Cytoarchitecture of the olfactory bulb in the laggard mutant mouse.

The laggard (lag) mutant mouse, characterized by hypomyelination and cerebellar ataxia, is a spontaneously occurring mutant mouse caused by mutation in the Kif14 gene. In this mutant mouse, the laminated structures such as the cerebral and cerebellar cortices and the dentate gyrus are cytoarchitecturally abnormal. Macroscopically, the olfactory bulb of the lag mutant mouse is smaller in size and more transparent than the normal counterpart. Hematoxylin-eosin staining reveals that the mutant olfactory bulb has normal lamination in general, but detailed analysis has demonstrated that olfactory periglomerular cells and granule cells are reduced in number. In the mutant, olfactory glomeruli are cytoarchitecturally disorganized and mitral cells are arranged in multiple cell layers instead of being arranged in a single layer. The rostral migratory stream in the mutant becomes gradually thinner or obliterated during early postnatal days. Some of mitral cells and periglomerular cells are multinucleated, suggesting that Kif14 mutation leads to an abnormal cell division. In the mutant, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the subventricular zone of the lateral ventricle are increased in number, especially at perinatal age, suggesting that the decreased population of granule cells in the lag mutant mouse is caused by the increased apoptotic cell death. The olfactory input appears to be intact, as indicated by anterograde labeling of olfactory nerves with an injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the olfactory mucosa. In conclusion, the olfactory bulb of the lag mutant mouse is cytoarchitecturally affected, suggesting that the causal gene for lag mutation, i.e., Kif14, has multiple effects on the development of laminated structures in the central nervous system in addition to the myelin formation.

Roles of nectins in cell adhesion, signaling and polarization.

Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which constitute a family of four members. Nectins homophilically and heterophilically trans-interact and cause cell-cell adhesion. This nectin-based cell-cell adhesion plays roles in the organization of adherens junctions in epithelial cells and fibroblasts and synaptic junctions in neurons in cooperation with cadherins. The nectin-based cell-cell adhesion plays roles in the contacts between commissural axons and floor plate cells and in the organization of Sertoli cell-spermatid junctions in the testis, independently of cadherins. Nectins furthermore regulate intracellular signaling through Cdc42 and Rac small G proteins and cell polarization through cell polarity proteins. Pathologically, nectins serve as entry and cell-cell spread mediators of herpes simplex viruses.

Requirement of interaction of nectin-1alpha/HveC with afadin for efficient cell-cell spread of herpes simplex virus type 1.

We recently found a novel cell-cell adhesion system at cadherin-based adherens junctions (AJs), consisting at least of nectin, a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin is associated with cadherin through afadin and alpha-catenin. The cadherin-catenin system increases the concentration of nectin at AJs in an afadin-dependent manner. Nectin constitutes a family consisting of three members: nectin-1, -2, and -3. Nectin-1 serves as an entry and cell-cell spread mediator of herpes simplex virus type 1 (HSV-1). We studied here a role of the interaction of nectin-1alpha with afadin in entry and/or cell-cell spread of HSV-1. By the use of cadherin-deficient L cells overexpressing the full length of nectin-1alpha capable of interacting with afadin and L cells overexpressing a truncated form of nectin-1alpha incapable of interacting with afadin, we found that the interaction of nectin-1alpha with afadin increased the efficiency of cell-cell spread, but not entry, of HSV-1. This interaction did not affect the binding to nectin-1alpha of glycoprotein D, a viral component mediating entry of HSV-1 into host cells. Furthermore, the cadherin-catenin system increased the efficiency of cell-cell spread of HSV-1, although it also increased the efficiency of entry of HSV-1. It is likely that efficient cell-cell spread of HSV-1 is caused by afadin-dependent concentrated localization of nectin-1alpha at cadherin-based AJs.

Importance of spatial activation of Cdc42 and rac small G proteins by frabin for microspike formation in MDCK cells.

BACKGROUND: Frabin is an actin filament (F-actin)-binding protein that shows GDP/GTP exchange activity for Cdc42 small G protein (Cdc42). Frabin furthermore induces indirect activation of Rac small G protein (Rac) in intact cells. We have recently shown that in nonepithelial cells, frabin induces the formation of both filopodia- and lamellipodia-like processes through the activation of Cdc42 and Rac, respectively. In epithelial cells such as MDCK cells, Cdc42 and Rac regulate cell-cell adherens junctions (AJs) via the accumulation of F-actin and E-cadherin, although neither Cdc42 nor Rac induces the formation of filopodia or lamellipodia. In this study, we have examined the effects of frabin on the reorganization of the actin cytoskeleton in MDCK cells. RESULTS: Frabin induces the formation of microspikes at the basal area of the lateral membranes through the activation of Cdc42 and Rac in MDCK cells, although a dominant active mutant of Cdc42 or Rac alone, or both, did not induce the formation of microspikes. Furthermore, frabin weakly increased the accumulation of F-actin and E-cadherin at cell-cell AJs and the formation of stress fibres through the activation of Cdc42 and Rac, under conditions where the dominant active mutant of Cdc42 or Rac markedly showed these effects. The Cdc42- and Rac-induced formation of stress fibres was dependent on the activation of Rho small G protein. CONCLUSION: These results indicate that the frabin-dependent spatial activation of Cdc42 and Rac is important for the formation of microspikes.

Ankycorbin: a novel actin cytoskeleton-associated protein.

BACKGROUND: Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity and cytokinesis. Many actin filament (F-actin)-binding proteins have been isolated and implicated in the maintenance and reorganization of actin cytoskeleton structures. RESULTS: We purified here a novel protein with a molecular mass of about 125 kDa (p125) from rat liver. We cloned its cDNA from a mouse kidney cDNA library and determined its nucleotide and deduced amino acid sequences. p125 was a protein of 979 amino acids with a calculated Mr of 108 847. p125 contained six ankyrin repeats in the N-terminal region and a domain predicted to form a coiled-coil structure in the C-terminal region. We named p125 ankycorbin (ankyrin repeat- and coiled-coil structure-containing protein). Northern blot analysis indicated that ankycorbin was ubiquitously expressed in all the tissues examined. Immunofluorescence and immunoelectron microscope analyses revealed that ankycorbin was associated with the cortical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibres. However, ankycorbin did not directly bind to F-actin as estimated by the F-actin co-sedimentation assay. CONCLUSIONS: These results indicate that ankycorbin is indirectly associated with the actin cytoskeleton structures, presumably through an unidentified factor and suggest that it is involved in their maintenance and/or reorganization.

Similar and differential behaviour between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells.

BACKGROUND: We have recently identified a novel cell-cell adhesion system, named NAP system, which is localized at cadherin-based cell-cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin-like cell adhesion molecule. Afadin is an actin filament-binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin-binding protein which furthermore binds to vinculin and provides a possible linkage of nectin-afadin to cadherin-catenin through vinculin. We compared here the behaviour of the NAP and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. RESULTS: At the early stage of the formation of the polarized junctional alignment in MTD-1 A cells, primordial spot-like junctions were formed at the tips of thin cellular protrusions radiating from adjacent cells. Nectin, afadin, ponsin, cadherin and catenin were simultaneously recruited to these junctions. As the cell polarization proceeded, the spot-like junctions were gradually fused to form belt-like AJs where all these proteins were concentrated. The disruption of cell-cell AJs in MDCK cells by culturing at a low Ca2+ concentration caused rapid endocytosis of cadherin, but not that of nectin or afadin. Addition of 12-O-tetradecanoylphorbol-13-acetate to the cells formed a tight junction-like structure where nectin and afadin, but not cadherin, accumulated. CONCLUSION: These results indicate that the NAP and cadherin-catenin systems show similar and differential behaviour during the formation and disruption of the polarized junctional alignment in epithelial cells.

Coendocytosis of cadherin and c-Met coupled to disruption of cell-cell adhesion in MDCK cells--regulation by Rho, Rac and Rab small G proteins.

Both E-cadherin, a cell-cell adhesion molecule, and c-Met, the hepatocyte growth factor (HGF)/scatter factor (SF) receptor, were colocalized at cell-cell adhesion sites of MDCK cells. HGF/SF or a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced disruption of cell-cell adhesion, which was accompanied by endocytosis of both E-cadherin and c-Met. Reduction of medium Ca2+ to a micromolar range showed the same effects. Re-increase in medium Ca2+ to a millimolar range formed cell-cell adhesion, which was accompanied by exocytosis of E-cadherin and c-Met, followed by their re-colocalization at the cell-cell adhesion sites. These results suggest that E-cadherin and c-Met are colocalized at cell-cell adhesion sites and undergo co-endo-exocytosis. We have previously shown that TPA does not induce disruption of cell-cell adhesion and subsequent scattering of MDCK cells stably expressing a dominant active mutant of RhoA or Rac1 small G protein or a dominant negative mutant of Rab5 small G protein. In these cell lines, the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met was inhibited, but the coendocytosis of E-cadherin and c-Met in response to reduction of medium Ca2+ was not affected. Wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, inhibited the HGF-induced disruption of cell-cell junction and endocytosis of E-cadherin and c-Met, but not the TPA-induced ones. These results suggest that disruption of cell-cell adhesion is involved in the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met in MDCK cells, and that the Rho and Rab family members indirectly regulate this coendocytosis. In addition, coendocytosis of E-cadherin and c-Met in response to HGF is partly mediated by PI 3-kinase. The cross-talk between cell-cell and cell-matrix adherens junctions is discussed.

Different behavior of l-afadin and neurabin-II during the formation and destruction of cell-cell adherens junction.

We have recently isolated two novel actin filament-binding proteins, l-afadin and neurabin-II and shown that they are localized at cell-cell adherens junction (AJ) in epithelial cells. We found here that l-afadin, neurabin-II, ZO-1, and E-cadherin showed similar and different behavior during the formation and destruction of cell-cell AJ in MDCK cells. In MDCK cells, the accumulation of both l-afadin and E-cadherin, but not that of ZO-1, changed in parallel depending on Rac small G protein activity. Dissociation of MDCK cells by culturing the cells at 2 microM Ca2+ caused rapid endocytosis of E-cadherin, but not that of l-afadin or ZO-1. Addition of phorbol 12-myristate 13-acetate to these dissociated cells formed a tight junction-like structure where ZO-1 and l-afadin, but not neurabin-II or E-cadherin, accumulated. We furthermore found that, in non-epithelial EL cells, which expressed E-cadherin and attached to each other, l-afadin, neurabin-II, ZO-1 and E-cadherin were all localized at AJ. In cadherin-deficient L cells, I-afadin was mainly localized at cell-cell contact sites, but ZO-1 was mainly localized at the tip area of cell processes. Neurabin-II did not accumulate at the plasma membrane area. Neither l-afadin nor neurabin-II significantly interacted with alpha-, beta-catenin, E-cadherin, ZO-1 or occludin.

Three splicing variants of tomosyn and identification of their syntaxin-binding region.

We have recently isolated a neural tissue-specific syntaxin-1-binding protein, named tomosyn, which is capable of dissociating Munc18/n-Sec1/rbSec1 from syntaxin-1 to form a 10S tomosyn complex, an intermediate complex converted to the 7S SNARE complex. We isolated here two splicing variants of tomosyn: one had 36 amino acids (aa) insertion and another had 17 aa deletion. We named original one m-tomosyn, big one b-tomosyn, and small one s-tomosyn. s-Tomosyn as well as m-tomosyn was mainly expressed in brain whereas b-tomosyn was ubiquitously expressed. All the isoforms bound to syntaxin-1, but not to syntaxin-2, -3, or -4, and had a region highly homologous to VAMP, another syntaxin-binding protein. This region was necessary but not sufficient for high-affinity binding of tomosyn to syntaxin-1.

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.