Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference

To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.     

Accessory protein recruitment motifs in clathrin-mediated endocytosis

Clathrin-mediated endocytosis depends upon the interaction of accessory proteins with the alpha-ear of the AP-2 adaptor. We present structural characterization of these regulatory interactions. DPF and DPW motif peptides derived from eps15 and epsin bind in type I beta turn conformations to a conserved pocket on the alpha-ear platform. We show evidence for a second binding site that is DPW motif specific. The structure of a complex with an AP-2 binding segment from amphiphysin reveals a novel binding motif that we term FxDxF, which is engaged in an extended conformation by a unique surface of the platform domain. The FxDxF motif is also used by AP180 and the 170 kDa isoform of synaptojanin and can be found in several potential endocytic proteins, including HIP1, CD2AP, and PLAP. A mechanism of clathrin assembly regulation is suggested by three different AP-2 engagement modes.     

Snap-shots of clathrin-mediated endocytosis

Clathrin-mediated endocytosis is one of the major entry routes into a eukaryotic cell. It is driven by protein components that aid the selection of cargo and provide the mechanical force needed to both deform the plasma membrane and detach a vesicle. Clathrin-coated vesicles were first observed by electron microscopy in the early 1960s. In subsequent years, many of the characteristic intermediates generated during vesicle formation have been trapped and observed. A variety of electron microscopy techniques, from the analysis of sections through cells to the study of endocytic intermediates formed in vitro, have led to the proposition of a sequence of events and of roles for different proteins during vesicle formation. In this article, these techniques and the insights gained are reviewed, and their role in providing snap-shots of the stages of endocytosis in atomic detail is discussed.     

Chemical proteomics reveals bolinaquinone as a clathrin-mediated endocytosis inhibitor

The emerging field of mass spectrometry-based chemical proteomics provides a powerful instrument in the target discovery of bioactive small-molecules, such as drugs or natural products. The identification of their macromolecular targets is required for a comprehensive understanding of their bio-pharmacological role and for unraveling their mechanism of action. We report the application of a chemical proteomics approach to the analysis of the cellular interactome of the marine metabolite bolinaquinone (BLQ). BLQ was linked to an opportune α,ω-diamino polyethylene glycol chain and then immobilized on a matrix support. The modified beads were then used as a bait for fishing the potential partners of BLQ in a THP-1 macrophage cell lysate. Surprisingly, we identified clathrin, a protein involved in the cell internalization of proteins, viruses and other biologically relevant macromolecules, as a specific and major BLQ partner. In addition, we verified the biochemical role of BLQ testing its ability to inhibit the clathrin-mediated endocytosis of albumin. This finding indicates BLQ as a new biotechnological tool for cell endocytosis studies and paves the way to further investigation on its potential role in modulating internalization process.     

Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo

The budding of clathrin-coated vesicles is essential for protein transport. After budding, clathrin must be uncoated before the vesicles can fuse with other membranous structures. In vitro, the molecular chaperone Hsc70 uncoats clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as auxilin. However, there is little evidence that either Hsc70 or auxilin is essential in vivo. Here we show that C. elegans has a single auxilin homologue that is identical to mammalian auxilin in its in vitro activity. When RNA-mediated interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes show markedly reduced receptor-mediated endocytosis of yolk protein tagged with green fluorescent protein (GFP). In addition, most of these worms arrest during larval development, exhibit defective distribution of GFP-clathrin in many cell types, and show a marked change in clathrin dynamics, as determined by fluorescence recovery after photobleaching (FRAP). We conclude that auxilin is required for in vivo clathrin-mediated endocytosis and development in C. elegans.     

Capturing the mechanics of clathrin-mediated endocytosis

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.     

Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis

Intersectin 1 (ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin-mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled-coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co-localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.     

Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis

Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K(+) depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-beta-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.     

The evolution of dynamin to regulate clathrin-mediated endocytosis: speculations on the evolutionarily late appearance of dynamin relative to clathrin-mediated endocytosis

Whereas clathrin-mediated endocytosis (CME) exists in all eukaryotic cells, we first detect classical dynamin in Ichthyosporid, a single-cell, metazoan precursor. Based on a key functional residue in its pleckstrin homology domain, we speculate that the evolution of metazoan dynamin coincided with the specialized need for regulated CME during neurotransmission.     

From uncertain beginnings: Initiation mechanisms of clathrin-mediated endocytosis

Clathrin-mediated endocytosis is a central and well-studied trafficking process in eukaryotic cells. How this process is initiated is likely to be a critical point in regulating endocytic activity spatially and temporally, but the underlying mechanisms are poorly understood. During the early stages of endocytosis three components—adaptor and accessory proteins, cargo, and lipids—come together at the plasma membrane to begin the formation of clathrin-coated vesicles. Although different models have been proposed, there is still no clear picture of how these three components cooperate to initiate endocytosis, which may indicate that there is some flexibility underlying this important event.

Introduction

Clathrin-mediated endocytosis is the process by which cargo-containing clathrin-coated vesicles bud off from the plasma membrane and are taken up into the cell. This process is critical for intercellular signaling, uptake of nutrients, and membrane recycling. Endocytosis requires the coordinated assembly of a large number of proteins at the plasma membrane, the timing and composition of which are very regular (Kaksonen et al., 2005; Taylor et al., 2011). Despite the large amount of research dedicated to the understanding of this assembly sequence, the way the assembly is triggered is not well understood.Although the budding of clathrin-coated vesicles in vitro has recently been shown to only require the presence of clathrin, an adaptor protein, and dynamin (Dannhauser and Ungewickell, 2012), endocytosis in vivo is a lot more complex. To gain a full understanding of how vesicle budding is regulated in vivo we need to determine how the initiation of this process is controlled.The location or timing of endocytosis can be tightly regulated, as highlighted in the following examples: In budding yeast Saccharomyces cerevisiae, a larger number of endocytic events occur in the growing bud compared with the mother cell (Fig. 1 A). This polarization of endocytosis in yeast is important for maintaining overall cellular polarity (Bi and Park, 2012). Another example is the neuronal synapse, where the level of endocytosis is tightly coupled to exocytic activity in a process called synaptic vesicle recycling, in order to maintain the pool of synaptic vesicles (Fig. 1 B; Murthy and De Camilli, 2003). Also, during entry into a host cell, viruses can exploit the mechanisms of endocytic initiation in order to induce their own uptake (Fig. 1 C; Sieczkarski and Whittaker, 2002).Open in a separate windowFigure 1.Examples of regulation of the initiation of endocytosis. (A) In yeast Saccharomyces cerevisiae, clathrin-mediated endocytosis is polarized to the growing bud. (B) After signal propagation at the synapse, exocytosis of the neurotransmitter is accompanied by a compensatory increase in clathrin-mediated endocytosis to maintain synaptic plasma membrane area and recycle synaptic vesicle components. (C) Many viruses enter the host cell by clathrin-mediated endocytosis. The virus particle may exploit the cell’s regulation of endocytosis in order to induce its own uptake.In this review we will discuss studies addressing the question of how endocytic sites are initiated. Although lots of data are available, there are a number of inconsistent results that have led to uncertainty about the mechanisms of initiation. When studying the initiation of endocytosis we need to focus on the earliest stages of the sequence of protein assembly. The key players involved in these stages are clathrin adaptors and accessory proteins, cargo, and lipids (Fig. 2). We will discuss how these components have been suggested to function in the initiation of endocytosis.Open in a separate windowFigure 2.The key players involved in the initiation of endocytosis. Adaptor proteins, cargo, lipids, and clathrin accumulate at the plasma membrane to make up the nascent endocytic site. Adaptor proteins, including the AP2 complex, bind to the inner leaflet of the plasma membrane. The adaptors interact with the plasma membrane via lipid-binding domains, many of which bind specifically to PIP₂. The tails of adaptor proteins bind and recruit clathrin triskelions to the plasma membrane where they polymerize to form the clathrin coat. Cargo—consisting of transmembrane receptors and extracellular soluble ligands—is taken up by clathrin-coated vesicles. Adaptors bind to signaling motifs on the intracellular side of the receptors.

Adaptors: Bringing everything together

Clathrin, the main coat component of the endocytic machinery, is unable to directly interact with the lipids or proteins of the plasma membrane (Maldonado-Báez and Wendland, 2006). Therefore, adaptor proteins are required to link the clathrin coat to the membrane (see Taylor et al., 2011; Carroll et al., 2012), making them good candidates to play a role in initiation. Additionally, their ability to interact with other endocytic proteins could be important for recruitment of further components to the plasma membrane at the early stages of endocytosis. Although two-color live-cell microscopy has been used to arrange the endocytic process into a temporal sequence, it has proved challenging to determine one protein that consistently arrives first at the site (Kaksonen et al., 2005; Henne et al., 2010; Taylor et al., 2011; Carroll et al., 2012). Nevertheless, it is plausible that there could be one factor that is essential for the initiation of endocytosis.

Table 1.

Adaptor and accessory proteins involved in endocytosis

Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference

Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes. mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission. To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi). Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression. In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified. In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited. This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.     

Cargo recognition during clathrin-mediated endocytosis: a team effort

Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.     

The ubiquitin-interacting motifs target the endocytic adaptor protein epsin for ubiquitination

The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.     

Soapwort saponins trigger clathrin-mediated endocytosis of saporin, a type I ribosome-inactivating protein

Saporin, a type I ribosome-inactivating protein (RIP), removes adenine residues from the 28S ribosomal RNA as part of a process that leads to inhibition of protein synthesis. However, as shown in this study, neither saporin nor his-tagged saporin (both 0.6-6 pM) exert toxicity on several human cell lines including H-2171, SK-N-SH, HEP-G2, MOLT-3, THP-1, HL-60 and ECV-304. Saporin and his-tagged saporin became highly cytotoxic when they were used in a combined treatment with Soapwort saponins (SA). When combined with SA (2-4 microg/ml) saporin became as cytotoxic as the highly toxic type II RIP rViscumin reflected by an IC50 of 42.5x10(-12) M for saporin and 21.5x10(-12) M for rViscumin. We demonstrated that saporin was internalized via clathrin-mediated endocytosis, followed by the release into the endosomal transport system. Our results indicate that SA triggers this endocytic event rendering the otherwise cell membrane impermeable type I RIP saporin a potent cytotoxin. This effect was not cell line-specific suggesting that saporin exploits a common SA-dependent mechanism to enter cells.     

RNA interference to target lipid disorders

PURPOSE OF REVIEW: This review focuses on proof-of-principle experiments providing validation of new targets for the development of RNA interference-based therapeutics for dyslipidemia. RECENT FINDINGS: Over the past few years, RNA interference has become an accepted approach to manipulate gene expression in mammalian systems. Advantage has been taken of the relative tissue specificity of adenovirus for liver, and the genetic specificity of short hairpin RNA-mediated RNA interference to create liver-specific downregulation of different genes. A different approach to target liver has been through the administration of chemically modified short interfering RNAs. For example, apolipoprotein B messenger RNA has been silenced in liver and jejunum resulting in decreased plasma levels of apolipoprotein B and total cholesterol. SUMMARY: RNA interference has aroused great interest as a powerful experimental tool and a potential therapeutic strategy. Successful animal studies indicate that RNA interference might be useful for the treatment of various human diseases. Clinical studies will soon begin to assess the use of this new class of therapeutics to treat dyslipidemia.     

Selective inhibition of hepatitis B virus replication by RNA interference

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.     

Molecular mechanism and physiological functions of clathrin-mediated endocytosis

Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.     

Temperature-controlled atmospheric-pressure plasma treatment induces protein uptake via clathrin-mediated endocytosis in tobacco cells

Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N₂ plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N₂ plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.