Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin- and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity.

BSTRACTChloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to b1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum-or LPA-induced recycling of b1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of b1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum-and LPAdependent integrin trafficking.

Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by Arp2/3 complex. Although regulation of Arp2/3-complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an EGF-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments activating the Arp2/3 complex and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium/ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.

Neuroblastoma is a pediatric embryonal malignancy characterized by impaired neuronal differentiation. A better understanding of neuroblastoma differentiation is essential for developing new therapeutic approaches. GDE2 (encoded by GDPD5) is a six-transmembrane-domain glycerophosphodiesterase that promotes embryonic neurogenesis. We find that high GDPD5 expression is strongly associated with favorable outcome in neuroblastoma. GDE2 induces differentiation of neuroblastoma cells, suppresses cell motility, and opposes RhoA-driven neurite retraction. GDE2 alters the Rac-RhoA activity balance and the expression of multiple differentiation-associated genes. Mechanistically, GDE2 acts by cleaving (in cis) and releasing glycosylphosphatidylinositol-anchored glypican-6, a putative co-receptor. A single point mutation in the ectodomain abolishes GDE2 function. Our results reveal GDE2 as a cell-autonomous inducer of neuroblastoma differentiation with prognostic significance and potential therapeutic value.

The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.