Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.     

Arp2/3 mediates early endosome dynamics necessary for the maintenance of PAR asymmetry in Caenorhabditis elegans

The widely conserved Arp2/3 complex regulates branched actin dynamics that are necessary for a variety of cellular processes. In Caenorhabditis elegans, the actin cytoskeleton has been extensively characterized in its role in establishing PAR asymmetry; however, the contributions of actin to the maintenance of polarity before the onset of mitosis are less clear. Endocytic recycling has emerged as a key mechanism in the dynamic stabilization of cellular polarity, and the large GTPase dynamin participates in the stabilization of cortical polarity during maintenance phase via endocytosis in C. elegans. Here we show that disruption of Arp2/3 function affects the formation and localization of short cortical actin filaments and foci, endocytic regulators, and polarity proteins during maintenance phase. We detect actin associated with events similar to early endosomal fission, movement of endosomes into the cytoplasm, and endosomal movement from the cytoplasm to the plasma membrane, suggesting the involvement of actin in regulating processes at the early endosome. We also observe aberrant accumulations of PAR-6 cytoplasmic puncta near the centrosome along with early endosomes. We propose a model in which Arp2/3 affects the efficiency of rapid endocytic recycling of polarity cues that ultimately contributes to their stable maintenance.     

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.     

OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos

Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.     

From the inside out: Ion fluxes at the centre of endocytic traffic

Endocytic traffic is a complex and elegant operation involving cargo sorting, membrane budding and tubulation, generation of force, and the formation of organellar contacts. The role of specific proteins and lipids in these processes has been studied extensively. By comparison, precious little is understood about the contribution of the endocytic fluid to these events, despite much evidence that alteration of the contents can severely affect membrane traffic along the endocytic pathway. In particular, it has long been appreciated that dissipation of ionic gradients arrests endosome-to-lysosome maturation. How cells sense inorganic ions and transmit this information have remained largely enigmatic. Herein, we review the experimental findings that reveal an intimate association between luminal ions, their transport, and endocytic traffic. We then discuss the ionic sensors and the mechanisms proposed to convert ion concentrations into protein-based trafficking events, highlighting the current paucity of convincing explanations.     

Remodeling Epithelial Cell Organization: Transitions Between Front–Rear and Apical–Basal Polarity

Polarized epithelial cells have a distinctive apical–basal axis of polarity for vectorial transport of ions and solutes across the epithelium. In contrast, migratory mesenchymal cells have a front–rear axis of polarity. During development, mesenchymal cells convert to epithelia by coalescing into aggregates that undergo epithelial differentiation. Signaling networks and protein complexes comprising Rho family GTPases, polarity complexes (Crumbs, PAR, and Scribble), and their downstream effectors, including the cytoskeleton and the endocytic and exocytic vesicle trafficking pathways, together regulate the distributions of plasma membrane and cytoskeletal proteins between front–rear and apical–basal polarity. The challenge is to understand how these regulators and effectors are adapted to regulate symmetry breaking processes that generate cell polarities that are specialized for different cellular activities and functions.     

Endocytosis and trafficking of human lactoferrin in macrophage-like human THP-1 cells (1)

Different cell types have been reported to internalize lactoferrin (Lf) by specific or nonspecific receptors. Our studies focused on the endocytic pathway of human Lf in macrophage-like THP-1 cells. Lactoferrin was found to be internalized by THP-1 cells differentiated with phorbol myristate acetate. Incubation of cells with chlorpromazine and dansylcadaverine, inhibitors of clathrin-dependent endocytosis, led to a 50% inhibition of Lf internalization compared with untreated cells. Bafilomycin A1 and NH(4)Cl treatment also resulted in 40%-60% inhibition, respectively, suggesting that the internalization of Lf may partly be mediated by acidic endosome-like organelles. Endocytic uptake of Lf was also cholesterol-dependent, as shown by methyl-β-cyclodextrin or nystatin treatment of the cells prior to internalization. Partial colocalization of Lf and EEA-1, a marker specific for early endosomes, could be observed. Colocalization of Lf with a specific endoplasmic reticulum marker was also detected. Our results suggest that Lf is internalized mainly by the clathrin-dependent pathway in THP-1 cells and targets the ER. The physiological consequences of this intracellular trafficking will be the subject of future investigations.     

Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans

AbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 +/- 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.     

Energetic adaptations: Metabolic control of endocytic membrane traffic

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP‐activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell.     

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.     

Evidence for pore-forming ability by Legionella pneumophila

Legionella pneumophila is the cause of Legionnaires' pneumonia. After internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L . pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high-multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high-molecular-weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane-impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L . pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.     

The MreB and Min cytoskeletal-like systems play independent roles in prokaryotic polar differentiation

Establishment of an axis of cell polarity and differentiation of the cell poles are fundamental aspects of cellular development in many organisms. We compared the effects of two bacterial cytoskeletal-like systems, the MreB and MinCDE systems, on these processes in Escherichia coli. We report that the Min proteins are capable of establishing an axis of oscillation that is the initial step in establishment of polarity in spherical cells, in a process that is independent of the MreB cytoskeleton. In contrast, the MreB system is required for establishment of the rod shape of the cell and for polar targeting of other polar constituents, such as the Shigella virulence factor IcsA and the aspartate chemoreceptor Tar, in a process that is independent of the Min system. Thus, the two bacterial cytoskeletal-like systems act independently on different aspects of cell polarization.     

Signal transduction and endocytosis: close encounters of many kinds

Binding of hormones, growth factors and other cell modulators to cell-surface receptors triggers a complex array of signal-transduction events. The activation of many receptors also accelerates their endocytosis. Endocytic transport is important in regulating signal transduction and in mediating the formation of specialized signalling complexes. Conversely, signal-transduction events modulate specific components of the endocytic machinery. Recent studies of protein tyrosine kinases and G-protein-coupled receptors have shed new light on the mechanisms and functional consequences of this bidirectional interplay between signalling and membrane-transport networks.     

Dorsal ventral polarity and pattern formation in the Drosophila embryo

The establishment of polarity along the dorsal-ventral axis of the Drosophila embryo requires the graded distribution of the dorsal morphogen. Several maternal genes are responsible for the formation of the gradient and their products act in an ordered series of events that begins during oogenesis and involves two different cell types, the oocyte and the follicle cells. The last step in the series results in selective nuclear localization of dorsal proteins, dorsal is thought to regulate the expression of zygotic genes in a concentration dependent way. The zygotic genes determine cell fates in specific regions of the embryo and direct other genes involved in the processes of differentiation.     

Deconvolving the roles of Wnt ligands and receptors in sensing and amplification

Establishment of cell polarity is crucial for many biological processes including cell migration and asymmetric cell division. The establishment of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified and maintained by intracellular signaling networks usually using positive feedback regulation. Generally, these two processes are intertwined and it is challenging to determine which proteins contribute to the sensing or amplification process, particularly in multicellular organisms. Here, we integrated phenomenological modeling with quantitative single‐cell measurements to separate the sensing and amplification components of Wnt ligands and receptors during establishment of polarity of the Caenorhabditis elegans P cells. By systematically exploring how P‐cell polarity is altered in Wnt ligand and receptor mutants, we inferred that ligands predominantly affect the sensing process, whereas receptors are needed for both sensing and amplification. This integrated approach is generally applicable to other systems and will facilitate decoupling of the different layers of signal sensing and amplification.     

Rsp5-dependent endocytosis and degradation of the arsenite transporter Acr3 requires its N-terminal acidic tail as an endocytic sorting signal and arrestin-related ubiquitin-ligase adaptors

The yeast plasma membrane transporter Acr3 mediates efflux of toxic arsenite and antimonite. Here, we investigated the mechanisms of Acr3 turnover. We found that after arrival and residence at the plasma membrane, Acr3 is subjected to internalization followed by proteolysis in the vacuole. Endocytic degradation of Acr3 is promoted by the ubiquitin ligase Rsp5 and requires polyubiquitination of Acr3 at multiple lysine residues via lysine 63-linked ubiquitin chains. The turnover of Acr3 also depends on two arrestin-related proteins, Art3/Aly2 and Art4/Rod1, that enable recruitment of Rsp5 to its targets. Finally, we found that a short acidic patch located in the N-terminal tail of Acr3 is needed for its ubiquitination and internalization. We propose that this motif serves as an endocytic signal that facilitates binding of the arrestin-Rsp5 complexes to the Acr3 cargo.     

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.     

Numb controls E-cadherin endocytosis through p120 catenin with aPKC

Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity.     

CLIP‐170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus‐end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward‐directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP‐170, as well as the association of CLIP‐170 to MT plus‐ends. In response to HGF, entry of Met into Rab4‐positive endosomes results in Golgi‐localized γ‐ear‐containing Arf‐binding protein 3 (GGA3) and CLIP‐170 recruitment to an activated Met RTK complex. We conclude that CLIP‐170 co‐ordinates the recycling and the transport of Met‐positive endocytic vesicles to plus‐ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration.