Different multicellular organisms undergo cell-cell fusion to form functional syncytia that support specialized functions necessary for proper development and survival. For years, monitoring the structural consequences of this process using live-cell imaging has been challenging due to the unpredictable timing of cell fusion events in tissue systems. Here we present a triggered vesicular stomatitis virus G-protein (VSV-G)-mediated cell-cell fusion assay that can be used to synchronize fusion between cells. This allows the study of cellular changes that occur during cell fusion. The process is induced using a fast wash of low pH isotonic buffer, promoting the fusion of plasma membranes of two or more adjacent cells within seconds. This approach is suitable for studying mixing of small cytoplasmic molecules between fusing cells as well as changes in organelle distribution and dynamics. © 2018 by John Wiley & Sons, Inc.

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.