Molecular Sciences - Posters L
László Fazekas1,2,3, Nada Al-Sheraji1, Fabian Dehne1,3, Benoit Roux1,3, Diána Kaszás1,2,3, Ágnes Enyedi4, Balázs Enyedi1,2,3
1Department of Physiology, Semmelweis University, Budapest,2MTA-SE Lendület Tissue Damage Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, 3HCEMM-SE Inflammatory Signaling Research Group, Department of Physiology, Semmelweis University, Budapest, 4Department of Transfusiology, Semmelweis University, Budapest
Calcium signals are among the earliest signaling events triggered by epithelial wounding. They are essential in mediating the very first tissue-protective responses including leukocyte recruitment and wound closure. Despite the importance of these signaling events, little is known about their molecular regulation. In a zebrafish tail fin wounding model, we have previously identified wound margin localized persistent, tissue-penetrating wave-like, and cell swelling-dependent oscillatory forms of Ca2+ signals. In seeking regulators of these Ca2+ signaling patterns, we turned our attention to plasma membrane Ca2+-ATPases (PMCA).
Using Western blot and immunostaining we first identified zebrafish PMCA4 as the major PMCA isoform in the tail fin of the larvae. Zebrafish PMCA4 is orthologous to human PMCA4, but its function has not yet been described in detail. To investigate the role of this ATPase, we expressed the zebrafish PMCA4 in HEK293A cells. Our measurements showed that fish PMCA4 is capable of similar cellular Ca2+ export as its human counterpart. We then moved to in vivo experiments in zebrafish larvae. In order to measure Ca2+ level changes, we created transgenic fish lines ubiquitously expressing a green fluorescent Ca2+ sensor, GCaMP7s. Using spinning disk confocal microscopy, we could then follow in real-time the rapid Ca2+ level changes after amputating the tip of the tail fin or by using a pulsed UV-laser ablation, which allows us to visualize signaling events during the moments of wounding.
To delineate the role of PMCA4 in wound-induced Ca2+ signaling, we used morpholino oligonucleotides to transiently knock it down. We observed that this resulted in diminished wound-induced oscillatory Ca2+-signaling and an increase in the Ca2+ levels measured at the wound margin. Furthermore, we also measured altered leukocyte wound migration in the knockdown animals with decreased speed of cellular motion and impaired wound orientation. We are currently working on finding a mechanistic explanation for the later results which we also confirmed by Crispr-mediated F0 knockout tools. In these experiments, 3 different guide RNAs are used to eliminate the expression of the gene of interest directly in the injected larvae. Further experiments on CRISPR/Cas9-mediated full PMCA4 knockout animals are also in progress.
Our results so far provide opportunities for a more detailed understanding of tissue-injury induced Ca2+ signaling, and for mapping the role of the PMCA4 protein in the process.