Yuko Ikegami

Cell Death-Associated Translocation of Plasma Membrane Components Induced by CTL

In the very early stages of target cell apoptosis induced by CTL, we found that fluorescence of labeling probes of the target plasma membrane, such as N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridinium dibromide (FM1-43), was translocated into intracellular membrane structures including nuclear envelope and mitochondria. This translocation was associated with the execution of CTL-mediated killing, because neither the CTL-target conjugation alone nor the binding of noncytotoxic Th2 clone with target cell was sufficient to provoke the process. Although FM1-43 translocation was observed in perforin-mediated cytotoxicity, examinations with several other dyes failed to detect the evidence for membrane damages that may cause influx of the dye. Moreover, the translocation was also observed in Fas-dependent apoptosis. These data indicate that the translocation precedes the damage of plasma membrane and intracellular organella in the course of apoptotic cell death and may represent the existence of a membrane trafficking that mediates the translocation of plasma membrane components in the early onset of apoptotic cell death.

Genetic interactions of pokkuri with seven in absentia, tramtrack and downstream components of the sevenless pathway in R7 photoreceptor induction in Drosophila melanogaster

The sevenless (sev) cascade plays an inductive role in formation of the R7 photoreceptor, whilst the pokkuri (pok) and tramtrack (ttk) gene products are known to repress R7 induction in developing ommatidia of Drosophila melanogaster. To elucidate how these positive and negative signalling mechanisms co-operate in the normal fate determination of R7, genetic interactions of mutations in the pok locus with ttk and downstream elements of sev including Gap1, raf1, rolled (r1) and seven in absentia (sina) were examined. The eye phenotype of a weak hypomorph, pok15, was enhanced dominantly by Gap1-mip, a recessive mutation in a gene encoding a down-regulator of Ras1, producing multiple R7 in ommatidia. Ras1 has been reported to activate r1-encoded mitrogen-activated protein (MAP) kinase via Raf1 that is associated physically with Rasl. Ommatidia of raf1c110 and rl2/rlEMS64 typically lacked R7 and a few outer photoreceptors. The pok1 mutation suppressed dominantly the raflc110 rl2/rlEMS64 eye phenotypes, allowing single R7 cells to develop in ommatidia. The raflc110 mutation improved adult viability of pok1 homozygotes. An in vitro experiment demonstrated that MAP kinase phosphorylates Pok protein. Ttk is a transcriptional repressor which binds to the regulatory sequence upstream of the fushi-tarazu (ftz), even skipped (eve) and engrailed (en) coding region. A reduced activity in ttk resulted in enhancement of the pok phenotype. ttk mutations produced extra R7 cells even in sina homozygotes whilst the pok mutation did not. This result indicates that Ttk represses R7 induction downstream of the sites where Pok and Sina function.