellular matrix regulate cellular migration, differentiation, and growth. They also contribute actively to the pathogenesis of diseases characterized by aberrant adhesion, including cancer. One of the most well characterized adhesion factors is the glycoprotein fibronectin; biochemical analyses of this molecule have substantially improved our understanding of how cells adhere, migrate, and invade, and it is now thought that fibronectin plays a key role in the tissue remodeling and cell migration events that occur during normal embryonic development and adult wound healing. In this review, we describe how recent studies, focusing on the basic question of precisely how a cell adheres to fibronectin at the molecular level, have led to insights into the mechanisms of cellular migration by normal and malignant cells and to the development of a novel, synthetic inhibitor of experimental metastasis. We also critically discuss the future prospects for antiadhesive agent...

Analysis of N-myc amplification in relation to disease stage and histologic types in human neuroblastomas

Both untreated and treated primary neuroblastomas from 52 patients were analyzed to determine the correlation between the amplification of N‐myc oncogene and various prognostic factors. Amplification of N‐myc was observed in eight of 28 untreated cases and in 12 of 24 treated cases. As a whole, 12 of 18 tumors (67%) in Stage IV had N‐myc amplification, but there were fewer cases in the unadvanced disease stage, as reported previously by others. Furthermore, the authors detected N‐myc amplification in three of nine tumors in Stage IV‐S, although the amplification was less than 50 copies. Analysis of progression‐free survival at 24 months revealed that amplification of N‐myc was associated with the worst prognosis (P < 0.001). In the untreated group, no amplification of N‐myc was detected in any of two ganglioneuromas and four ganglioneuroblastomas, whereas amplification of N‐myc was observed in all two round‐cell and six of 20 rosette fibrillary neuroblastomas. On the other hand, the authors detected amplification of N‐myc in three of eight less differentiated ganglioneuroblastomas in the treated group and observed the worst prognosis in these three patients. The total percentage of the cases from both untreated and treated groups suggest that amplification of N‐myc may occur more frequently in undifferentiated types of neuroblastomas than in less malignant types. In conclusion, the amplification of N‐myc in neuroblastomas was closely associated with the worst prognosis, which was suggested by both disease stage and histologic characteristics.

Unique expression patterns of matrix metalloproteinases in regenerating newt limbs.

The process of regeneration of urodele limbs includes a drastic remodeling of extracellular matrices (ECMs) that is induced by matrix metalloproteinases (MMPs) and is thought to be one of the triggers of the regeneration. We studied this remodeling in limbs of Japanese newt, Cynops pyrrhogaster, by using five genes of newt MMPs (nMMPs) as probes: nMMP9, nMMP3/10‐a, nMMP3/10‐b, and nMMP13 that had been characterized previously, and nMMPe that was newly cloned in the present study. nMMPe was 502 amino acid residues long and showed a low homology to other known vertebrate MMPs. Reverse transcriptase‐polymerase chain reactions analysis localized the transcript of nMMPe in the apical epidermal cap (AEC) and the non–blastemal wound epidermis but not in the blastemal mesenchyme or the normal epidermis. Northern blot analysis localized the transcripts of nMMP9, nMMP3/10‐a, and nMMP13 in the bone of regenerating limbs, whereas those of nMMP3/10‐b in AEC. mRNA in situ hybridization experiments identified the nMMP‐expressing cells. nMMP9 gene was strongly expressed in chondrocytes of the cartilage of epiphysis. Of interest, basal cells of AEC, but not those of the normal skin, expressed nMMP3/10‐b intensely. Immunohistochemical analysis showed that the nMMP9 proteins synthesized by chondrocytes were secreted and distributed widely in the basement membrane of bone and ECMs of the amputation plane. These nMMPs characterized in the present study might cooperatively work to remodel ECMs of regenerating limbs. Developmental Dynamics 226:366–376, 2003.

Human carcinoma cells synthesize and secrete tenascin in vitro.

Tenascin is an extracellular matrix glycoprotein produced in response to epithelial‐mesenchymal interactions that initiate fetal organogenesis, and it is also found in the stroma of benign and malignant neoplasms. Thirty‐five human cell lines representing a variety of cancers were examined by im‐munoprecipitation and polyacrylamide gel electrophoresis of radiolabeled tenascin proteins from conditioned media. Two forms of tenascin with relative molecular masses of 190,000 and 250,000 were identified. Eight cell lines produced both forms. With the exception of myeloid and lymphoid leukemias and Burkitts lymphoma, all of the mesodermal and neuroectodermal tumor lines were found to synthesize tenascin. Unexpectedly, tenascin was secreted by several mammary and colonic adenocarcinomas as well as by a line derived from normal mammary epithelial cells, and in some cases increased production was induced by transforming growth factor beta in serum‐free medium. Cells producing fibronectin but not tenascin attached and spread on plastic culture dishes, while those producing tenascin alone remained suspended in the medium or were rarely attached. Tenascin also inhibited fibronectin‐mediated adhesion of MCF7 breast carcinoma cells in vitro. The results suggest that tenascins synthesized and secreted by some cancer cells, especially those of epithelial origin, may have specific roles in determining tumor cell adhesion and ultimately the ability to form invasive outgrowths.

Characterization of human stellate cell activation-associated protein and its expression in human liver.

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.

Lineage of anuran epidermal basal cells and their differentiation potential in relation to metamorphic skin remodeling

The anuran remodels the larval epidermis into the adult one during metamorphosis. Larval and adult epidermal cells of the bullfrog were characterized by determining the presence of huge cytoplasmic keratin bundles and the expression profiles of specific marker genes, namely colα1 (collagen α1 (I)), rlk (larval keratin) and rak (adult keratin). We identified four types of epidermal basal cells: (i) basal skein cells that have keratin bundles and express colα1 and rlk; (ii) rak+‐basal skein cells that have keratin bundles and express colα1, rlk, and rak; (iii) larval basal cells that express rlk and rak; and (iv) adult basal cells that express rak. These traits suggested that these basal cells are on the same lineage in which basal skein cells are the original progenitor cells that consecutively differentiate into rak+‐basal skein cells into larval basal cells, and finally into adult basal cells. To directly verify the differentiation potential of larval basal cells into adult ones, the mono‐layered epidermis composed of larval basal cells was cultured in the presence of aldosterone and thyroid hormone. In this culture, larval basal cells differentiated into adult basal cells that reconstituted the adult epidermis. Thus, it was concluded that larval basal cells are the direct progenitor cells of the adult epidermal stem cells.

Typing of hepatic nonparenchymal cells using fibulin-2 and cytoglobin/STAP as liver fibrogenesis-related markers

Fibulin-2 and cytoglobin/stellate cell activation-associated protein (Cygb/STAP) are considered to be markers of hepatic myofibroblasts (MFs) and stellate cells (HSCs), respectively. The aim of the present study was to characterize the nonparenchymal cells (NPCs) of normal rat livers and carbon tetrachloride-induced fibrotic rat livers with respect to the expression of these two proteins. NPCs in normal (Glisson’s capsules) and fibrotic (fibrotic septa) connective tissues were immunohistochemically categorized into four cell types in terms of the expression of fibulin-2 and Cygb/STAP: fibulin-2 and Cygb/STAP double-positive (Fib+/STAP+); fibulin-2-positive and Cygb/STAP-negative (Fib+/STAP−); Fib−/STAP+; and Fib−/STAP−. The Glisson’s capsules had Fib+/STAP+ and Fib−/STAP− cell occupancy rates of 45.5% and 54.5%, respectively, but did not contain Fib+/STAP− or Fib−/STAP+ cells. On the other hand, the fibrotic septa contained Fib+/STAP+, Fib−/STAP+, and Fib−/STAP− cells at occupancy rates of 35.0%, 50.5%, and 9.1%, respectively, but did not contain Fib+/STAP− cells. Thus, fibrosis is characterized by a dramatic increase in Fib−/STAP+ NPCs, and a dramatic decrease in Fib−/STAP− NPCs. Fib+/STAP+ NPCs are located uniformly in Glisson’s capsules and peripherally in fibrotic septa. The present study strongly suggests that Fib+/STAP+ and Fib−/STAP+ NPCs correspond to MFs and activated HSCs, respectively, both of which may contribute to liver fibrogenesis.