Feasibility of imaging intracellular tension probes in multicellular aggregates

Luni Hu; Julian Ong; Margarida Barroso; Nada N. Boustany

doi:10.1117/12.2655401

17 March 2023 Feasibility of imaging intracellular tension probes in multicellular aggregates

Luni Hu, Julian Ong, Margarida Barroso, Nada N. Boustany

Proceedings Volume 12398, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XIV; 1239804 (2023) https://doi.org/10.1117/12.2655401
Event: SPIE BiOS, 2023, San Francisco, California, United States

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.

Conference Presentation

Citation Download Citation

Luni Hu, Julian Ong, Margarida Barroso, and Nada N. Boustany "Feasibility of imaging intracellular tension probes in multicellular aggregates", Proc. SPIE 12398, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XIV, 1239804 (17 March 2023); https://doi.org/10.1117/12.2655401

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available