Regulation of raft-dependent endocytosis

Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveolae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1.     

Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages

SR-A (class A macrophage scavenger receptor) is a transmembrane receptor that can bind many different ligands, including modified lipoproteins that are relevant to the development of vascular diseases. However, the precise endocytic pathways of SR-A/mediated ligands internalization are not fully characterized. In this study, we show that the SR-A/ligand complex can be endocytosed by both clathrin- and caveolae-dependent pathways. Internalizations of SR-A-lipoprotein (such as acLDL) complexes primarily go through clathrin-dependent endocytosis. In contrast, macrophage apoptosis triggered by SR-A-fucoidan internalization requires caveolae-dependent endocytosis. The caveolae-dependent process activates p38 kinase and JNK signaling, whereas the clathrin-mediated endocytosis elicits ERK signaling. Our results suggest that different SR-A endocytic pathways have distinct functional consequences due to the activation of different signaling cascades in macrophages.     

Mechanism of p38 MAPK–induced EGFR endocytosis and its crosstalk with ligand-induced pathways

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.     

Endocytosis in Drosophila: progress, possibilities, prognostications.

By many outside the field, endocytosis is often perceived as a "house-keeping" function performed via identical mechanisms in yeast and man. Recent discoveries have done much to reduce this misperception. (1) Endocytosis occurs via different mechanisms and different pathways in different cellular contexts. (2) Molecular mechanisms that regulate homologous pathways in unicellular and multicellular organisms show considerable variance. (3) Temporally controlled endocytosis of specific regulatory molecules underlies several important and intricate biological processes including synapse formation, synaptic plasticity, cell fate determination, and morphogen gradient formation. Interactions between endocytosis and cytoskeletal and signaling pathways have been particularly revealing. In this intellectual context, Drosophila has become invaluable as a metazoan genetic model in which to understand the many faces of endocytosis. This review discusses two aspects of work in Drosophila: (a) its contributions toward understanding fundamental mechanisms that underlie the operation of endocytic pathways; (b) how analyses in Drosophila provide insights into varied biological processes regulated by endocytosis. In addition, while offering our commentary on merits and limitations of Drosophila work, we speculate on likely areas for contributions and future research on endocytosis in Drosophila.     

Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.     

病毒入胞机制研究方法及其研究进展

多数病毒家族利用胞吞作为入侵宿主细胞的途径。胞吞既可以介导病毒内化,也可以将病毒运输到复制位点。已知的胞吞途径包括:网格蛋白依赖型内吞、小窝蛋白依赖型内吞、巨胞饮和网格蛋白、小窝蛋白非依赖型内吞。随着对胞吞过程中各组分结构和功能了解的日趋深入,研究胞吞过程以及病毒入侵过程的手段也变得更有效,特异性更高。目前,化学抑制剂的使用仍十分普遍,但该方法常非特异性地阻断细胞某些功能。一些分子抑制方法,如过表达显性负突变体和siRNA技术等,因其对单一途径的特异性阻断,使得应用分子型抑制剂逐渐取代了化学抑制剂。本文主要分析了研究病毒入侵途径时所使用的实验方法,并列举了一些实例。     

Molecular and cellular mechanisms of receptor-mediated endocytosis.

In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors and ligands are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of certain viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Although endocytosis is common to all nucleated eukaryotic cells, the factors that regulate these receptor-mediated endocytic pathways are not fully understood. Defective receptors that are not capable of undergoing normal endocytosis can lead to certain disease states, as in the case of familial hypercholesteremia (FH). This review has three objectives: (i) to describe the different routes that receptors and ligands follow after internaliation; (ii) to describe the potential mechanisms which regulate the initiation and subsequent sorting of receptors and ligands so they reach their final destination; and (iii) to describe the potential functions of receptor-mediated endocytosis.     

Multiple pathways for ligand internalization in rat hepatocytes. II: Effect of hyperosmolarity and contribution of fluid-phase endocytosis.

In a companion report (Moss and Ward: J. Cell. Physiol 149:313-318, 1991) evidence was presented for multiple pathways for insulin internalization based on differences between the internalization of insulin and that of two other ligands, asialofetuin (Afet) and epidermal growth factor (EGF), in the presence of several perturbations of endocytosis. In the present study we have explored the characteristics of three internalization pathways and the contribution of each to overall insulin uptake. Freshly isolated hepatocytes were incubated with radiolabeled ligands in the presence of hyperosmolar sucrose, treatment that is thought to inhibit the coated pit pathway of endocytosis. Insulin internalization was decreased approximately 39%, but much greater decreases were observed with Afet (86%) and EGF (62%). Competition between uptake of radiolabeled and unlabeled insulin was observed in hyperosmolar-treated cells, suggestive of endocytosis by a receptor-mediated noncoated-pit pathway. Uptake of radiolabeled insulin that persisted in the presence of hyperosmolarity and high concentrations of unlabeled insulin suggested a third uptake pathway: fluid-phase endocytosis. A rate of fluid-phase endocytosis of 7.2 microL/hr/10(6) cells was determined from the uptake of the fluid-phase marker lucifer yellow. At high insulin concentrations (greater than or equal to 250 ng/ml), fluid-phase endocytosis appears to be the predominant pathway for insulin uptake, but at lower insulin concentrations (physiological) the coated pit and noncoated pit pathways are the primary routes for insulin internalization.     

Regulation of EGF-stimulated EGF receptor endocytosis during M phase

It has been generally accepted that endocytosis is inhibited during mitotic phase (M phase) as a means to insulate the cell from outside influences. Many endocytic/trafficking proteins are present during M phase, but are associated with partners that are distinct from those involved in trafficking pathways. These findings have led to the 'moonlighting' hypothesis. However, all these findings are based on the study of fluid-phase and constitutive endocytosis. Here, we used epidermal growth factor receptor (EGFR) as a model system to study ligand-induced receptor endocytosis in M phase. We found that EGF-induced EGFR endocytosis still occurs during M phase, but follows different kinetics. EGF-induced EGFR endocytosis is delayed/inhibited for a few minutes and is slower in M phase, especially at metaphase. However, consistent with previous reports, transferrin endocytosis is inhibited under the same conditions. We further showed that EGFR endocytosis is differentially regulated during the cell cycle: dependent on EGFR kinase activation in M phase, but independent of EGFR kinase activation in interphase. We conclude that cells have adopted a system for selective endocytosis in M phase.     

The pathways for fluid phase and receptor mediated endocytosis in rat hepatocytes are different but thermodynamically equivalent

In isolated rat hepatocytes fluid phase endocytosis, determined by the uptake of the fluorescent dye lucifer yellow (LY), and receptor mediated endocytosis, determined using a ligand for the asialoglycoprotein receptor (asialo-orosomucoid; ASOR), are different pathways based on their different sensitivities to hyperosmolarity induced by sucrose (Oka and Weigel, J. Cell. Biol. 105, 311a, 1987). LY uptake was unaffected by 0.2 M sucrose at all temperatures tested between 12 degrees and 37 degrees C whereas the uptake of 125I-ASOR was completely inhibited at any temperature. Since the two probes are taken up by different pathways it was possible to determine independently the activation energies (Ea) for the fluid phase versus the receptor mediated coated pit endocytic process. The Ea was 26.4 +/- 3.5 and 25.8 +/- 1.9 kcal/mole for, respectively, receptor mediated and fluid phase endocytosis. These values are not significantly different, and we conclude that the fluid phase and receptor mediated pathways are thermodynamically equivalent even though they are independent.     

Endocytosis and signaling cascades: a close encounter.

Internalization of receptors and other cell surface components is well known to occur via clathrin-mediated endocytosis, although other less well characterized pathways are also involved. Internalized receptors are then delivered to early endosomes, where they are sorted to be recycled back to the plasma membrane for reutilization or transported to late endosomes/lysosomes for degradation. Endocytosis has long been considered as a constitutive, housekeeping function of animal cells that occurs independently of the cellular environment in contrast to regulated secretion. Here, we will discuss recent studies that are uncovering the existence of cross-talk between signaling molecules and components of the transport machinery, indicating that endocytosis can be modulated by signaling pathways.     

Endocytosis and post-endocytic sorting of connexins

The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells in solid tissues to directly exchange ions and small molecules. These channels assemble into distinct plasma membrane domains known as gap junctions. Gap junction intercellular communication plays critical roles in numerous cellular processes, including control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are dynamic plasma membrane domains, and there is increasing evidence that modulation of endocytosis and post-endocytic trafficking of connexins are important mechanisms for regulating the level of functional gap junctions at the plasma membrane. The emerging picture is that multiple pathways exist for endocytosis and sorting of connexins to lysosomes, and that these pathways are differentially regulated in response to physiological and pathophysiological stimuli. Recent studies suggest that endocytosis and lysosomal degradation of connexins is controlled by a complex interplay between phosphorylation and ubiquitination. This review summarizes recent progress in understanding the molecular mechanisms involved in endocytosis and post-endocytic sorting of connexins, and the relevance of these processes to the regulation of gap junction intercellular communication under normal and pathophysiological conditions. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Mechanisms regulating membrane trafficking of G protein-coupled receptors in the endocytic pathway

Endocytic membrane trafficking plays multiple roles in GPCR signaling and regulation. In the past several years much has been learned about molecular mechanisms that mediate and regulate endocytic trafficking of cloned GPCRs expressed in transfected cell lines, and there is accelerating progress toward elucidating the membrane trafficking of GPCRs in native tissues. Current views regarding ligand-induced endocytosis of adrenergic catecholamine and opioid neuropeptide receptors will be reviewed, focusing on recent data suggesting the existence of additional machinery controlling the endocytosis of specific GPCRs via clathrin-coated pits. Evidence that GPCRs are selectively 'sorted' between divergent downstream pathways after endocytosis will be discussed, focusing on recent insight to mechanisms controlling receptor sorting between distinct recycling and non-recycling membrane pathways.     

Exocytosis,endocytosis, and development

During development, metazoans are faced with the daunting task of generating many different cell types in a temporally and spatially precise manner. This orderly process of cell generation relies on creating localized signals that activate or inhibit specific cellular pathways. Recent work has shown that some of these localized signals require the targeted secretion of proteins, or their uptake by endocytosis. The importance of these protein trafficking pathways in localized signal generation is further substantiated by endo- and exocytosis mutants which can phenocopy many developmental mutants. Genetic and molecular techniques that increase our ability to inhibit exocytosis and endocytosis in a temporal and cell-type specific manner are likely to further elucidate the complexities of development.     

Encapsulated group B Streptococcus modulates dendritic cell functions via lipid rafts and clathrin‐mediated endocytosis

Group B Streptococcus (GBS) capsular type III is an important agent of life‐threatening invasive infections. It has been previously shown that encapsulated GBS is easily internalized by dendritic cells (DCs) and can persist inside these immune cells. The mechanisms underlying these processes are unknown. Here, colocalization studies and the use of endocytosis inhibitors and caveolin^?/? mice, demonstrated that GBS uses multiple endocytosis mechanisms to enter mouse DCs. The capsular polysaccharide (CPS) selectively drives GBS internalization via caveolae‐independent but lipid raft‐dependent pathways. Non‐encapsulated bacteria failed to engage lipid rafts. GBS internalization by DCs also occurs via clathrin‐mediated endocytosis in a process independent of bacterial CPS. Albeit caveolae are not required for GBS internalization, signalling events through caveolin‐1 are involved in production of the inflammatory chemokine CCL2 by DCs infected with encapsulated GBS only. This study addresses for the first time endocytosis pathways implicated in DC internalization of encapsulated GBS and suggests a complex interplay between GBS and DCs, which was selectively modulated by the presence of CPS.     

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.     

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.     

Functional entry of baculovirus into insect and mammalian cells is dependent on clathrin-mediated endocytosis

Entry of the budded virus form of baculoviruses into insect and mammalian cells is generally thought to occur through a low-pH-dependent endocytosis pathway, possibly through clathrin-coated pits. This insight is primarily based on (immuno)electron microscopy studies but requires biochemical support to exclude the use of other pathways. Here, we demonstrate using various inhibitors that functional entry of baculoviruses into insect and mammalian cells is primarily dependent on clathrin-mediated endocytosis. Our results further suggest that caveolae are somehow involved in baculovirus entry in mammalian cells. A caveolar endocytosis inhibitor, genistein, enhances baculovirus transduction in these cells considerably.     

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization.     

Endocytosis without clathrin coats

Endocytosis is involved in an enormous variety of cellular processes. To date, most studies on endocytosis in mammalian cells have focused on pathways that start with uptake through clathrin-coated pits. Recently, new techniques and reagents have allowed a wider range of endocytic pathways to begin to be characterized. Various non-clathrin endocytic mechanisms have been identified, including uptake through caveolae, macropinosomes and via a separate constitutive pathway. Many markers for clathrin-independent endocytosis are found in detergent-resistant membrane fractions, or lipid rafts. We will discuss these emerging new findings and their implications for the nature of lipid rafts themselves, as well as for the potential roles of non-clathrin endocytic pathways in remodeling of the plasma membrane and in regulating the membrane composition of specific intracellular organelles.