A defect in cell-to-cell adhesion via integrin-fibronectin interactions in a highly metastatic tumor cell line.

We investigated the role of integrin-fibronectin (FN) interactions in tumor cell adhesion. Two cloned tumor cell lines designated OV-LM (low-metastatic) and OV-HM (high-metastatic) were isolated from a murine ovarian carcinoma, OV2944. OV-LM and OV-HM cells exhibited high and low RGDS-sequence-dependent adhesiveness to FN, respectively.

Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve.

The localization of synapsin I, a synaptic vesicle-associated protein, was investigated immunocyto-chemically in normal nerve fibers and regenerating axonal sprouts following crush-injuries to the rat sciatic nerve. In normal myelinated axons, weak synapsin I immunoreactivity was found in the axoplasmic/smooth endoplasmic domains, but not in the cytoskeletal domains comprising neurofilaments and microtubules. In non-myelinated axons without dense cytoskeletal structures, moderate immunoreactivity was distributed diffusely throughout the axoplasm.

Phase specific association of heterotrimeric GTP-binding proteins with the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Subcellular distribution of heterotrimeric GTP-binding proteins during thrombin receptor-mediated platelet activation was examined, revealing two phases of translocation to the cytoskeleton. A part of Gi2 alpha and Gs alpha shows first phase translocation to the low-speed pellet (15000 x g pellet) within 1 min after activation, suggesting involvement in platelet shape change or granule secretion. In the second phase, Gi2 alpha, Gs alpha, Gq alpha, and G beta translocate to the low-speed pellet, depending on platelet aggregation.

Translocation of cortactin (p80/85) to the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Cortactin (p80/85) was discovered as a src kinase substrate and an actin filament binding protein. We investigated translocation of cortactin to the cytoskeleton during thrombin receptor-mediated platelet activation. Only a few percent of total cortactin (minor cortactin pool) translocates to the cytoskeleton as early as 5 s after platelet activation, while about 40% of total cortactin (major cortactin pool) is thereafter recovered in the cytoskeleton during platelet aggregation.

Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.

The intracellular protein tyrosine kinase FAK (focal adhesion kinase) was originally identified gy its high level of tyrosine phosphorylation in v-src-transformed cells. FAK is also highly phosphorylated during early development. In cultured cells it is localized to focal adhesion contacts and becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix, suggesting an important role in cell adhesion and/or migration.

Transcriptional regulation of the chicken caldesmon gene. Activation of gizzard-type caldesmon promoter requires a CArG box-like motif.

Caldesmon, which plays a vital role in the actomyosin system, is distributed in smooth muscle and non-muscle cells, and its isoformal interconversion between a high M(r) form and low M(r) form is a favorable molecular event for studying phenotypic modulation of smooth muscle cells. Genomic analysis reveals two promoters, of which the gizzard-type promoter displays much higher activity than the brain-type promoter. Here, we have characterized transcriptional regulation of the gizzard-type promoter.

Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin (nonerythroid spectrin or fodrin) by calpain.

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet.

Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones.

Neuronal growth cones move actively, accompanying changes in intracellular Ca2+ concentration. The movement of growth cones may partly depend on the actomyosin system, considering the presence of actin and myosin II. Yet, Ca(2+)-sensitive regulatory proteins for the actomyosin system have not been identified in growth cones.

Annexin VI binds to a synaptic vesicle protein, synapsin I.

Annexin VI bound to > 14 species of proteins in the whole homogenate of rat forebrain in a Ca2+/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M(r) 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I.

Annexin VI-binding proteins in brain. Interaction of annexin VI with a membrane skeletal protein, calspectin (brain spectrin or fodrin).

Identification of annexin VI-binding proteins is essential to elucidate the physiological functions of annexin VI. Here, we developed the methods to identify an annexin VI-binding protein and characterized the binding. Annexin VI bound to about 14 species of proteins in the whole homogenate of rat forebrain, when examined with 125I-annexin VI using blots of SDS-polyacrylamide gels.

Identification of two distinct promoters in the chicken caldesmon gene.

Caldesmon (CaD) is a suitable molecular marker for phenotypic modulation of smooth muscle cells. Chicken CaD gene is composed of 17 exons with a whole length of 100-150 kilobases (kb). Exons 1a-1, 1a-2, and 1a-3 encode the 5'-terminal sequence specific to mRNA for gizzard type CaD, and exon 1b encodes the sequence specific to brain type CaD mRNA.

Localization and characterization of gelsolin in nervous tissues: gelsolin is specifically enriched in myelin-forming cells.

Gelsolin is a Ca(2+)-sensitive actin filament-severing protein. To elucidate the role of gelsolin in nervous tissues, we have investigated localization and expression of gelsolin in rat CNS and PNS using biochemical and morphological methods with a polyclonal antibody against the COOH-terminal fragment of plasma gelsolin. Immunohistochemical study showed that gelsolin was specifically enriched in oligodendrocytes and Schwann cells, and was also detected in myelin sheath, especially around the Ranvier's nodes.

Gelsolin is localized in neuronal growth cones.

Gelsolin, a Ca(2+)-sensitive actin filament-severing protein, is involved in actin turnover. Immunocytochemical study with anti-gelsolin antibody revealed that the protein is localized in both filopodia and the body part of growth cones of differentiated PC12 cells with nerve growth factor (NGF) and of rat dorsal root ganglion neurons. Here, we identified gelsolin as one of the molecular bases for the Ca(2+)-dependent movement of growth cones.

Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery.

Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluorescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions.

Common structural and expressional properties of vertebrate caldesmon genes.

We have determined the genomic structure of chicken caldesmon (CaD) gene. The gene, 100-150 kilobases long, is composed of 17 exons. Exons 1a-1, 1a-2, and 1a-3 encode the 5'-terminal sequence specific to the mRNAs for CaDs expressed in gizzard.

Actin-based cytoskeleton in growth cone activity.

A growing tip of neurite, the neuronal growth cone, is a highly motile and adhesive form of cytoarchitecture. The growth cone plays vital roles for navigation, elongation and maintenance of neurites. One major constituent of growth cones, regulated by the intracellular Ca2+ signal, is the actin-based cytoskeleton.

Morphological and biochemical analyses of contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal and transformed cells.

The expression and intracellular distribution of four contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal fibroblasts and their transformed counterparts by Rous or avian sarcoma virus were compared. By analyzing the isoformal expression of actin, caldesmon and tropomyosin using two-dimensional gel electrophoresis, only tropomyosin showed significant alteration in its isoformal expression accompanied by transformation. Morphological study revealed that in normal cells, myosin, caldesmon and tropomyosin were distributed periodically along stress fibers, but were excluded from focal adhesions (adhesion plaques), at which stress fibers terminate.

Genomic structure of the human caldesmon gene.

The high molecular weight caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for isoform diversity of human CaDs, l-CaD cDNAs were cloned from HeLa S3 cells.

The synapsin I brain distribution in ischemia.

We examined the distribution of synapsin I in the gerbil brain and investigated ischemic damage of presynaptic terminals immunohistochemically by using this protein as a marker protein of synaptic vesicles. The reaction for synapsin I in normal gerbil brain is exclusively localized in the neuropil, and other brain structures such as neuronal soma, dendrites, axon bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, ischemic loss of synapsin I immunoreactivity in the affected hemisphere was confined to the area exhibiting overt infarction, where the breakdown of this protein was also confirmed by the immunoblot analysis, and noted much later than that of microtubule-associated protein 2 immunoreactivity, which was demonstrated in neuronal soma and dendrites.

The characteristics of blood-brain barrier in three different conditions--infarction, selective neuronal death and selective loss of presynaptic terminals--following cerebral ischemia.

We investigated the extravasation of serum albumin using immunohistochemistry in three different conditions, i.e., infarction, selective neuronal death and selective loss of presynaptic terminals following cerebral ischemia in gerbils.

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1.

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin.

Structural and functional relationships between h- and l-caldesmons.

Two different Mr forms of caldesmon as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr values in the range of 120,000-150,000, h-caldesmon and 70,000-80,000, l-caldesmon) have been already identified. h-Caldesmon is predominantly expressed in smooth muscle cells, whereas l-caldesmon widely distributes in non-muscle cells. Most recently, the molecular cloning of h-caldesmon has been reported (Hayashi, K.

Developmental changes of synapsin I subcellular localization in rat cerebellar neurons.

Synapsin I, one of the major synaptic proteins, is thought to associate with synaptic vesicles and to play a regulatory role in neurotransmitter release. In mature neurons, it is concentrated almost exclusively in presynaptic nerve endings. Here, we studied the subcellular localization of synapsin I during the development of rat cerebellar cortices by immunocytochemistry, using anti-synapsin I antibodies and found that during the development of rat cerebellar cortices it tentatively exists in the dendritic growth cones of immature internal granule cells and in the axonal growth cones of mossy fibers as well as mature presynaptic endings.