Using Live Imaging Techniques to Measure the Membrane Tension of Cells in Xenopus laevis 3D Tissues

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Poster Session Two
Ms Iona Norwood (1), Dr Emma Johns (1), Dr Joseph Hetmanski (1), Dr Patrick Caswell (1), Prof Oliver Jensen (2), Dr Sarah Woolner (1)
1. Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester
2. School of Mathematics, University of Manchester

Membrane tension, live imaging, FLIM, FLIPPER-TR, Membrane proximal actin, MPAct, Mechanobiology, Developmental biology

Abstract text


Cells within our tissues are constantly subjected to internal and external mechanical forces and must detect and respond to these forces appropriately. Responses to mechanical force result in physical, geometric, and behavioural changes – such as changes in cell division rate and orientation1. These mechano-responses are likely to play a key role in maintaining tissue homeostasis, morphogenesis, and the prognosis of many common diseases, such as cancer2. Furthermore, our preliminary results indicate that the rate at which a tissue experiences mechanical force can have an important impact on the resultant behaviour of a tissue.

The exact method by which cells can detect forces is not fully understood. One possible mechanism is through the direct detection of changes in membrane tension – which has been shown to be involved in processes such as cell fate determination and cell migration3-4. However, it is difficult to measure membrane tension in tissues without disrupting the integrity of cells and tissues. Therefore, we aimed to develop less invasive protocols to measure changes in membrane tension in live 3D tissues and during cell division using the fluorescent membrane tension probe, FLIPPER-TR5, and a probe for the membrane proximal actin, MPAct4.


To test whether it would be feasible to use FLIPPER-TR to measure membrane tension in 3D tissues, we carried out preliminary experiments using adherent RPE-1 cells, plated on either cell derived matrix or grown to confluence on glass. The RPE-1 cells were then treated with FLIPPER-TR and Fluorescent Lifetime (FLIM) images were used to determine the lifetime of decay of the FLIPPER-TR probe.

To explore the use of FLIPPER-TR in a 3D tissue, we used animal cap tissue dissected from Xenopus laevis early embryos. The Xenopus animal cap provides a multi-layered, vertebrate epithelial tissue that is highly amenable to live imaging. Animal cap tissue was adhered to glass, treated with FLIPPER-TR and then imaged by FLIM.

To explore a possible alternative to measuring membrane tension by FLIPPER-TR, we are also investigating the use of MPAct in cells and tissues. RPE-1 cells were transfected with a fluorescent marker of MPAct and a membrane marker (Caax). The cells were plated on glass and confocal time-lapse images were taken of cells that were about to undergo mitosis. The ratio of MPAct to the membrane marker (MPAct/Caax) was calculated where high MPAct/Caax was predicted to be correlated with high membrane tension. 


Fluorescent lifetime images of RPE-1 cells and animal caps indicated that FLIPPER-TR lifetime varies throughout all the cell membranes and there are similar variations in patterns of membrane tension in cultured cells and 3D tissues. However, in both cultured cells and 3D tissues, imaging FLIPPER-TR negatively impacted the health of cells, meaning that long-term imaging is not possible.

With the MPAct probe, we were able to identify spatial and temporal differences in MPAct/Caax in cells undergoing cell division with an increase in MPAct/Caax at the equator prior to cytokinesis. We are now exploring how membrane tension measured by FLIPPER-TR correlates with MPAct/Caax. 


We have developed protocols which have the potential to measure membrane tension in 3D tissues with FLIM and laser confocal microscopy. We aim to implement these in experiments where animal cap tissues are subjected to different rates of tensile forces to determine whether direct detection of the membrane tension plays a role in the mechano-responses of cells in 3D.

  1. Nestor-Bergmann, A., Goddard, G., Woolner, S., & Jensen, O. E. (2018). Relating cell shape and mechanical stress in a spatially disordered epithelium using a vertex-based model. Mathematical medicine and biology: a journal of the IMA, 35, i1–i27.
  2. Moruzzi, M., Nestor-Bergmann, A., Goddard, G. K., Tarannum, N., Brennan, K., & Woolner, S. (2021). Generation of anisotropic strain dysregulates wild-type cell division at the interface between host and oncogenic tissue. Current Biology, 31 (15), 3409–3418.
  3. Berget, M., Lembo, S., Sharma, S., Russo, L., Milovanovic, D., Gretarsson, K. H., Bormel, M., Neveu, P. A., Hackett, J. A., Petsalaki, E., et al. (2021). Cell surface mechanics gate embryonic stem cell differentiation. Cell Stem Cell, 28 (2), 2.
  4. Bisaria, A., Hayer, A., Garbett, D., Cohen, D. and Meyer, T., 2020. Membrane-proximal F-actin restricts local membrane protrusions and directs cell migration. Science, 368(6496), pp.1205-1210.
  5. Colom, A., Derivery, E., Soleimanpour, S., Tomba, C., Molin, M.D., Sakai, N., González-Gaitán, M., Matile, S. and Roux, A., 2018. A fluorescent membrane tension probe. Nature chemistry, 10(11), pp.1118-1125.