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 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 import and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporin, 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 drives extracellular nutrient scavenging under cell starvation

“…Previous studies have shown that secreted βγ-CAT promotes cell import through endolysosomal pathways probably via inducing pinocytosis/macropinocytosis in distinct cell context (13, 21, 24). This raises the possibility that the PFP protein complex participates in macromolecule intake for cellular nutrient supply and metabolic flexibility under variations in nutrient availability (13, 23, 24).…”

“…Previous studies have shown that secreted βγ-CAT promotes cell import through endolysosomal pathways probably via inducing pinocytosis/macropinocytosis in distinct cell context (13, 21, 24). This raises the possibility that the PFP protein complex participates in macromolecule intake for cellular nutrient supply and metabolic flexibility under variations in nutrient availability (13, 23, 24). To test this hypothesis, we used a starvation model of toad cells cultured under three nutrient conditions including glucose/glutamine-containing medium (Glc + /Gln + ), glucose/glutamine-depleted medium (Glc - /Gln - ), and glucose-containing but glutamine-depleted medium (Glc + /Gln - ).…”

“…1C). Biologically active βγ-CAT in culture supernatants was analyzed by its hemolytic activity on human erythrocytes, a sensitive method to determine the existence of βγ-CAT (13, 17). First, we verified that the various culture media did not affect the hemolytic activity of βγ-CAT (Fig.…”

“…This PFP complex targets cell surface acidic glycosphingolipids in lipid rafts via a double-receptor binding model (16) to act along cell endocytic and exocytic pathways with channel formation on endolysosomes, which have been shown to play roles in immune defense and tissue repair (18-22). Thus, the PFP and its regulatory network define an unknown secretory endolysosomal channel (SELC) pathway, representing a novel PFP-driven cell vesicular delivering system (13, 23). The newly defined βγ-CAT pathway is able to mediate cell import and export of extracellular materials and/or plasma membrane components through endolysosomal systems, making the PFP complex a versatile multiple functional protein machine depending on the cell context and surroundings.…”

“…The newly defined βγ-CAT pathway is able to mediate cell import and export of extracellular materials and/or plasma membrane components through endolysosomal systems, making the PFP complex a versatile multiple functional protein machine depending on the cell context and surroundings. βγ-CAT drives macropinocytosis to facilitate water maintaining in toad osmoregulatory organs in response to osmotic stress (13). Toad blood βγ-CAT is an immediate responsive element under animal fasting, which has been proposed to mediate transcellular transport of albumin-bound fatty acids for nutrient supply of tissue parenchymal cells (24).…”