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Kirchhausen and colleagues show that actin is required for clathrin-mediated endocytosis at membranes under tension—such as apical surfaces of polarized cells. Actin engages with Hip1R bound to clathrin light chain to complete the deformation of a clathrin-coated pit into an endocytic vesicle. Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between ‘actin dependent and ‘actin independent’. We also find that light-chain-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

Using Caenorhabditis elegans genetic screens, we identified receptor‐mediated endocytosis (RME)‐4 and RME‐5/RAB‐35 as important regulators of yolk endocytosis in vivo . In rme‐4 and rab‐35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME‐4 and RAB‐35 function downstream of clathrin, upstream of RAB‐7, and act synergistically with recycling regulators RAB‐11 and RME‐1. We find that RME‐4 is a conserved DENN domain protein that binds to RAB‐35 in its GDP‐loaded conformation. GFP–RME‐4 also physically interacts with AP‐2, is enriched on clathrin‐coated pits, and requires clathrin but not RAB‐5 for cortical association. GFP–RAB‐35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme‐4 mutants. We propose that RME‐4 functions on coated pits and/or vesicles to recruit RAB‐35, which in turn functions in the endosome to promote receptor recycling.

A cell-free system has been developed to image vesicle budding and fission events. This method reveals an important role for the F-BAR protein FBP17 in regulating tubulation and clathrin-dependent budding. Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical and molecular mechanisms involved in membrane transport 1 , 2 , 3 . To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) 4 , 5 , 6 , revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPγS. Triggering fission by replacing GTPγS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17-dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.

While tracheid size of conifers is often a good proxy of water transport efficiency, correlations between conifer wood structure and transport safety remain poorly understood. It is hypothesized that at least some of the variation in bordered pit and tracheid structure is associated with both transport efficiency and embolism resistance. Stem and root samples from three boreal Pinaceae species were collected to test this hypothesis. Tracheid and pit anatomy were studied using light microscopy as well as scanning and transmission electron microscopy. While tracheid size explained at least 90% of the variation in specific conductivity for stem and root samples, the strongest correlations with embolism resistance occurred at the pit level. Both torus thickness and depth of the pit chamber showed a linear increase with greater vulnerability to cavitation. Greater embolism resistance was correlated with increasing wood density and tracheid wall reinforcement. A thinner torus may be more flexible and better able to seal the pit aperture. The pit chamber depth is proportional to the distance that the margo needs to deflect for pit aspiration.

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits. Clathrin-dependent endocytosis is one of the mechanisms used by cells to internalize specific proteins (cargo) from their surface. First, the cargo interacts with adaptor proteins that help cluster them in the cell's outer membrane, called the plasma membrane. This causes the protein clathrin to assemble into a lattice at the cytosolic side of the plasma membrane and deform the membrane into a pit. The pit grows deeper over time as more clathrin molecules assemble, eventually resulting in a deeply invaginated clathrin-coated pit that encloses the cargo to be taken up by the cell. The clathrin-coated pit then pinches off inside the cell in a process called fission to form a bubble-like structure called a vesicle, which transports the molecule to its destination. The deep invagination of clathrin-coated pits that leads to fission is assisted by actin, a protein that assembles into filaments that are suggested to generate the forces needed for this process. Many other factors are also involved. One of them is epsin, the collective name for a family of three very similar proteins in mammalian cells. Epsin binds to several other proteins implicated in clathrin-dependent endocytosis, including clathrin itself, and to plasma membrane proteins specifically ‘tagged’ for internalization. In addition, a portion of the epsin molecule can insert into the plasma membrane and help it to curve, which is important for forming the invaginated pit. However, due to the number of possible functions epsin could perform, its main role has remained elusive. Messa et al. created mouse cells that lack all three epsin proteins. Although these cells can form clathrin-coated pits, they struggle to develop into vesicles. The normal linking of the actin filaments to the clathrin coat does not occur, and another protein called Hip1R that also participates in clathrin-mediated endocytosis and links clathrin to actin, no longer accumulates at the clathrin-coated pits. Messa et al. also find that epsins can bind directly to actin. Overall, these results suggest that a main role of epsin is to help actin interact with the clathrin-coated pits and generate the force required for a pit to develop into a vesicle. However, epsin also performs many other roles, including recruiting a membrane protein (a so-called SNARE) that directs the fate of the vesicle to the clathrin-coated pit. Additionally, Messa et al. find that cells lacking all three epsins have problems dividing correctly. More research is required to establish whether this effect is also due to epsin's interaction with the cell's actin cytoskeleton.

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P 2 in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P 2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

The effect of increasing pressure difference (deltaP) on intervessel pit membrane porosity was studied in two angiosperm tree species with differing pit architecture. Fraxinus americana L. possesses typical angiosperm bordered pit structure while Sophora japonica L. exhibits well-developed vestures in intervessel pit chambers. It was hypothesized (a) that large deltaP across intervessel pits would cause the deflection of pit membranes in the stems of F. americana resulting in significant increases in porosity and thus lower cavitation thresholds, and (b) that the presence of vestures would prevent the deflection of pit membranes in S. japonica. To determine if the porosity of pit membranes increased under mechanical stress, suspensions of colloidal gold, 5 nm and 20 nm in diameter, were perfused across intervessel pit membranes at deltaP ranging from 0.25 MPa to 6.0 MPa. The effect of increasing deltaP on membrane porosity was also tested by comparing air seeding thresholds (P(a)) in stems perfused with water or a solution with lower surface tension. Air seeding and colloidal gold experiments indicated that pit membrane porosity increased significantly with deltaP in F. americana. In S. japonica, increases in permeability to colloidal gold with deltaP were small and maximum pore diameters predicted from P(a) were independent of deltaP, suggesting that vestures limited the degree to which the membrane can be deflected from the centre of the pit cavity. This provides the first experimental evidence that vestures reduce the probability of air seeding through pit membranes.

Symptom development of Pierce's disease (PD) in grapevine (Vitis vinifera) depends largely on the ability of the bacterium Xylella fastidiosa to use cell wall-degrading enzymes (CWDEs) to break up intervessel pit membranes (PMs) and spread through the vessel system. In this study, an immunohistochemical technique was developed to analyze pectic and hemicellulosic polysaccharides of intervessel PMs. Our results indicate that PMs of grapevine genotypes with different PD resistance differed in the composition and structure of homogalacturonans (HGs) and xyloglucans (XyGs), the potential targets of the pathogen's CWDEs. The PMs of PD-resistant grapevine genotypes lacked fucosylated XyGs and weakly methylesterified HGs (ME-HGs), and contained a small amount of heavily ME-HGs. In contrast, PMs of PD-susceptible genotypes all had substantial amounts of fucosylated XyGs and weakly ME-HGs, but lacked heavily ME-HGs. The intervessel PM integrity and the pathogen's distribution in Xylella-infected grapevines also showed differences among the genotypes. In pathogeninoculated, PD-resistant genotypes PM integrity was well maintained and Xylella cells were only found close to the inoculation site. However, in inoculated PD-susceptible genotypes, PMs in the vessels associated with bacteria lost their integrity and the systemic presence of the X. fastidiosa pathogen was confirmed. Our analysis also provided a relatively clear understanding of the process by which intervessel PMs are degraded. All of these observations support the conclusion that weakly ME-HGs and fucosylated XyGs are substrates of the pathogen's CWDEs and their presence in or absence from PMs may contribute to grapevine's PD susceptibility.

‘Coincidence-detecting’ phosphoinositide sensors are used to study changes in the phosphoinositide lipid species found in membranes during the development and maturation of endocytic clathrin-coated vesicles. Changing composition of cell membranes The traffic within cells is busy. At any given time, many vesicles bud off the membrane of one organelle and travel to fuse with another membrane elsewhere. Which characteristics identify the donor and acceptor membranes is an intriguing question. The answer seems to be the lipid and protein composition of the membranes, specifically the presence and relative abundance of the seven species of phosphoinositide lipids, as well as GTP-bound GTPases. Tom Kirchhausen and colleagues describe a new generation of phosphoinositide sensors. They used these sensors to study the phosphoinositide composition of clathrin-associated membranes, which are involved in the process of endocytosis. The findings provide information on how the composition of the membrane changes from the time it is coated with clathrin to form pits, to when the pits close into vesicles, and, once the vesicles bud off, to when they lose their clathrin coating and fuse with endosomes. Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes 1 , 2 . Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments 2 , 3 . It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe ‘coincidence-detecting’ sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.