Yasunori Murakami

Plexin-Neuropilin-1 Complexes Form Functional Semaphorin-3A Receptors

Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins. We find that the two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes.

Plexin: A novel neuronal cell surface molecule that mediates cell adhesion via a homophilic binding mechanism in the presence of calcium ions

Plexin (previously referred to as B2) is a neuronal cell surface molecule that has been identified in Xenopus. cDNA cloning reveals that plexin has no homology to known neuronal cell surface molecules but possesses, in its extracellular segment, three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. The exogenous plexin proteins expressed on the surfaces of L cells by cDNA transfection mediate cell adhesion via a homophilic binding mechanism, under the presence of calcium ions. Plexin is expressed in the receptors and neurons of particular sensory systems. These findings indicate that plexin is a novel calcium-dependent cell adhesion molecule and suggest its involvement in specific neuronal cell interaction and/or contact.

Differential expression of plexin-A subfamily members in the mouse nervous system.

Plexins comprise a family of transmembrane proteins (the plexin family) which are expressed in nervous tissues. Some plexins have been shown to interact directly with secreted or transmembrane semaphorins, while plexins belonging to the A subfamily are suggested to make complexes with other membrane proteins, neuropilins, and propagate chemorepulsive signals of secreted semaphorins of class 3 into cells or neurons. Despite that much information has been gathered on the plexin‐semaphorin interaction, the role of plexins in the nervous system is not well understood. To gain insight into the functions of plexins in the nervous system, we analyzed spatial and temporal expression patterns of three members of the plexin‐A subfamily (plexin‐A1, ‐A2, and ‐A3) in the developing mouse nervous system by in situ hybridization analysis in combination with immunohistochemistry. We show that the three plexins are differentially expressed in sensory receptors or neurons in a developmentally regulated manner, suggesting that a particular plexin or set of plexins is shared by neuronal elements and functions as the receptor for semaphorins to regulate neuronal development.

Identification and characterization of a novel mouse plexin, plexin-A4 ☆

Plexins belonging to the plexin-A subfamily form complexes with neuropilins and propagate signals of class 3 semaphorins into neurons, even though they do not directly bind the semaphorins. In this study, we identified a new member of the plexin-A subfamily in the mice, plexin-A4, and showed that it was expressed in the developing nervous system with a pattern different to that of other members of the plexin-A subfamily (plexin-A1, plexin-A2 and plexin-A3). COS-7 cells coexpressing plexin-A4 with neuropilin-1 were induced to contract by Sema3A, a member of the class 3 semaphorin. Ectopic expression of plexin-A4 in mitral cells that are originally insensitive to Sema3A resulted in the collapse of growth cones in the presence of Sema3A. These results suggest that plexin-A4 plays a role in the propagation of Sema3A activities.

ISOLATION OF BLEPHARISMIN‐BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step‐up photophobic response) in response to light stimulation. A profile of a gel‐permeation of a crude detergent‐solubilized sample of the cells resulted in several red‐colored fractions. Among these blepharismin‐containing fractions, the fractions III‐V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step‐up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin‐protein complex) is responsible for the step‐up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore‐protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin‐layer chromatography‐purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore‐protein complex.

Function of a Cell Adhesion Molecule, Plexin, in Neuron Network Formation

Plexin is a type I membrane protein which was identified in Xenopus nervous system by hybridoma technique. Molecular cloning studies demonstrated that the extracellular segment of the plexin protein possesses three internal repeats of cysteine cluster which are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. A cell aggregation test revealed that the plexin protein mediated cell adhesion via a homophilic binding mechanism, in the presence of calcium ions. Plexin was expressed in the neuronal elements composing particular neuron circuits in Xenopus CNS and PNS. These findings indicate that plexin is a new member of the Ca(2+)-dependent cell adhesion molecules, and suggest that the molecule plays an important role in neuronal cell contact and neuron network formation.

PHOTORECEPTOR PIGMENT IN Blepharisma: H+ RELEASE FROM RED PIGMENT

In faded cells of Blepharisma kept in a standard saline solution containing bacteria which had been cultured on agar plates containing glucose and polypepton, threshold light intensity for step‐up photophobic response elevated. This result suggests that red pigment (blepharismin) contained in Blepharisma cells is involved in the step‐up photophobic response. The pH of the aqueous solution of the red pigment was found to decrease when light was applied, indicating that the pigment releases H+ in response to light stimulation. However, faded pigment preparation by light irradiation did not show pH decrease. In the living cells faded by light irradiation, threshold light intensity for the step‐up photophobic response was raised. Results suggest that H+ release from the red pigment induced by light irradiation might be responsible for the step‐up photophobic response of the cells.

The photoreceptor pigment of the unicellular organism Blepharisma generates hydroxyl radicals

Abstract The quinone pigment blepharismin isolated from Blepharisma, which is believed to be a photoreceptor pigment mediating photobehaviour (P. Scevoli, F. Bisi, G. Colombetti, F. Ghetti, F. Lenci and V. Passarelli, Photomotile responses of Blepharisma japonicum. I. Action spectra determination and time-resolved fluorescence of photoreceptor pigments, J. Photochem. Photobiol. B: Biol., 1 (1987) 75–84; T. Matsuoka, S. Matsuoka, Y. Yamaoka, T. Kuriu, Y. Watanabe, M. Takayanagi, Y. Kato and K. Taneda, Action spectra for step-up photophobic response in Blepharisma, J. Protozool., 39 (1992) 498–502), killed mammalian cells (L cells) when activated by light. Electron spin resonance (ESR) spectroscopy demonstrated that light-activated blepharismin generated hydroxyl radicals (.OH). Light-activated blepharismin pigment promoted lipid peroxidation of L cells, which was partially suppressed in the presence of the singlet oxygen quencher sodium azide (NaN3). The results suggest that the photodynamic action of blepharismin is correlated with lipid peroxidation which may be caused by hydroxyl radicals and/or singlet oxygen (1O2) produced via reactions sensitized by the pigment.