SummaryClathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.DOI: http://dx.doi.org/10.7554/eLife.04137.001

p53-upregulated modulator of apoptosis (PUMA) plays an essential role in p53-dependent apoptosis following DNA damage. PUMA also mediates apoptosis independent of p53. In this study, we investigated the role and mechanism of PUMA induction in response to serum starvation in p53-deficient cancer cells. Following serum starvation, the binding of Sp1 to the PUMA promoter significantly increased, whereas inhibition of Sp1 completely abrogated PUMA induction. p73 was found to be upregulated by serum starvation and mediate PUMA induction through the p53-binding sites in the PUMA promoter. Sp1 and p73beta appeared to cooperatively activate PUMA transcription, which is inhibited by the phosphoinsitide 3-kinase (PI3K)-protein kinase B (AKT) pathway. Furthermore, knockdown of PUMA suppressed serum starvation-induced apoptosis in leukemia cells. Our results suggest that transcription factors Sp1 and p73 mediate p53-independent induction of PUMA following serum starvation to trigger apoptosis in human cancer cells.

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded ␣ subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NE-CAP1, binds a second interaction site on the bilobal ␣ appendage, located on the N-terminal ␤ sandwich subdomain. Both ␣ appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the ␣ appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.Clathrin-coated vesicles are a major portal of entry into eukaryotic cells, carrying macromolecular nutrients, ligands, select transmembrane proteins, and even viruses into the cell interior from the plasma membrane (1, 2). Cargo selectivity of these short-lived transport intermediates is often thought to be governed by a central triad of proteins, the cargo receptors, clathrin, and the heterotetrameric AP-2 adaptor complex. Cargo receptors contain cytosolic internalization sequences, such as the YXXØ motif (where X is any amino acid and Ø represents a residue with a bulky hydrophobic side chain), found within proteins like the receptor for the endocytosed iron transport protein transferrin. Several distinct internalization sequences or tags are known, each specifying internalization by promoting preferential incorporation into assembling clathrincoated vesicles (3). Clathrin functions as a trimer of heterodimers, composed of three 192-kDa heavy and three 20 -25-kDa light chains, that polymerize to form the characteristic polyhedral clathrin lattice (4,5). Assembled clathrin appears to act as a mechanical scaffold during the process of bud invagination. AP-2, the archetypical sorting adaptor, is composed of two large (ϳ100 kDa) subunits (␣ and ␤2), a 50-kDa medium 2 subunit, and a 17-kDa small 2 chain (2, 6). AP-2 binds physically to both clathrin, through the hinge and appendage domains of the ␤2 subunit (7), and to YXXØ-type internalization sequences, via the 2 subunit, in a phosphorylation-regulated manner (6, 8 -10). AP-2 is therefore a multifunctional protein that couples coat assembly with cargo selection. Surprisingly, after small interfering RNA silencing of either the AP-2 ␣ or 2 subunit mRNA in HeLa cells to deplete cellular AP-2 adaptor levels, certain transmembrane receptors, like the epidermal growth factor and low ...

Phosphoinositides play a fundamental role in clathrin-coat assembly at the cell surface. Several endocytic components and accessory factors contain independently folded phosphoinositide-binding modules that facilitate, in part, membrane placement at the bud site. As the clathrin-coat assembly process progresses toward deeply invaginated buds, focally synthesized phosphoinositides are dephosphorylated, principally through the action of the phosphoinositide polyphosphatase synaptojanin 1. Failure to catabolize polyphosphoinositides retards the fission process and endocytic activity. The long-splice isoform of synaptojanin 1, termed SJ170, contains a carboxyl-terminal extension that harbors interaction motifs for engaging several components of the endocytic machinery. Here, we demonstrate that in addition to DPF and FXDXF sequences, the SJ170 carboxyl terminus contains a novel AP-2 binding sequence, the WXXF motif. The WXXF sequence engages the independently folded ␣-subunit appendage that projects off the heterotetrameric AP-2 adaptor core. The endocytic protein kinases AAK1 and GAK also contain functional WXX(FW) motifs in addition to two DPF repeats, whereas stonin 2 harbors three tandem WXXF repeats. Each of the discrete SJ170 adaptor-interaction motifs bind to appendages relatively weakly but, as tandemly arrayed within the SJ170 extension, can cooperate to bind bivalent AP-2 with good apparent affinity. These interactions likely contribute to the appropriate targeting of certain endocytic components to clathrin bud sites assembling at the cell surface.The major intracellular pool of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 is found, at steady state, within the cytosolic leaflet of the plasma membrane. PtdIns(4,5)P 2 -specific probes, such as the phospholipase C-␦1 pleckstrin homology domain fused to the green fluorescent protein, reveal a highly selective distribution of this important regulatory phospholipid over the cell surface (1, 2). This same PtdIns(4,5)P 2 -binding pleckstrin homology domain prevents receptor-mediated endocytosis in vitro by interfering with clathrin-coated vesicle assembly (3) underscoring the inter-relationship between phosphoinositides and clathrin-dependent internalization. PtdIns(4,5)P 2 regulates coat assembly in part by physically interacting with several of the components of the endocytic clathrin machinery, including the heterotetrameric AP-2 adaptor complex (4 -6), AP180 (7-9), epsin (10 -13), HIP1 (11), Dab2 (12), and the ␤-arrestins (14). The activity of type I phosphatidylinositol 4-phosphate 5-kinases promotes AP-2-containing coat assembly upon membrane templates in vitro (15,16) where the generated PtdIns(4,5)P 2 appears to act as an organizing principle to promote proper placement of endocytic components at the bud site (9,(11)(12)(13)17). In fact, overexpression of phosphatidylinositol 4-phosphate 5-kinase ␤ promotes AP-2 association with the plasma membrane, increases the number of clathrin-coated pits, and potentiates transferrin uptake (18).At the n...

Metabolic syndrome is a complex disease that involves multiple organ systems including a critical role for the liver. Non-alcoholic fatty liver disease (NAFLD) is a key component of the metabolic syndrome and fatty liver is linked to a range of metabolic dysfunctions that occur in approximately 25% of the population. A panel of experts recently agreed that the acronym, NAFLD, did not properly characterize this heterogeneous disease given the associated metabolic abnormalities such as type 2 diabetes mellitus (T2D), obesity, and hypertension. Therefore, metabolic dysfunction-associated fatty liver disease (MAFLD) has been proposed as the new term to cover the heterogeneity identified in the NAFLD patient population. Although many rodent models of NAFLD/NASH have been developed, they do not recapitulate the full disease spectrum in patients. Therefore, a platform has evolved initially focused on human biomimetic liver microphysiology systems that integrates fluorescent protein biosensors along with other key metrics, the microphysiology systems database, and quantitative systems pharmacology. Quantitative systems pharmacology is being applied to investigate the mechanisms of NAFLD/MAFLD progression to select molecular targets for fluorescent protein biosensors, to integrate computational and experimental methods to predict drugs for repurposing, and to facilitate novel drug development. Fluorescent protein biosensors are critical components of the platform since they enable monitoring of the pathophysiology of disease progression by defining and quantifying the temporal and spatial dynamics of protein functions in the biosensor cells, and serve as minimally invasive biomarkers of the physiological state of the microphysiology system experimental disease models. Here, we summarize the progress in developing human microphysiology system disease models of NAFLD/MAFLD from several laboratories, developing fluorescent protein biosensors to monitor and to measure NAFLD/MAFLD disease progression and implementation of quantitative systems pharmacology with the goal of repurposing drugs and guiding the creation of novel therapeutics.

The recent recovery of mutations in vesicular trafficking genes causing congenital heart disease (CHD) revealed an unexpected role for the endocytic pathway. We now show that mice with a C4232R missense mutation in Low density lipoprotein receptor related protein 1 (LRP1) exhibit atrioventricular septal defects with double outlet right ventricle. Lrp1m/m mice exhibit shortened outflow tracts (OFT) and dysmorphic hypocellular cushions with reduced proliferation and increased apoptosis. Lrp1m/m embryonic fibroblasts show decreased cell motility and focal adhesion turnover associated with retention of mutant LRP1 in endoplasmic reticulum and reduced LRP1 expression. Conditional deletion of Lrp1 in cardiac neural crest cells (CNC) replicates the full CHD phenotype. Cushion explants showed defective cell migration, with gene expression analysis indicating perturbation of Wnt and other signaling pathways. Thus, LRP1 function in CNCs is required for normal OFT development with other cell lineages along the CNC migratory path playing a supporting role.

During oogenesis in Drosophila, the phagocytic engulfment protein Ced-6 recognizes the atypical endocytic sorting signal within the vitellogenin receptor Yolkless. Because Ced-6 displays all of the features of an authentic clathrin adaptor, an unrecognized clathrin dependence for Ced-6/Gulp operation during phagocytosis is possible.