A stress-responsive p38 signaling axis in choanoflagellates

Abstract

Choanoflagellates, the closest living relatives of animals, express diverse animal genes and may thereby provide insights into the ancestral roles of genes essential for modern animal cell biology and development. While efforts to study conserved gene families in choanoflagellates have been constrained by the relative inefficiency of currently available genetic tools, small molecule approaches are readily available and may provide a complementary and scalable approach for studying protein function. To study the physiological roles of choanoflagellate kinases, including animal kinase homologs, we established a high-throughput platform to screen the model choanoflagellate Salpingoeca rosetta with a curated library of human kinase inhibitors. We identified 36 diverse kinase inhibitors that disrupt S. rosetta cell proliferation. By exploring structure-activity relationships of one inhibitor, sorafenib, we identified a p38 kinase as a stress-activated regulator of cell proliferation in S. rosetta. This finding indicates a conserved p38 function between choanoflagellates, animals and fungi. Moreover, this study demonstrates that existing kinase inhibitors can serve as powerful tools to examine the ancestral roles of kinases that regulate modern animal development.

Cell differentiation controls iron assimilation in a choanoflagellate

“…In a previous paper 86 , we developed a lysis buffer to extract RNA from S. rosetta that has subsequently been used in other work for RNA [88][89][90] and protein extraction 91 . Here we show that this buffer was developed to preferentially lyse S. rosetta in cultures that have an abundance of the prey bacterium E. pacifica .…”

“…The membrane was washed 3x in PBST (PBS with 0.2% Tween-20) and then imaged in the 800 nm channel with a 10 minute exposure. This western protocol was adapted from a previous study 91 .…”

What is it like to be a choanoflagellate? Sensation, processing and behavior in the closest unicellular relatives of animals

“…Although none of the ligands of those receptors have yet been identified, that extensive molecular diversity might suggest a role in discriminating surrounding species in the biotic environment of choanoflagellates. Excitingly, functional characterization of the choanoflagellate complete set of protein kinases (a.k.a., the kinome) has recently been initiated (Rutaganira et al 2022 ), and has already shown activation of p38 kinase in S. rosetta by environmental stressors (i.e., heat shock and oxidative stress).…”

“…The pathways that control apical constrictions in choanoflagellates remain unknown but seem to involve cyclic guanosine monophosphate (cGMP) in C. flexa . Intriguingly, a recent study in S. rosetta demonstrated that biochemical inhibition of p38 kinase transiently induces an apical constriction-like behavior (Rutaganira et al 2022 ). This suggests that kinase signaling might be involved in the control of choanoflagellate apical constriction, which remains to be tested in response to environmental stimuli.…”