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bioRxiv · 10.1101/2025.02.17.638627

Actin contractility and endocytosis create apico-basal tension gradient in HeLa cells

Ghosh, T.·Majhi, A.·Kundu, A.·Banerjee, A.·Sinha, B.
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Abstract

The functions of the plasma membrane of a cell are coupled to its mechanical state. To understand the players that contribute to the tight regulation of membrane mechanics with its local heterogeneities, the tension of the cell membrane requires to be mapped out from the basal to the apical membrane. In this work, optical-trap-based tension measurements performed at two axial (z) planes per cell reveal an apico-basal gradient, that is understood better on comparison with basal membrane mechanics and membrane compaction measurements by a combination of high resolution microscopy (IRM) and fluorescence lifetime (Flipper-TR) measurements, respectively. While the apico-basal gradient in the apparent tension (low to high) was found to depend on an intact actin cytoskeleton, analysing the colocalization of the cortical actin with the motor protein myosin and the actin-membrane linker ezrin suggested that cytoskeleton contractility is a major determinant of the patterned tension. Tension-sensitive lifetime of Flipper-TR, validated the gradient but failed to catch the dependence on actin due to the dual and opposite effects of tension on lifetime. Finally, measurements utilizing fluorescently tagged transferrin (Tf) demonstrated that the functional state of the membrane also showed similar height dependence. Thus, planes where tension maximized showed a peak in accumulation of Tf colocalizing with clathrin indicative of the presence of more pits. This enabled us to demonstrate that cells exist in tightly regulated patterned tension states that is coupled to their cytoskeleton as well membrane functions like endocytosis. Significance StatementThe cell membrane is protective but also highly dynamic - performing a diverse set of functions usually via membrane shape changes. These functions affect membrane mechanics but are also mechano-regulated. Mapping the state of cell surface mechanics or tension of live cells is therefore of fundamental importance. Interestingly, recent works have suggested that membrane tension can be non-uniform. However, no single tension-measuring technique can truly reach every part of the cell. Our work addresses this issue where we map tension in live HeLa cells using multifarious approaches including the measurement of tether forces, membrane fluctuations, and lipid compaction. We show that at the top/apical surface, the membrane-tension is lower than that at the midplane. The observed gradient resembles that of the state of endocytosis and controlled acto-myosin contractility.

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Ghosh, T., Majhi, A., Kundu, A., Banerjee, A., Sinha, B.. 2025-02-21. Actin contractility and endocytosis create apico-basal tension gradient in HeLa cells. https://doi.org/10.1101/2025.02.17.638627

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