Feasibility of imaging intracellular tension probes in multicellular aggregates

Abstract

Multicellular aggregates constitute important 3-dimenstional (3D) models for investigating cellular behaviors, including proliferation and migration, that are relevant to neoplastic growth and progression. Studies point to the role of the aggregates' biomechanical environment in modulating cell proliferation, segregation, and migration. To obtain measurements of mechanical responses at the cellular level, we investigate the use of molecular tension probes in multicellular aggregates. While these tension probes have been used in 2-dimensional monolayers of cells, the feasibility of their application in 3D multicellular aggregates has not been demonstrated yet. In this paper, we utilize a previously described, frequency-domain fluorescence lifetime microscope (FLIM), to test the feasibility of measuring FRET using the Vinculin Tension Sensor (Vinculin TS), in multicellular aggregates of CHO-K1 cells. Our data suggest that the probes can be expressed in the spheroids and that we can measure FLIM-FRET signal from calibration constructs that are diffusely distributed within the cells. When the cells express Vinculin TS, we were able to discern changes in fluorescence distribution, compared with the calibration constructs, with evidence of punctate staining suggestive of localization at adhesion sites. The fluorescence lifetime of puncta expressing Vinculin TS was 2.46 +/-0.12 ns, longer than 2.21 +/-0.103 ns, the lifetime of puncta expressing Tail-less Vinculin TS (VinTL), which lacks the actin-binding domain. The longer lifetime is consistent with higher tension across vinculin TS compared with the unloaded VinTL control. Work is underway to fully characterize these puncta.