Yuichi Takada

Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

Lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) activated Fyn tyrosine kinase and induced stress fiber formation, which was blocked by pharmacological inhibition of Fyn, gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn localized at both ends of F‐actin bundles and triggered stress fiber formation, only the latter of which was abolished by Rho‐kinase (ROCK) inhibition. SPC, but not LPA, induced filopodia‐like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts.

Sphingosylphosphorylcholine induces stress fiber formation via activation of Fyn-RhoA-ROCK signaling pathway in fibroblasts.

Sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, has recently been reported to modulate actin cytoskeleton rearrangement. We have previously demonstrated Fyn tyrosine kinase is involved in SPC-induced actin stress fiber formation in fibroblasts. However, Fyn-dependent signaling pathway remains to be elucidated. The present study demonstrates that RhoA-ROCK signaling downstream of Fyn controls stress fiber formation in SPC-treated fibroblasts. Here, we found that SPC-induced stress fiber formation was inhibited by C3 transferase, dominant negative RhoA or ROCK. SPC activated RhoA, which was blocked by pharmacological inhibition of Fyn activity or dominant negative Fyn. Constitutively active Fyn (ca-Fyn) stimulated stress fiber formation and localized with F-actin at the both ends of stress fibers, both of which were prevented by Fyn translocation inhibitor eicosapentaenoic acid (EPA). In contrast, inhibition of ROCK abolished only the formation of stress fibers, without affecting the localization of ca-Fyn. These results allow the identification of the molecular events downstream SPC in stress fiber formation for a better understanding of stress fiber formation involving Fyn.

Light-induced and apoptosis-like cell death in the unicellular eukaryote, Blepharisma japonicum

The unicellular eukaryote, Blepharisma japonicum, is a light‐sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step‐up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300 W m−2) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3–30 W m−2) only induced pigment extrusion and photooxidation. We have found that moderate light (5 W m−2) induced apoptosis‐like cell death. Microscopically it was found that >3 h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3 h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light‐irradiated cells contained DNA fragments of 180–200 and 360–400 bp, which were seen as apoptosis ladders.

Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum.

A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian alpha-, micro-, pi-, sigma-, theta-, zeta-, kappa-, and omega-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to alpha-, micro-, pi-, and sigma-class GSTs rather than the other class of GSTs.