How the membrane environment informs when and where actin is polymerized in clathrin-mediated endocytosis is unclear. Daste et al. show that high membrane curvature stimulates PI(3,4)P2 dephosphorylation by INPP4A and that PI(3)P recruits SNX9 in conjunction with both PI(4,5)P2 and high membrane curvature. Furthermore, they find that Lowe syndrome mimics this membrane microenvironment with the aberrant formation of a PI(4,5)P2/PI(3)P intermediate, giving rise to actin comets.

Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis owing to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 (also known as PRKCDBP) is targeted to caveolae by cavin1 (also known as PTRF) where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 (also known as SDPR) region, showing that the cavins form distinct subcomplexes through their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae that are only present at the membrane for a short time. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

The epidermal growth factor receptor (EGFR) is an important regulator of normal growth and differentiation, and it is involved in the pathogenesis of many cancers. Endocytic downregulation is central in terminating EGFR signaling after ligand stimulation. It has been shown that p38 MAPK activation also can induce EGFR endocytosis. This endocytosis lacks many of the characteristics of ligand-induced EGFR endocytosis. We compared the two types of endocytosis with regard to the requirements for proteins in the internalization machinery. Both types of endocytosis require clathrin, but while epidermal growth factor (EGF)-induced EGFR internalization also required Grb2, p38 MAPK-induced internalization did not. Interestingly, AP-2 knock down blocked p38 MAPK-induced EGFR internalization, but only mildly affected EGF-induced internalization. In line with this, simultaneously mutating two AP-2 interaction sites in EGFR affected p38 MAPK-induced internalization much more than EGF-induced EGFR internalization. Thus, it seems that EGFR in the two situations uses different sets of internalization mechanisms.