Membrane tension as an integrating mechanism in cell division and migration

Abstract

Many vital cellular processes such as migration and division require a change in cell shape. To migrate through the extracellular matrix, a cell contracts its rear end to push itself forward and this action has been found to be regulated by plasma membrane tension through RhoA. Cell division also requires massive shape transformations as cells enter, undergo, and exit mitosis. This project aims to investigate the mechanical role of membrane tension in integrating processes, particularly migration and division, using A2780 (human ovarian cancer) and RPE1 (human retinal pigment epithelial-1) cell lines as models. To identify membrane sensitive proteins, an initial thirteen membrane-related proteins were screened to determine if these localised at the rear during 3D migration on cell-derived matrix and re-localised during hypo-osmotic shock. From these, 8 responded to tension changes from the osmotic shock and were also shown to affect migration speed and directionality when knocked down. Membrane tension analysis of dividing cells using the Flipper-TR probe by FLIM surprisingly revealed fluctuations around the membrane regardless of mitotic phase, and a decreasing trend of global lifetime dependent on mitotic stage that is lowest during early cleavage furrow formation in anaphase. Global perturbation by osmotic shock completely abrogated division in synchronised A2780s and drastically reduced division in RPE1s. Local membrane perturbations using constitutively active ezrin and the inert membrane-cortex attachment linker (iMC linker, a signaling-inert linker that forcibly tethers the membrane to the cortex) on synchronised cells also led to decreased division rates and delays in mitosis. A second screening of the migrating rear-localising 'membrane sensitive' candidates revealed protein knockdowns to result in decreased division success and mitotic time defects, with cavin-1, caveolin-1, and Myo1e knockdowns causing the strongest effects. Immunofluorescence and live-cell imaging showed these candidates to localise to the membrane during mitosis. Imaging the Raichu-RhoA biosensor revealed that RhoA activity fluctuated around the cell periphery during mitotic rounding, and that increasing membrane tension with hypo-osmotic shock opposed this. Finally, knockdown of cavin-1, caveolin-1, and Myo1e resulted in increased RhoA activity in rounding cells, suggesting that they are required to maintain the correct balance of RhoA activity, and resulting actin cortex contractility, during mitosis. Altogether, these findings shed light into the regulation of cell division by the plasma membrane and how membrane tension integrates vital functions like migration and division through shared machineries.

Date of Award	6 Oct 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jean-Marc Schwartz (Co Supervisor), Patrick Caswell (Main Supervisor) & Joe Swift (Co Supervisor)