Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis

Abstract

Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission.

Cdc42 negatively regulates endocytosis during apical plasma membrane maintenance and development in mouse tubular organs in vivo

“…However, whereas in vitro Cdc42 has been shown to enhance endocytic pathways via the regulation of the actin cytoskeleton (Georgiou et al, 2008;Leibfried et al, 2008;Sathe et al, 2018), in the acinar cells of salivary glands in vivo we found that it negatively regulates this process. Indeed, downregulation of Cdc42 induced a robust internalization of the membrane-targeted reporter mGFP, that is localized both at the basolateral and apical PM under control conditions.…”

Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells

“…5a ). In contrast, AP2 shRNA treatment of AGS cells that exhibit a bonafide CG pathway 38 only reduced TfR uptake while fluid uptake was increased (Supplementary Fig. 5b ).…”

“…5c ). GBF1 is recruited to the plasma membrane as punctae in CHO cells 34 and in AGS 38 cells that exhibit robust CG endocytosis. However, Hela cells do not show an obvious recruitment of GBF1 to the plasma membrane compared to CHO cells (Supplementary Fig 5d/e ).…”

“…We recently found that IRSp53, an I-BAR domain protein, is necessary for the functioning of the CG pathway 38 . Cells lacking this protein show reduced fluid uptake compared to IRSp53-null cells restored with a wild-type version of IRSp53.…”

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166

“…Specific function of endoA3 in CIE of CD166. In order to further characterize the molecular mechanisms of CD166 uptake, we focused on the endophilin-A (endoA) family of BAR domain proteins, which together with other members of the BAR domain protein superfamily, have already been implicated in CIE [2][3][4][25][26][27] . Interestingly, a strong inhibition of CD166 uptake was observed upon depletion of the A3 isoform of endophilin-A (endoA3) in HeLa cells, while no effect was observed upon depletion of the two other isoforms ( Fig.…”