Cargo Recognition in Clathrin-Mediated Endocytosis

The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.The internalization of a subset of plasma membrane proteins by clathrin-mediated endocytosis is one the best-characterized sorting processes that takes place in the endomembrane system of eukaryotic cells (Kirchhausen 2014). Selection of transmembrane proteins (referred to as “cargo”) for internalization by clathrin-mediated endocytosis involves recognition of endocytic signals in the cytosolic domains of the proteins by adaptors located in the inner layer of clathrin coats. Signal–adaptor interactions lead to concentration of the transmembrane proteins within clathrin-coated pits that eventually bud into the cytoplasm as clathrin-coated vesicles (Kirchhausen 2014). Transmembrane proteins that have endocytic signals are thus rapidly delivered to endosomes, whereas those that lack signals remain at the plasma membrane. This article summarizes recent progress in the elucidation of the mechanisms of signal recognition in clathrin-mediated endocytosis, with additional reference to related intracellular sorting events. Further information on this topic can be found in previous reviews (Bonifacino and Traub 2003; Traub 2009; Kelly and Owen 2011).     

Rhesus Rhadinovirus Establishes a Latent Infection in B Lymphocytes In Vivo

Recent DNA sequence analysis indicates that rhesus rhadinovirus (RRV) is a member of the lymphotropic gamma-2 herpesvirus family. To determine if RRV is lymphotropic, peripheral blood mononuclear cells from naturally infected monkeys were separated by immunomagnetic bead depletion and analyzed for the presence of RRV by virus isolation and nested PCR. The recovery and consistent detection of RRV in the CD20(+)-enriched fraction clearly demonstrates that B lymphocytes are a major site of virus persistence.     

The Initiation of Clathrin-Mediated Endocytosis Is Mechanistically Highly Flexible

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Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

Up to 60 different proteins are recruited to the site of clathrin-mediated endocytosis in an ordered sequence. These accessory proteins have roles during all the different stages of clathrin-mediated endocytosis. First, they participate in the initiation of the endocytic event, thereby determining when and where endocytic vesicles are made; later they are involved in the maturation of the clathrin coat, recruitment of specific cargo molecules, bending of the membrane, and finally in scission and uncoating of the nascent vesicle. In addition, many of the accessory components are involved in regulating and coupling the actin cytoskeleton to the endocytic membrane. We will discuss the different accessory components and their various roles. Most of the data comes from studies performed with cultured mammalian cells or yeast cells. The process of endocytosis is well conserved between these different organisms, but there are also many interesting differences that may shed light on the mechanistic principles of endocytosis.Receptor-mediated endocytosis is the process by which eukaryotic cells concentrate and internalize cell surface receptors from the plasma membrane into small (∼50 nm– ∼100 nm diameter) membrane vesicles (Chen et al. 2011; McMahon and Boucrot 2011; Weinberg and Drubin 2012). This mechanism has been studied extensively in mammalian tissue culture cells and in yeast, and despite the evolutionary distance between yeast and mammalian cells the mechanism of receptor-mediated endocytosis in the respective cell types show remarkable similarities. Indeed many of the ∼60 endocytic accessory proteins (EAPs) found in yeast have homologs in mammalian cells, although both cell types also have unique EAPs (McMahon and Boucrot 2011; Weinberg and Drubin 2012).In the following, we briefly describe known yeast and mammalian EAPs (Sigismund et al. 2012; see also Bökel and Brand 2013; Cosker and Segal 2014; Di Fiore and von Zastrow 2014).

Table 1.

Key endocytic proteins in mammals and in yeast

Imaging and Modeling the Dynamics of Clathrin-Mediated Endocytosis

Clathrin-mediated endocytosis (CME) plays a central role in cellular homeostasis and is mediated by clathrin-coated pits (CCPs). Live-cell imaging has revealed a remarkable heterogeneity in CCP assembly kinetics, which can be used as an intrinsic source of mechanistic information on CCP regulation but also poses several major problems for unbiased analysis of CME dynamics. The backbone of unveiling the molecular control of CME is an imaging-based inventory of the full diversity of individual CCP behaviors, which requires detection and tracking of structural fiduciaries and regulatory proteins with an accuracy of >99.9%, despite very low signals. This level of confidence can only be achieved by combining appropriate imaging modalities with self-diagnostic computational algorithms for image analysis and data mining.Clathrin-mediated endocytosis (CME) drives the uptake of diverse receptor-bound macromolecules and is one of the main endocytic mechanisms constitutively active in all mammalian cells. Clathrin-coated vesicles (CCVs) were the first transport vesicles to be isolated (Pearse 1975), which subsequently led to the identification of clathrin and the heterotetrameric adaptor protein AP2 as the major coat components (Pearse 1976, 1978). Further research in this area was spurred by the discovery that familial hypercholesterolemia is caused by a single substitution of a cysteine for a tyrosine in the cytoplasmic tail of the low-density lipoprotein receptor (LDLR), which disrupts its endocytic internalization motif and prevents its concentration in clathrin-coated pits (CCPs) (Anderson et al. 1977). In the following decades, biochemistry combined with molecular biology and electron microscopy (EM) have revealed much about the molecular players involved in CME (reviewed by Conner and Schmid 2003; Schmid and McMahon 2007; McMahon and Boucrot 2011; Boettner et al. 2012). Today, we know that CME is initiated via assembly of clathrin and AP2 to form CCPs and that receptor–ligand complexes (referred to as “cargo”) are concentrated in CCPs via direct interactions between endocytic motifs within their cytoplasmic domains and adaptor molecules that recruit clathrin. With the aid of a multitude of endocytic accessory proteins (EAPs)—many with as-yet poorly defined functions—CCPs undergo stabilization, maturation, and invagination. Finally, membrane fission, catalyzed by the GTPase dynamin, pinches off the CCV carrying its cargo into the cell.Although powerful and invaluable, bulk biochemical assays can only report cumulative and ensemble-averaged effects on CME, whereas EM only provides static snapshots of highly dynamic structures. Both approaches are not sufficient to resolve critical, rate-limiting stages of CCP maturation and alternative outcomes that prevent CCV internalization. They are also not sufficient to probe the frequently overlapping functions of individual components in CCP formation and maturation. Perturbation of molecular players in a system with such redundancy may lead to no detectable shifts, or to detectable shifts that merely represent system adaptation, and thus may not reveal the actual function of the targeted component itself. Moreover, perturbing CME may globally interfere with cell homeostasis, which can also elicit phenotypes unrelated to the actual functions of the target. To remedy these issues, it is necessary to follow the dynamics of CME at the level of individual CCPs and to correlate these behaviors with differential patterns of cargo and EAP recruitment and activity.These goals became approachable with the “GFP revolution” in the 1990s, which was paralleled by leaps in the sensitivity of digital light microscopy. For CME, the power of these technologies was first shown by Keen and colleagues, who used a green fluorescent protein (GFP) fusion of the clathrin light chain (CLC) to image clathrin dynamics by time-lapse wide-field epifluorescence microscopy (Gaidarov et al. 1999). Since then, numerous live-cell imaging studies have revealed remarkable heterogeneity in CCP assembly kinetics and internalization (Rappoport and Simon 2003; Ehrlich et al. 2004; Keyel et al. 2004; Merrifield et al. 2005; Loerke et al. 2009; Taylor et al. 2011). Although the physiological and molecular bases for this heterogeneity remain to be uncovered, the working hypothesis is that CCP heterogeneity arises from variations in molecular composition, in cortical membrane mechanics, and in differences between cell types. More recent advances in imaging and computational image data analyses have made it possible to determine the order in which EAPs are incorporated or released from growing CCPs. Thus, multidimensional live-cell imaging and mathematical models, in combination with very mild chemical, molecular, and mechanical perturbations, may uncover how the molecular composition of an assembling CCP affects its behavior. In the following we describe the developments of imaging modalities and image analysis methods that have led to the current state of the art in quantitative imaging of CME.     

Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection

The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.     

Rhadinovirus Host Entry by Co-operative Infection

Rhadinoviruses establish chronic infections of clinical and economic importance. Several show respiratory transmission and cause lung pathologies. We used Murid Herpesvirus-4 (MuHV-4) to understand how rhadinovirus lung infection might work. A primary epithelial or B cell infection often is assumed. MuHV-4 targeted instead alveolar macrophages, and their depletion reduced markedly host entry. While host entry was efficient, alveolar macrophages lacked heparan - an important rhadinovirus binding target - and were infected poorly ex vivo. In situ analysis revealed that virions bound initially not to macrophages but to heparan⁺ type 1 alveolar epithelial cells (AECs). Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not. Rather bound virions were acquired by macrophages; epithelial infection occurred only later. Thus, host entry was co-operative - virion binding to epithelial cells licensed macrophage infection, and this in turn licensed AEC infection. An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages. By contrast an antibody block of membrane fusion was effective. Therefore co-operative infection extended viral tropism beyond the normal paradigm of a target cell infected readily in vitro; and macrophage involvement in host entry required neutralization to act down-stream of cell binding.     

Different Rotavirus Strains Enter MA104 Cells through Different Endocytic Pathways: the Role of Clathrin-Mediated Endocytosis

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins α2β1 and αvβ3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.Endocytosis is a cellular process that involves the formation of a vesicle whose cargo is transported from the extracellular milieu to the interior of the cell. Several endocytic pathways have been described, and all of them have been shown to be used by viruses during cell entry. These pathways include clathrin-mediated endocytosis, uptake via caveolae, macropinocytosis, phagocytosis, and a novel non-clathrin-, non-caveola-mediated pathway that is currently not well characterized (32). While detailed information about the entry of several enveloped viruses is now available (4, 35, 49, 53, 56), the mechanism by which nonenveloped viruses enter cells is not well understood. Two general mechanisms have been proposed to be used by these viruses to reach the cell''s cytoplasm: direct penetration at the cell surface, during which the viral particles are directly translocated from the external milieu into the cytoplasm, or internalization through endocytic processes (55).Rotaviruses, members of the family Reoviridae, are the leading etiologic agent of viral gastroenteritis in infants and young children worldwide, being responsible for an estimated 500,000 deaths each year (41). These nonenveloped viruses are formed by three concentric layers of protein that surround the viral genome, formed by 11 segments of double-stranded RNA. The outermost layer of the virion is formed by two proteins, VP4 and VP7, which are involved in the early interactions of the virus with its host cell (7, 27). VP4 is involved in receptor binding and cell penetration. The role of VP7 is less clear, although it has been shown that it interacts with the cell surface molecules at a postattachment step (17). After binding to the cell surface, the virus penetrates the plasma membrane to productively infect the cell. This penetration depends on the trypsin treatment of the virus, which results in the specific cleavage of VP4 to polypeptides VP8 and VP5. This cleavage promotes VP4 rearrangements in the viral particles that rigidify the spikes (7, 11).Despite the fact that, in vivo, rotaviruses primarily infect the mature enterocytes of the small intestine, studies of the infection of this type of cells have been limited due to the lack of established intestinal cell lines of small intestine origin. Given the absence of a better model, most of the studies on the entry and replication cycle of rotavirus have been conducted either in the epithelial monkey kidney cell line MA104 or in the human colon carcinoma cell line Caco-2, which are highly permissible to these viruses.Using as a model MA104 cells and the simian rotavirus RRV, we have proposed that rotavirus cell entry is a complex multistep process that involves the two virus surface proteins and several cell receptors, including sialic acids, gangliosides, integrins α2β1, α4β1, αvβ3, and αxβ2, and the heat shock cognate protein hsc70 (22). We have also shown that depletion of cholesterol from the cellular membrane severely impairs the infectivity of rotavirus (19, 50) and have suggested that sphingolipid- and cholesterol-enriched membrane lipid microdomains might be involved in rotavirus cell entry, since the virus and its receptors associate with these domains at early times during infection (23). However, there are some rotavirus strains that may not use all these molecules; some rotavirus strains are resistant to the neuraminidase (NA) treatment of the cell, and thus, they have been classified as NA resistant (6, 34). Additionally, the infectivity of some viral strains is not blocked by anti-integrin antibodies, suggesting the existence of rotavirus strains that are integrin independent (Table ​(Table1)1) (17).

TABLE 1.

Cellular receptor requirements of different rotavirus strains

Identification of the Rhesus Macaque Rhadinovirus Lytic Origin of DNA Replication

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.     

Absorption of Proteoglycan via Clathrin-Mediated Endocytosis in the Small Intestine of Rats

An alginate was isolated from commercially cultured Nemacystus decipiens which had been harvested in Yonashiro Town (Okinawa, Japan). The yield of the alginate was 1.6% (w/w of wet alga), and the uronic acid, ash and moisture contents of the alginate were 86.0%, 12.0%, and 2.3% (w/w), respectively. The molecular mass of the alginate was estimated to be about 1.5×10⁵. The infrared spectrum and optical rotation of the alginate were in agreement with those of the standard alginate. D-Mannuronic acid and L-guluronic acid were identified by ¹H- and ¹³C-NMR spectroscopy, the molar ratio of both sugar residues being estimated to be 0.72:1.00.     

High-Affinity Dkk1 Receptor Kremen1 Is Internalized by Clathrin-Mediated Endocytosis

Kremens are high-affinity receptors for Dickkopf 1 (Dkk1) and regulate the Wnt/β-catenin signaling pathway by down-regulating the low-density lipoprotein receptor-related protein 6 (LRP6). Dkk1 competes with Wnt for binding to LRP6; binding of Dkk1 inhibits canonical signaling through formation of a ternary complex with Kremen. The majority of down-regulated clathrin-mediated endocytic receptors contain short conserved regions that recognize tyrosine or dileucine sorting motifs. In this study, we found that Kremen1 is internalized from the cell surface in a clathrin-dependent manner. Kremen1 contains an atypical dileucine motif with the sequence DXXXLV. Mutation of LV to AA in this motif blocked Kremen1 internalization; as reported previously for other proteins, the aspartic acid residue in Kremen1 is not critical. Inhibition of expression of the adaptor protein 2 (AP-2) or inhibition of clathrin by pitstop 2 also blocked Kremen1 internalization. The novel amino acid sequence identified in Kremen1 is similar to the motif previously identified in hydra, yeast, and other organisms known to signal from the trans-Golgi network to the endosomal compartment.     

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin.     

Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.