Histone Methyltransferase DOT1L is Involved in Larval Molting and Second Stage Nymphal Feeding in Ornithodoros Moubata.

Epigenetic mechanisms have not been characterized in ticks despite their importance as vectors of human and animal diseases worldwide. Our investigation identifies and functionally characterizes the orthologue of S-adenosylmethionine (SAM) binding methyltransferase enzyme, disruptor of telomeric silencing 1-like (DOT1L) in Ornithodoros moubata (OmDOT1L), a soft tick vector for the relapsing fever pathogen Borrelia duttonii and the African swine fever virus. The OmDOT1L tertiary structure was predicted and compared to the Homo sapiens DOT1L which had been co-crystalized with SGC0946, a DOT1L-specific inhibitor. The amino acid residues crucial for SAM and SGC0946 binding conserved in most DOT1L sequences available, are also conserved in OmDOT1L. Quantitative PCR of Omdot1l during O. moubata life stages showed that transcripts were significantly upregulated in first-stage nymphs. O. moubata larvae exposed to SGC0946 displayed high mortality during molting to first-stage nymphs. Furthermore, a significant decrease in weight was observed in second-stage nymphs fed on recombinant OmDOT1L-immunized rabbits. In contrast, artificial blood feeding supplemented with SGC0946 did not affect survival and reproductive performance of adult female ticks. We concluded that OmDOT1L plays an essential role in the regulation of larval molting and the feeding of O. moubata second-stage nymphs.

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos.

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1-8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 µm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.

Turnover of the actomyosin complex in zebrafish embryos directs geometric remodelling and the recruitment of lipid droplets.

Lipid droplets (LDs), reservoirs of cholesterols and fats, are organelles that hydrolyse lipids in the cell. In zebrafish embryos, the actomyosin complex and filamentous microtubules control the periodic regulation of the LD geometry. Contrary to the existing hypothesis that LD transport involves the kinesin-microtubule system, we find that their recruitment to the blastodisc depends on the actomyosin turnover and is independent of the microtubules. For the first time we report the existence of two distinct states of LDs, an inactive and an active state, that occur periodically, coupled weakly to the cleavage cycles. LDs are bigger, more circular and more stable in the inactive state in which the geometry of the LDs is maintained by actomyosin as well as microtubules. The active state has smaller and irregularly shaped LDs that show shape fluctuations that are linked to actin depolymerization. Because most functions of LDs employ surface interactions, our findings on the LD geometry and its regulation bring new insights to the mechanisms associated with specific functions of LDs, such as their storage capacity for fats or proteins, lipolysis etc.