EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments

Dileucine-based motifs have been shown to regulate endosomal sorting of a number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates.     

Cortical patches on the move

Endocytosis is the primary means by which eukaryotic cells internalize materials from the environment. However, while many components of the endocytic machinery are known, the timing of molecular events leading to endocytosis remains undefined. In this issue of Cell, Kaksonen et al. use real-time microscopy to define the temporal assembly of components of the endocytic machinery in the yeast S. cerevisiae. They also provide new insight into how the actin cytoskeleton is coordinated with the endocytic machinery.     

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.     

The endocytic machinery in nerve terminals surrounds sites of exocytosis

In most models of endocytosis, the endocytic machinery is recruited from the cytoplasm by cytoplasmic tails of the plasma membrane proteins that are to be internalized. This does not appear to be true at synapses where the endocytic machinery required for synaptic vesicle recycling is localized to membrane-associated 'hot spots' [1] [2]. In Drosophila neuromuscular junctions, the multi-domain protein Dap160 is also localized to hot spots [3] and has some characteristics expected of an anchoring protein. Anchoring the endocytic machinery to the plasma membrane might help contribute to the remarkable speed of synaptic vesicle recycling [4]. Here, we report that the endocytic machinery surrounds sites that are believed to be sites of exocytosis. We propose that the radial distribution of the synaptic vesicle recycling machinery already present on the plasma membrane in unstimulated nerve terminals is a fundamental unit of pre-synaptic organization and allows the nerve terminal to extract maximum recycling efficiency out of conventional endocytic machinery.     

Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand- receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet- Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X- 100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu- Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G- protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet- Leu-Phe-associated G-protein.     

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis

The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Oncogenic breakdowns in endocytic adaptor proteins

Endocytosis is a versatile tool to regulate the intensity, localization, half-life and function of signaling complexes (signalosomes) that form in cells upon binding of growth factors, cytokines and morphogens to their cognate receptors. Endocytic adaptors are non-catalytic proteins that assemble effectors and structural components of the endocytic machinery around the trafficking cargo and serve as scaffolds for signalosomes, which in turn modify their location and activity by various post-translational modifications. We discuss how breakdowns in the function of endocytic adaptors might facilitate impairment of tissue homeostasis and consequent tumor development.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

The endocytic machinery at an interface with the actin cytoskeleton: a dynamic,hip intersection

Clathrin-mediated endocytosis is the major mechanism by which proteins and membrane lipids gain access into cells. Over the past several years, an array of proteins has been identified that define the molecular machinery regulating the formation of clathrin-coated pits and vesicles. This article focuses on how the identification of this machinery has begun to reveal a molecular basis for a link between endocytosis and the actin cytoskeleton--a link that had long been suspected to exist in mammalian cells but which had remained elusive. In particular, I discuss the relationship between actin and three components of the endocytic machinery--dynamin, HIPs (huntingtin-interacting proteins) and intersectin.     

Hijacking the endocytic machinery by microbial pathogens

Understanding the mechanisms that microbes exploit to invade host cells and cause disease is crucial if we are to eliminate their threat. Although pathogens use a variety of microbial factors to trigger entry into non-phagocytic cells, their targeting of the host cell process of endocytosis has emerged as a common theme. To accomplish this, microbes often rewire the normal course of particle internalization, frequently usurping theoretical maximal sizes to permit entry and reconfiguring molecular components that were once thought to be required for vesicle formation. Here, we discuss recent advances in our understanding of how toxins, viruses, bacteria, and fungi manipulate the host cell endocytic machinery to generate diseases. Additionally, we will reveal the advantages of using these organisms to expand our general knowledge of endocytic mechanisms in eukaryotic cells.     

RLIP controls receptor-ligand signaling by regulating clathrin-dependent endocytosis

RLIP (Ral-interacting protein) is a multifunctional protein that couples ATP hydrolysis with the movement of substances. Its primary function appears to be in the plasma membrane, where it catalyzes the ATP-dependent efflux of glutathione-conjugates (GS-Es), as well as un-metabolized drugs and toxins. In the plasma membrane, its interaction with the clathrin adaptor protein AP2 localizes it to endocytic vesicle, where its GS-E-stimulated ATPase and transport activity are required for clathrin-dependent endocytosis (CDE). CDE is an essential mechanism for internalizing ligand-receptor complexes that signal proliferation (EGF, insulin, IGF1), apoptosis (TNFα, TRAIL, Fas-L), and differentiation and morphogenesis (TGFβ, WNT, Notch, SHH). Aberrant functioning of these pathways appears crucial for most cancer cells to evade apoptosis, invade surrounding tissues, and metastasize. Internalization of receptor-ligand complexes by CDE begins a sequence of events that can terminate, initiate, or modulate downstream signaling; the consequences of signaling through these downstream pathways may be inherently different in cancer and normal cells, a view supported by numerous basic and clinical observations. In this review, we will discuss the GS-E transport activity of RLIP, which determines the rate of ligand endocytosis, and how the inhibition and/or depletion of RLIP globally disrupts in ligand-receptor signaling.     

Biochemical features and functional activity of the endocytic compartment in the liver cell

When many ligands, polypeptidic hormones, growth factors, metabolic carriers, plasma glycoproteins, etc., bind to cell-surface receptors, ligand-receptor complexes are internalized by a process called receptor-mediated endocytosis towards the endocytic compartment. The endocytic compartment is an extensive network of anastomosing vesiculo-tubular membranes that differs biochemical and functionally from other intracellular organelles. Endosome fractions were prepared and antibodies raised against endosome membrane proteins. In addition to a detailed biochemical study of proteins and glycoproteins the antibodies were used to immunolocalize the endocytic structures in the hepatic cell. These studies aided to demonstrate the involvement of endocytic compartment not only in the sorting of proteins to specific domains of the plasma membrane but in the identification of "resident" endosome components.     

The Cell Biology of the Endocytic System from an Evolutionary Perspective

Evolutionary cell biology can afford an interdisciplinary comparative view that gives insights into both the functioning of modern cells and the origins of cellular systems, including the endocytic organelles. Here, we explore several recent evolutionary cell biology studies, highlighting investigations into the origin and diversity of endocytic systems in eukaryotes. Beginning with a brief overview of the eukaryote tree of life, we show how understanding the endocytic machinery in a select, but diverse, array of organisms provides insights into endocytic system origins and predicts the likely configuration in the last eukaryotic common ancestor (LECA). Next, we consider three examples in which a comparative approach yielded insight into the function of modern cellular systems. First, using ESCRT-0 as an example, we show how comparative cell biology can discover both lineage-specific novelties (ESCRT-0) as well as previously ignored ancient proteins (Tom1), likely of both evolutionary and functional importance. Second, we highlight the power of comparative cell biology for discovery of previously ignored but potentially ancient complexes (AP5). Finally, using examples from ciliates and trypanosomes, we show that not all organisms possess canonical endocytic pathways, but instead likely evolved lineage-specific mechanisms. Drawing from these case studies, we conclude that a comparative approach is a powerful strategy for advancing knowledge about the general mechanisms and functions of endocytic systems.The endomembrane system mediates transport of lipids, proteins, and other molecules to the various locations in the eukaryotic cell. It also underlies the interactions with the extracellular environment, presenting material at the cell surface as well as secreting and internalizing material. In modern cells, these latter aspects are important for signal transduction, surface remodeling, and nutrient acquisition. Just as these abilities are crucial to modern cells, they were likely equally important for the very first eukaryotes as they underwent speciation from prokaryotic-like ancestors via niche competition in the ancient world (Cavalier-Smith 2002). Understanding the events and biological processes involved in the evolution of the membrane-trafficking system in general, and the endocytic system in particular, gives us insights into landmark events in our cellular past.Evolutionary insight about cellular phenomenon is derived from two basic types of comparative study: from molecular cell biological analyses of increasingly tractable model organisms across the diversity of eukaryotes, and by computational analyses of genomic information (i.e., the genes encoding the membrane-trafficking machinery). Whereas the information gathered from taking this comparative, or evolutionary cell biology, approach (Brodsky et al. 2012) is valuable for evolutionary content, these same analyses are potentially highly valuable in understanding basic cell biology, a benefit that is perhaps less obvious and hence less appreciated. In this article, we frame what has been learned about the evolution of the endocytic system, in the dual context of what it tells us about ancient cells together with what it can tell us about modern ones. We begin with a brief introduction to eukaryotic diversity and the evolution of the membrane-trafficking system. We then delve into the evolution of specific endocytic factors to illustrate the ways in which cell biologists of all stripes can benefit from the emerging field of evolutionary cell biology.     

Membrane Mechanics of Endocytosis in Cells with Turgor

Endocytosis is an essential process by which cells internalize a piece of plasma membrane and material from the outside. In cells with turgor, pressure opposes membrane deformations, and increases the amount of force that has to be generated by the endocytic machinery. To determine this force, and calculate the shape of the membrane, we used physical theory to model an elastic surface under pressure. Accurate fits of experimental profiles are obtained assuming that the coated membrane is highly rigid and preferentially curved at the endocytic site. The forces required from the actin machinery peaks at the onset of deformation, indicating that once invagination has been initiated, endocytosis is unlikely to stall before completion. Coat proteins do not lower the initiation force but may affect the process by the curvature they induce. In the presence of isotropic curvature inducers, pulling the tip of the invagination can trigger the formation of a neck at the base of the invagination. Hence direct neck constriction by actin may not be required, while its pulling role is essential. Finally, the theory shows that anisotropic curvature effectors stabilize membrane invaginations, and the loss of crescent-shaped BAR domain proteins such as Rvs167 could therefore trigger membrane scission.     

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers

A number of recent studies have provided new insights into the complexity of the endocytic pathways originating at the plasma membrane of mammalian cells. Many of the molecules involved in clathrin coated pit internalization are now well understood but other pathways are less well defined. Caveolae appear to represent a low capacity but highly regulated pathway in a restricted set of tissues in vivo. A third pathway, which is both clathrin- and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluid. The relationship of this pathway, if any, to macropinocytosis or to the endocytic pathways of lower eukaryotes remains an interesting open question. Our understanding of the regulatory mechanisms and molecular components involved in this pathway are at a relatively primitive stage. In this review, we will consider some of the characteristics of different endocytic pathways in high and lower eukaryotes and consider some of the common themes in endocytosis. One theme which becomes apparent from comparison of these pathways is that apparently different pathways can share common molecular machinery and that pathways considered to be distinct actually represent similar basic pathways to which additional levels of regulatory complexity have been added.     

Endosome dynamics during development

Endocytosis has traditionally been studied in isolated cells. More recently, however, the analysis of protein trafficking in whole organisms has revealed that it plays exciting roles during development. Endocytic trafficking of cell adhesion molecules regulates epithelial polarity and cell migration. Developmental signaling pathways are regulated by the trafficking of receptors and their ligands through the endocytic pathway. Finally, impairment of the endocytic machinery can affect proliferation control and contribute to tumor development.     

Internalization and trafficking of guanylyl cyclase/natriuretic peptide receptor-A

One of the principal loci involved in the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), whose ligand-binding efficiency and GC catalytic activity vary remarkably in different target cells and tissues. In its mature form, NPRA resides in the plasma membrane and contains an extracellular ligand-binding domain, a single transmembrane region, and the intracellular protein kinase-like homology domain (KHD) and guanylyl cyclase (GC) catalytic domain. NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. Binding of ligand to NPRA triggers a complex array of signal transduction events and accelerates the endocytosis. The endocytic transport is important in regulating signal transduction, formation of specialized signaling complexes, and modulation of specific components of internalization events. The present review describes the experiments which reveal the internalization of ligand-receptor complexes of NPRA, receptor trafficking and recycling, and delivery of both ligand-receptor molecules into subcellular compartments. The ligand-receptor complexes of NPRA are finally degraded within the lysosomes. The experimental evidence provides a consensus forum, which establishes the endocytosis, cellular trafficking, sequestration, and metabolic processing of ANP/NPRA complexes in the intact cells. The discussion is afforded to address the experimental insights into the mechanisms that cells utilize in modulating the delivery and metabolic processing of ligand-bound NPRA into the cell interior.