Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells.

clathrin-coated structures form gradually without a major structural rearrangement. Currently, the endocytosis field is literally split between these two models due to the lack of experimental and analytical approaches that allow real time detection of conformational changes in clathrin coats with high resolution. In this study, using structured illumination microscopy in the total internal reflection mode, we demonstrate that curvature generation by clathrincoated pits can be detected in real time within cultured cells and and tissues of developing fruit fly embryos. We found that the footprint of clathrin coats increase monotonically until the formation of curved pits. These results show that the proposed flat-to-curved transition is not the mechanism through which clathrin pits invaginate. On the contrary, clathrin coats gain curvature at very early stages of their formation. Therefore, curvature generation by clathrin coats does not necessitate a dynamically unstable clathrin lattice.

24Plasma membrane tension is an important factor that regulates many key 25 cellular processes. Membrane trafficking is tightly coupled to membrane tension 26 and can modulate the latter by addition or removal of the membrane. However, 27the cellular pathway(s) involved in these processes are poorly understood. Here 28 we find that, among a number of endocytic processes operating simultaneously 29 at the cell surface, a dynamin and clathrin-independent pathway, the 30 CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon 31 reduction of tension. On the other hand, inhibition of the CG pathway results in 32 lower membrane tension, while up regulation significantly enhances membrane 33 tension. We find that vinculin, a well-studied mechanotransducer, mediates the 34 tension-dependent regulation of the CG pathway. Vinculin negatively regulates a 35 key CG pathway regulator, GBF1, at the plasma membrane in a tension 36 dependent manner. Thus, the CG pathway operates in a mechanochemical 37 feedback loop with membrane tension potentially leading to homeostatic 38 regulation of plasma membrane tension. 39

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