Small Cell-Cell Fusogens Activate a PI3K-mTORC2-Akt Signalling Axis and Macropinocytosis for Efficient Cell Fusion

Abstract

Cell-cell fusion is mediated by distinct types of protein fusogens. Class I, II and III enveloped virus fusogens facilitate fusion through interaction with receptors on an apposing membrane that trigger conformational transitions in the fusogen that provide the energy necessary to fuse the two apposed membranes. Small fusogens, such as the reovirus fusion-associated small transmembrane (FAST) proteins and the vertebrate muscle fusogen myomerger, have small extracellular domains that do not span the intermembrane aqueous layer and have not been observed to undergo large conformational changes to drive the fusion process. These small fusogens may co-opt cell machinery and processes to drive cell fusion. To investigate this possibility, I conducted genetic and drug kinase screens that identified the PI3K(p110α)-Akt kinase pathway as essential for syncytiogenesis mediated by the reovirus p15 and p14 FAST proteins and myomerger, but dispensable for SARS CoV2 spike syncytium formation. I demonstrate that small fusogen-mediated fusion increases PIP(3,4,5) production through PI3K(p110α), that PIP(3,4,5) is necessary for fusion, and that PIP(3,4,5) and p14 colocalize to membrane ruffles at the fusion synapse. Using genetic and small molecule kinase inhibitors, I also show that Akt phosphorylation by PDK1 and mTORC2 but not mTORC1 is essential for small fusogen-mediated cell fusion. Furthermore, I demonstrate that p14 upregulates membrane ruffling and macropinocytosis through the PI3K-Akt pathway, which colocalizes spatiotemporally with the cell fusion synapse. In combination with the inhibitory effect of mechanistically diverse macropinocytosis inhibitors on p14 and myomerger fusion, these findings indicate that macropinocytosis is essential for efficient small fusogen-mediated cell-cell fusion and may promote fusion by increasing membrane tension via macropinosome formation.