Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.

The transcription factor Bcl6 is essential for the development of germinal center (GC) B cells and follicular helper T (Tfh) cells. However, little is known about in vivo dynamics of Bcl6 protein expression during and after development of these cells. By using a Bcl6 reporter mouse strain, we found that antigen-engaged B cells upregulated Bcl6 before clustering in GCs. Two-photon microscopic analysis indicated that Bcl6 upregulation in pre-GC B cells contributed to sustaining their interactions with helper T cells and was required for their entry to GC clusters. Our data also suggested that Tfh cells gradually downmodulated Bcl6 protein over weeks after development. The Bcl6-low Tfh cells rapidly terminated proliferation and upregulated IL-7 receptor. These results clarify the role of Bcl6 in pre-GC B cell dynamics and highlight the modulation of Bcl6 expression in Tfh cells that persist in the late phase of the antibody response.

mDia2 induces the actin scaffold for the contractile ring and stabilizes its position during cytokinesis in NIH 3T3 cells.

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

Inactivation of Rho GTPases with Clostridium difficile toxin B impairs centrosomal activation of Aurora-A in G2/M transition of HeLa cells.

During G2 phase of cell cycle, centrosomes function as a scaffold for activation of mitotic kinases. Aurora-A is first activated at late G2 phase at the centrosome, facilitates centrosome maturation, and induces activation of cyclin B-Cdk1 at the centrosome for mitotic entry. Although several molecules including HEF1 and PAK are implicated in centrosomal activation of Aurora-A, signaling pathways leading to Aurora-A activation at the centrosome, and hence mitotic commitment in vertebrate cells remains largely unknown. Here, we have used Clostridium difficile toxin B and examined the role of Rho GTPases in G2/M transition of HeLa cells. Inactivation of Rho GTPases by the toxin B treatment delayed by 2 h histone H3 phosphorylation, Cdk1/cyclin B activation, and Aurora-A activation. Furthermore, PAK activation at the centrosome that was already present before the toxin addition was significantly attenuated for 2 h by the addition of toxin B, and HEF1 accumulation at the centrosome that occurred in late G2 phase was also delayed. These results suggest that Rho GTPases function in G2/M transition of mammalian cells by mediating multiple signaling pathways converging to centrosomal activation of Aurora-A.

An essential role of Cdc42-like GTPases in mitosis of HeLa cells.

Here we used RNA interference and examined possible redundancy amongst Rho GTPases in their mitotic role. Chromosome misalignment is induced significantly in HeLa cells by Cdc42 depletion and not by depletion of either one or all of the other four Cdc42-like GTPases (TC10, TCL, Wrch1 or Wrch2), four Rac-like GTPases or three Rho-like GTPases. Notably, combined depletion of Cdc42 and all of the other four Cdc42-like GTPases significantly enhances chromosomal misalignment. These observations suggest that Cdc42 is the primary GTPase functioning during mitosis but that the other four Cdc42-like GTPases can also assume the mitotic role in its absence.

Effect of anoxia on diapause termination in eggs of the false melon beetle, Atrachya menetriesi.

The effect of anoxia on diapause development in the leaf beetle Atrachya menetriesi was investigated to elucidate the role of oxygen in regulation of egg diapause. While anoxia alone had no effect on diapause termination, it decreased diapause intensity before chilling. Such an effect reached a maximum level when anoxia lasted for about 10 days. Anoxia applied during the pre-diapause stage also reduced diapause intensity. On the other hand, anoxia terminated diapause when the diapause intensity had been lowered by sufficient duration of chilling (50 days at 7.5 degrees C). The effect of anoxia was temperature dependent; the larger effect was elicited when anoxia was combined with a higher temperature. A 50-day chilling caused more than 20% of eggs to terminate diapause upon transfer to warm conditions. However, when this chilling period was interrupted on the 20th day by a 5-day exposure to a high temperature of 20, 25 or 30 degrees C, the effect of the former chilling was cancelled partially or completely, suggesting that warming reversed diapause development. This reversing effect of a high temperature, however, was not manifested when the warming was combined with anoxia. The results suggest that anoxia inhibits diapause reversal and facilitates a certain process of diapause development. The sequence of exposure to anoxia and chilling is not important.

Effects of temperature during chilling and pre-chilling periods on diapause and post-diapause development in a katydid, Eobiana engelhardti subtropica.

In Eobiana engelhardti subtropica, early laid eggs reach the diapause stage in early autumn. For long periods before winter, the eggs are exposed to temperatures higher than their theoretical lower threshold for development. In contrast, late-laid eggs cannot reach their diapause stage before winter. Our study showed that E. e. subtropica copes with these difficulties via the thermal response involving embryonic diapause. In this katydid, the almost fully developed embryo undergoes an obligatory diapause. When diapause eggs were maintained at a temperature of 20 degrees C or higher, diapause persisted for a long time. Diapause was effectively terminated by temperatures ranging from 1 to 11 degrees C, and hatching occurred successfully at temperatures from 11 to 15 degrees C. In addition to the chilling temperature, pre-chilling temperature modified diapause intensity and hatching time. Diapause eggs hatched earlier after chilling when the pre-chilling temperature was lower, within a range of 14.5-25 degrees C. Thus, the low-temperature requirement for diapause termination prevents early laid eggs from untimely hatching in autumn, and low temperatures before and during winter decrease diapause intensity and shorten the hatching time in the following spring. When eggs were chilled before diapause, they tolerated chilling and averted diapause. Thus, even if eggs encounter low temperatures before diapause, they can hatch in the following spring.