A pore-forming protein drives macropinocytosis to facilitate toad water maintaining

Abstract

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a secretory aerolysin family pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water intake and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporins, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

Pore-forming protein βγ-CAT promptly responses to fasting with capacity to deliver macromolecular nutrients

“…These results indicated that βγ-CAT not only was able to stimulate the cellular import of albumin-bound FAs but also mediated the export of albumin and FAs in the form of exosomes. These results were different from our previous findings that when toad B. maxima urinary bladder epithelial cells were treated with 50 nM purified βγ-CAT, augmented exosome release was determined but no enrichment of cargo molecules (dextran) was observed (Zhao et al, 2021). This discrepancy clearly reflected that when cells are treated with βγ-CAT, the specific biological outcomes are dependent on the type of cells and the cell surroundings.…”

“…We then assayed the ability of βγ-CAT to stimulate macropinocytosis in hCMEC/D3 and Caco-2 cells. Consistent with our previous findings (Zhao et al, 2021), βγ-CAT greatly increased the internalization of the micropinocytosis marker 70-kDa dextran, which was inhibited by addition of the macropinocytosis inhibitor wortmannin (WORT; a PI3K inhibitor) (Supplementary Fig. S2D).…”

“…Particularly, βγ-CAT promoted the internalization of ovalbumin (an antigen) in a murine DC model via pinocytosis (Deng et al, 2020). βγ-CAT has been shown to facilitates water maintaining in toad by driving macropinocytosis in toad osmoregulatory organs, including the skin and urinary bladder (Zhao et al, 2021). Experimental evidence obtained regarding βγ-CAT and its regulatory network led us to propose the hypothesis of the SELC pathway, which is a novel PFP-driven cell vesicular delivery and transport system (Zhang et al, 2021).…”

“…Previous studies have shown that βγ-CAT promoted pinocytosis in murine DC cells, as determined by Lucifer yellow (LY) uptake assays, which may enhance ovalbumin intake into the cells (Deng et al, 2020). In addition, βγ-CAT has been shown to stimulate macropinocytosis (> 200 nm) in toad skin and urinary bladder epithelial cells, as well as in mammalian MDCK cells under osmotic stress (Zhao et al, 2021). In the present study, the ability of βγ-CAT to promote macropinocytosis was also observed in mammalian endothelial hCMEC/D3 cells and epithelial Caco-2 cells (Supplementary Fig.…”

“…The complex acts on endocytic and exocytic systems to form channels in endolysosomes, which promotes cell material import and export through endolysosomal pathways (Zhang et al, 2021). These cellular effects have been shown to be involved in immune defense and tissue repair (Xiang et al, 2014; Li et al, 2017; Gao et al, 2019; Deng et al, 2020; Liu et al, 2021) as well as in toad water maintaining by driving macropinocytosis under osmotic stress (Zhao et al, 2021). Acting as a secretory endolysosome channel (SELC) protein, βγ-CAT and its regulatory network (βγ-CAT-pathway) act as a novel PFP-mediated cell vesicular delivery and transport system, playing multiple roles in the adaptation of toads to diverse environmental conditions (Zhang et al, 2021).…”