Biological processes in any physiological environment involve changes in cell shape, which must be accommodated by their physical envelope—the bilayer membrane. However, the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear. Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process. This process is passive, local, almost instantaneous, before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane. We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell–substrate interface. Once formed, cells reabsorb the invaginations through an active process with duration of the order of minutes.

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.

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.

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.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.

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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