Confocal frequency-domain fluorescence lifetime and Förster resonance energy transfer (FRET) microscopy of Chinese hamster ovary (CHO-K1) cells expressing the vinculin tension sensor (VinTS) is used to compare vinculin tension in three-dimensional (3D) multicellular aggregates and 2D cellular monolayers. In both 2D and 3D cultures, the FRET efficiency of VinTS is 5-6% lower than that of VinTL (p < 0.05), a tail-less control which cannot bind actin or paxillin. The difference between VinTS and VinTL FRET efficiency can be mitigated by treatment with the Rho-associated kinase inhibitor Y-27632, demonstrating that VinTS is under tension in both 2D and 3D cultures. However, there is an overall decrease in FRET efficiency of both VinTS and VinTL in the 3D multicellular aggregates compared with the 2D monolayers. Expression of VinTS in 2D and 3D cultures exhibits puncta consistent with cellular adhesions. While paxillin is present at the sites of VinTS expression in the 2D monolayers, it is generally absent from VinTS puncta in the 3D aggregates. The results suggest that VinTS experiences a modified environment in 3D aggregates compared with 2D monolayers and provide a basis for further investigation of molecular tension sensors in 3D tissue models.
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