Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show the μ-homology domain (μHD) of FCHO1/2 represents a novel endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μHD interacts with the Bmp receptor Alk8, uncovering a new endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus contradict the primacy of FCHO1/2 in coat initiation.

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

The independently folded appendages of the large alpha and beta2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta2 chain with the closely related endogenous beta1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta1 and beta2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta2-yellow fluorescent protein (YFP) expressed in the beta1 + beta2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta2 appendage site for proper cargo recruitment to clathrin bud sites.

The AP-2 clathrin adaptor differs fundamentally from the related AP-1, AP-3, and AP-4 sorting complexes because membrane deposition does not depend directly on an Arf family GTPase. Instead phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) appears to act as the principal compartmental cue for AP-2 placement at the plasma membrane as well as for the docking of numerous other important clathrin coat components at the nascent bud site. This PtdIns(4,5)P 2 dependence makes type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIs) lynchpin enzymes in the assembly of clathrin-coated structures at the cell surface. PIPKI␥ is the chief 5-kinase at nerve terminals, and here we show that the 26-amino acid, alternatively spliced C terminus of PIPKI␥661 is an intrinsically unstructured polypeptide that binds directly to the sandwich subdomain of the AP-2 ␤2 subunit appendage. An aromatic side chain-based, extended interaction motif that also includes the two bulky C-terminal residues of the short PIPKI␥635 variant is necessary for ␤2 appendage engagement. The clathrin heavy chain accesses the same contact surface on the AP-2 ␤2 appendage, but because of additional clathrin binding sites located within the unstructured hinge segment of the ␤2 subunit, clathrin binds the ␤2 chain with a higher apparent affinity than PIPKI␥661. A clathrin-regulated interaction with AP-2 could allow PIPKI␥661 to be strategically positioned for regional PtdIns(4,5)P 2 generation during clathrin-coated vesicle assembly at the synapse.The key regulatory activity of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 2 during clathrin-mediated endocytosis is firmly established (1, 2). The heterotetrameric AP-2 adaptor complex and numerous clathrin-associated sorting proteins (CLASPs) display dedicated surfaces or domains that engage PtdIns(4,5)P 2 with good selectively (3-5). PtdIns(4,5)P 2 , which is localized to the cell surface, thus biases the deposition and assembly of these coat components at the plasma membrane by synergizing with other low affinity interactions in a phenomenon termed coincidence detection (2, 4). Later acting endocytic regulatory proteins also bind to PtdIns(4,5)P 2 . The large GTPase dynamin contains a pleckstrin homology domain, which engages PtdIns(4,5)P 2 and is required for vesicle scission (6). Similarly the clathrin uncoating cofactor, auxilin, has a PTEN homology domain that also binds to phosphoinositides and is necessary for targeting of this J-domain protein to clathrin-coated membranes (7). The lipid binding features of all these endocytic components is in full accord with PtdIns(4,5)P 2 being necessary for both early and late stages of coated vesicle production (8).PtdIns(4,5)P 2 is a general, apparently ubiquitous marker of the plasma membrane, and the concept of functionally autonomous, stable PtdIns(4,5)P 2 -enriched microdomains within the cytosolic leaflet of the membrane has been challenged (9 -11). This raises the question of whether the prevailing PtdIns(4,5)P 2 concentration at the cell surf...

[1] In this paper, we attempt to clarify the relationship between Jovian hectometric (HOM) and non-Io-related decametric (non-Io-DAM) radio structure. For that purpose, we extend the analysis by including more data and investigating statistical properties of the Jovian DAM and HOM radio emissions based on Cassini and Voyager observations, especially below 16 MHz. We have investigated these emissions observed by the Cassini, Voyager 1, and Voyager 2 spacecraft for specific Jovigraphic latitudes in the range of À3.7°-7.3°a nd local times in the range of 0.76-21.4 hours. We show a statistical comparison of Cassini, Voyager 1, and Voyager 2 data for occurrence probability in Central Meridian Longitude (CML) versus Io phase and in CML versus Frequency. The main results are as follows: (1) the detailed frequency structures of non-Io-related components can be seen for different spacecraft's local time and Jovigraphic latitude, (2) the high frequency of HOM extends up to near 10 MHz, and (3) a new DAM component, named the non-Io-D, appears from 40°to 60°CML in the frequency range of 7-11 MHz. On the basis of additional information of different behaviors of non-Io-B and non-Io-A structures in longitude depending on pre-and post-encounter of Cassini data, we improved the DAM angular beaming model that shows the cone half-angle of the emitting cone decreases as a function of frequency. We conclude that the changing beaming angle is not affected by Jovigraphic latitude of the spacecraft, but rather due to different local time of the source regions.