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Highlights • S. pneumoniae invades brain endothelium through a novel dynamin independent endocytosis pathway. • Invasion through dynamin independent pathway is aided by SPN adhesin and host receptor interaction. • Entry through dynamin independent route promotes enhanced intracellular persistence., Adoption of an endocytosis route promoting safe intracellular trafficking is a pre-requisite for development of invasive diseases by Streptococcus pneumoniae (SPN). We aim to explore the contribution of various endocytic routes in internalization and survival of SPN in blood brain barrier (BBB), a key event in development of pneumococcal meningitis. Pneumococcal entry and survival in brain endothelial cells were evaluated following treatment with combinations of inhibitors to block multiple endocytosis pathways leaving a single entry port open. Entry of SPN into brain endothelium through a novel dynamin independent pathway dictates a separate downstream trafficking itinerary. This allows SPN to evade lysosomal degradation, potentially promoting safe transit across BBB, leading to development of meningitis., Graphical abstract Image, graphical abstract

Iron is an essential micronutrient for all bacteria but presents a significant challenge given its limited bioavailability. Furthermore, iron’s toxicity combined with the need to maintain iron levels within a narrow physiological range requires integrated systems to sense, regulate and transport a variety of iron complexes. Most bacteria encode systems to chelate and transport ferric iron (Fe(3+)) via siderophore receptor mediated uptake or via cytoplasmic energy dependent transport systems. Pathogenic bacteria have further lowered the barrier to iron acquisition by employing systems to utilize haem as a source of iron. Haem, a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such pathogenic bacteria have evolved sophisticated cell surface signaling (CSS) and transport systems to sense and obtain haem from the host. Once internalized haem is cleaved by both oxidative and non-oxidative mechanisms to release iron. Herein we summarize our current understanding of the mechanism of haem sensing, uptake and utilization in Pseudomonas aeruginosa, its role in pathogenesis and virulence, and the potential of these systems as antimicrobial targets.

Clathrin mediated endocytosis is one pathway for internalization of extracellular nano materials into cells [1, 2]. In this pathway, proteins attached to receptors and the internalized materials are encapsulated in clathrin coated membrane vesicles that subsequently fuse with or transform into intracellular compartments (early and late endosomes) as their contents are being directed to the lysosomes for degradation. The following proteins are commonly used to mark the pathway at various stages: Rab5 (early endosome), Rab7 (late endosome), and LAMP-1 (lysosome). In this work, we studied the distribution and co-localization of these marker proteins in two cell lines (C2C12 and A549) to determine whether these markers are unique for specific endosome types or whether they can co-exist with other markers. We estimate the densities and sizes of the endosomes containing the three markers, as well as the number of marker antibodies attached to each endosome. We determine that the markers are not unique to one endosome type but that the extent of co-localization is different for the two cell types. In fact, we find endosomes that contain all three markers simultaneously. Our results suggest that the use of these proteins as specific markers for specific endosome types should be reevaluated. This was the first successful use of triple image cross correlation spectroscopy to qualitatively and quantitatively study the extent of interaction among three different species in cells and also the first experimental study of three-way interactions of clathrin mediated endocytic markers.

Dendritic cells (DCs) play a complex role in the progression of myocardial infarction (MI). The impact of angiotensin-converting enzyme (ACE) inhibitor therapy, partly via affecting DCs maturation and recruitment, was tested on a MI mouse model. Furthermore, the cardioprotective effects of ACEI were enhanced through attenuating migration of DCs from the spleen into peripheral circulation, thereby inhibiting DCs maturation and tissue inflammation. ACEI repress DCs immune inflammatory response through down-regulating DCs maturation surface markers and regulating inflammatory cytokines, which led to a higher survival rate, improved function and remodeling through decreased inflammatory response after MI. However, inhibition of AT2R activation, resulted in a reduction of ACEI effects on DCs. The potent anti-inflammatory effect of ACEI can partially be attributed to its impact on DCs through activation of AT2R, which may provide a new target mechanism for ACEI therapy after MI.

SummarySelective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity.

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.

SummaryCells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.

The blood–brain barrier (BBB) remains a formidable challenge in the treatment of neurological diseases and cancers, limiting the permeability of therapeutics and preventing them from reaching their targets in effective concentrations. The BBB, formed at the level of the endothelial cells (ECs) of the cerebral capillaries by two plasma membranes in series, differs from capillaries in the periphery by increased mitochondrial content, a lack of fenestrations, minimal pinocytotic activity, and the presence of tight junctions. The ECs are surrounded by pericytes and astroglial foot processes, forming a continuous anatomical and enzymatic barrier. Understanding the physiology of the BBB and the mechanism of permeation across the BBB is critical in overcoming this bottleneck in development and in designing effective and safe treatments for the rising number of patients suffering from neurological diseases and cancers.

Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD–EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.

It was recently reported that liposomes modified with octaarginine (R8) and its analogue peptide (IRQRRRR: IRQ) are taken up by NIH3T3 cells by unique pathways, macropinocytosis and caveolae-mediated endocytosis, respectively. This study evaluated the topology of these peptides as it relates to the uptake routes of liposomes, where they are modified either directly on the surface, or on the edge of a polyethylene glycol (PEG) spacer. The uptake mechanism of peptide-modified liposomes and peptide-modified PEG-liposomes was investigated by confocal laser scanning microscopy. To determine the contribution of clathrin-mediated endocytosis, macropinocytosis and caveolar endocytosis to the uptake of liposomes, uptake was evaluated in the presence of some specific inhibitors of these processes. The uptake pathway changed from macropinocytosis to clathrin-mediated endocytosis when R8 was modified on the edge of a PEG spacer, indicating that the flexible display of R8 impaired the induction of macropinocytosis. However, the contribution of caveolae-mediated endocytosis increased when IRQ was conjugated to the distal end of the PEG chain, suggesting that flexible surface display enhanced IRQ recognition by the specific molecules in the caveolae. The present results demonstrate that topology control by the ligand affects the contribution of the entry pathway, depending on the uptake mechanism.

Endocytosis is responsible for the movement of materials from outside the cell into the cytoplasm. Cells use phagocytosis to engulf large particulate material. The case study, “Following the Fate of a Phagosome,” examines work by C.D. Blanchette, Y.-H. Woo, C. Thomas, N. Shen, T.A. Sulchek, and A.L. Hiddessen (Decoupling internalization, acidification and phagosomal-endosomal/lysosomal fusion during phagocytosis of InlA coated beads in epithelial cells. PLoS One. 2009;4:e6056) investigating the internalization, acidification, and fusion steps of phagocytosis. Receptor-mediated endocytosis is responsible for the uptake of extracellular materials based on receptor ligand binding. This type of endocytosis is associated with the formation of clathrin-coated pits that become clathrin-coated vesicles. The work by W. Boll, A. Gallusser, and T. Kirchhausen (Role of the regulatory domain of the EGF-receptor cytoplasmic tail in selective binding of the clathrin-associated complex AP-2. Curr Biol. 1995;5:1168–1178) is used in the case study, “Catching a Receptor by the Tail,” to investigate the role of the receptor in clathrin coat assembly. The case study, “Can Clathrin Bend a Membrane?” evaluates data from work by P.N. Dannhauser and E.J. Ungewickell (Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat Cell Biol. 2012;14:634–639) which supports the idea that clathrin alone can cause membrane deformation. Finally, the case study, “Modeling Membrane Fission,” looks at the role of the protein dynamin in endocytosis (Pucadyil TJ, Schmid SL. Real-time visualization of dynamin-catalyzed membrane fission and vesicle release. Cell. 2008;135:1263–1275).

Adenoviruses contain an icosahedral coat and a nucleoprotein core of viral deoxyribonucleic acid. Mastadenoviruses infecting mammals are composed of several hundred different types. Human adenoviruses are the most widely used vectors in clinical applications, mainly for killing cancer cells and vaccinations. They are physically and genetically stable and are safe for use in humans. We discuss the state of the art in adenovirus entry, a key process for infection and immunity. Entry requires hundreds of host factors, but mechanisms are known for only some of them. It involves coating the virus with soluble serum or tissue factors, using receptors that initiate stepwise uncoating, signaling, endocytic uptake, and lysis of the endosomal membrane. Cytoplasmic transport and immunity, together with complete uncoating, determine nuclear import and transcriptional activation of the viral genome. We conclude with perspectives and a discussion of outstanding questions in the field.

If one were able to sit inside a cell and look around, one would be witness to a bewildering array of every type of life component (proteins, lipids, nucleic acids, membrane vesicles, etc.) zipping around in all directions, heading to specific cellular locations. It would first appear that everything is in total chaos, but on closer examination, well-designed patterns would emerge. Movement of membrane-derived vesicles, referred to as “membrane trafficking,” is of particular interest as it forms the heart of the endo-membrane concept. Three major patterns will be discussed here: endo-membrane flow (for secretion), receptor-mediated endocytosis, and intracellular carrier proteins.

Clathrin is the prototype self-assembling protein that coats transport vesicles during membrane traffic. Its ability to polymerize into a polyhedral lattice contributes to organization and sorting of integral membrane proteins during receptor-mediated endocytosis, organelle biogenesis, and selected recycling pathways and degradation pathways. The morphology, structure, and biochemistry of clathrin is described with a focus on how these properties contribute to clathrin's cellular functions and their regulation. The interactions of the clathrin light chain subunits with actin-organizing proteins and with the central portion of the clathrin triskelion defines a role for these subunits in contributing stability and strength to the clathrin lattice, functions that expand the repertoire of clathrin-transported cargo and facilitate a role for the clathrin lattice in organizing the actin cytoskeleton. With the discovery of a second form of clathrin in humans and a non-membrane traffic role for clathrin at the mitotic spindle, the diversity of intracellular functions ascribed to clathrin proteins now extends to specific roles in human glucose metabolism and in mitosis, in addition to classical clathrin-mediated pathways.

Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.

Free to read at publisher's site. Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions.

Since amyloid beta-protein (A beta) is the primary component of both vascular and parenchymal amyloid deposits in Alzheimer's disease, information regarding its permeability at the blood-brain barrier (BBB) will help elucidate the contribution of circulating A beta to vascular and parenchymal A beta deposition in this disease and in brain aging. The permeability of the D- and L-enantiomers of A beta 1-40 and L-A beta 1-42 at the BBB was determined in the normal adult rat by quantifying the permeability coefficient-surface area product (PS) for each protein after correction for the residual plasma volume (Vp) occupied by the protein [labeled with a different isotope of iodine (125I vs 131I)] in blood vessels of different brain regions. After a single i.v. bolus injection, the plasma pharmacokinetics determined by TCA precipitation, paper chromatography, and SDS-PAGE were similar for both 125I-L-A beta 1-40 and 125I-L-A beta 1-42. The PS at the BBB for L-A beta 1-42 was significantly (1.4- to 1.8-fold) higher than for L-A beta 1-40 and ranged from 17.7 to 26.4 x 10(-6) ml/g/s for different brain regions. A comparison of the PS values at the BBB for L-A beta 1-40 showed no significant difference when determined at 15 or 30 min after i.v. bolus injection, times that reflect different levels of degradation in plasma (37.9% at 15 min and 65.5% at 30 min). The PS values obtained, therefore, were representative of the intact protein rather than degradation products. The PS values obtained for the all-D-enantiomer of A beta 1-40 were very low and comparable to that of albumin and IgG, whose mechanism of transport is by passive diffusion. Taken together, these data imply a stereoisomer-specific, ligand-receptor interaction at the BBB for the L-A beta proteins. The high PS values observed for L-A beta 1-40 and 1-42 compare to insulin, whose uptake is decidedly by a receptor-mediated transport process, and suggest a similar mechanism for L-A beta entry into the brain.

During hypothermic preservation of cells (0-4°C), metab¬olism is diminished and energy-dependent transport processes are arrested. The effect of hypothermic preservation of hepatocytes in endocytic transport following rewarming has not been previously reported. We evaluated the uptake of EGF (Epidermal Growth Factor) ligand conjugated to fluorescent Quantum Dots (QDs) probes in rat hepatocytes after 24 and 72 h cold storage in University of Wisconsin (UW) solution at 4°C. QDs uptake was visualized during rewarming to 37°C under air or, in a second approach, at the end of rewarming under 5% CO2. After 24 h in UW solution, QDs were internalized under both rewarming conditions similar to non-preserved hepatocytes and cells maintained a normal cytoskeleton distribution. However, in hepatocytes preserved 72 h none of the cells internalized QDs, which remained bound to the membranes. After rewarming, this group showed diminished actin staining while viability was maintained at ~70%. Our results present evidence that, hypothermic preservation for 72 h in UW solution at 4°C does not prevent EGFR (Epidermal Growth Factor Receptor) activation but irreversibly impairs endocytic uptake upon EGF stimulation; presumably due to actin cytoskeleton disassembling. Our approach can be applied on other membrane receptor systems and with other hypothermic preservation solutions to understand the effect of cooling in endocytic transport and to determine the optimal cold storage period. Fil: Hovanyecz, P.. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina Fil: Guibert, Edgardo Elvio. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pellegrino, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina Fil: Rodriguez, Joaquin Valentin. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina Fil: Sigot, Valeria. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina

Cell-free assays represent an important complement to studies in living cells for the elucidation of mechanisms underlying the dynamics of biological membranes, such as budding, fission, and fusion reactions. Here we describe a method for the reconstitution of endocytosis, the process through which cells internalize portions of the plasma membrane along with extracellular material, under conditions that allow the visualization of individual budding events with high spatial and temporal resolution. The method, which is based on the generation of planar plasma membrane sheets attached to a glass substrate and their subsequent incubation with a cytosolic extract, results in a very robust formation of endocytic buds, which upon appropriate conditions undergo fission. The synchronization of the endocytic events and the accessibility of the material to a variety of manipulations make this experimental system a powerful tool for the molecular dissection of endocytosis.

Activated cell surface receptors are rapidly removed from the plasma membrane through clathrin mediated endocytosis and transported to the endosome where they are either recycled or sorted to the lysosomal pathway to be degraded. Receptors, destined for degradation in the lysosome, are packaged into intraluminal vesicles (ILVs) of endosomes by a reaction that is topologically unrelated to other budding reactions in cells. First, receptors are clustered at the endosomal membrane and receptor-rich membrane patches then bud towards the lumen of the endosome. The nascent membrane buds are finally cleaved from the limiting membrane to release cargo-bearing vesicles into the endosomal interior. The molecular machinery that drives multivesicular body biogenesis, the endosomal sorting complex required for transport (ESCRT) machinery, has been identified through genetic screens. It consists of the cytoplasmic, hetero-multimeric complexes ESCRT-0, -I, -II, and -III, and of the Vps4/VtaI complex. Although the ESCRT machinery has been characterized extensively using cell-biological and biochemical approaches, the molecular mechanism of multivesicular body biogenesis remained unclear. In this chapter, I will present in vitro reconstitution systems that we used to study ESCRT-driven membrane remodeling reactions with purified components on artificial membranes. This includes generation of large and giant unilamellar liposomes, as well as in vitro reconstitution reactions of fluorescently labeled proteins on such membranes. I will discuss both, the potential of in vitro systems to analyze membrane-remodeling events and also their limitations.

Overlap of lipid composition exists among high-density lipoprotein (HDL), low-density lipoprotein (LDL), modified LDL, lipid-coated microbubble (LCM), and dispersed lipid-mesophase nanoparticle (LMN). This overlap suggests that LCM and/or dispersed LMN themselves can act as ligands for multiligand lipoprotein receptors. In particular, the multiligand scavenger receptor (i.e., SR-BI) is known to bind both HDL and LDL particles. Accordingly, SR-BI represents the most likely candidate involved in enhanced endocytosis of “LCM/nanoparticle-derived” lipid nanoemulsions (i.e., mostly dispersed LMN) into tumor cells. Moreover, the overlap of lipid composition between HDL, LDL, modified LDL, LCM, and LMN can partially mimic the known heterogeneity (i.e., subpopulations or subspecies) of LDL and HDL particles.

Besides tumors, some other (noncancerous) lesion/injury sites involving hyperproliferative disease processes (such as atherosclerosis as well as traumatic neuroinjury sites) often display an overexpression of cell-surface lipoprotein receptors (i.e., SR-BI and others). Accordingly, targeted drug-delivery therapy of that given hyperproliferative disease or injury site, via “LCM/nanoparticle-derived” nanoemulsions (such as Filmix ® nanoemulsion), becomes quite practicable. A rather similar therapeutic capability, therefore, would also be expected for a few other types of parenteral nanoemulsions known to actively target lipoprotein receptors. Finally, the related intellectual property, key lipid components, field of use, and/or commercialization paths of these separate technologies are also compared herein.

A renewed interest in targetable lipid-based drug-delivery systems concerns solubilizing lipophilic drugs. One example is a tocol nanoemulsion to deliver paclitaxel, without any toxic solvents; this paclitaxel formulation can passively accumulate in tumors (EPR effect). Also under clinical study is a parenteral cholesterol-rich/phospholipid nanoemulsion (termed “LDE”), which undergoes active uptake into cancer cells that overexpress lipoprotein receptors. However, LDE nanoemulsion requires derivatization of the drug to stabilize the LDE–paclitaxel complex. Another lipid nanoemulsion that actively targets tumors, after intravenous injection, is the “lipid-coated microbubble (LCM)/nanoparticle-derived” drug-delivery agent. It is produced by a commercial-scale method involving no chemical derivatization, of paclitaxel, at all.

As concerns the actual endocytic pathways likely to be involved in lipid-coated microbubble (LCM) uptake by tumors, a few reasonable candidates emerge upon reviewing parts of an extensive research literature describing significantly enhanced, receptor-mediated endocytosis in many different cancerous cells and solid tumors. Basically, the LCM lipid formulation actively targets drug (e.g., paclitaxel) to tumor cells that commonly overexpress certain surface receptors, that is, “lipoprotein receptors.” In addition, there are several other (noncancerous) lesion/injury sites involving hyperproliferative diseases (e.g., neuroinjury [gliosis] sites and atherosclerosis) which include overexpression of cell-surface lipoprotein receptors; therefore, these sites are also suitable for targeted chemotherapy via LCM.

Publisher Summary This chapter covers the cell biology evidence establishing the significance of actin dynamics for the correct execution of endocytosis and emphasizes the molecular connections between the endocytic and actin machineries. It also describes results showing how endocytosis itself controls various forms of actin dynamics, especially those required for the spatial restriction of signaling events. The minimal requirement to support cycles of actin polymerization and depolymerization have been identified through in vitro reconstitution experiments. These experiments showed that five different types of purified proteins are sufficient to generate forces for motility, including de novo actin nucleators and their regulators (Arp2/3 complex and N-WASP), actin severing/depolymerizing factors, barbed end cappers, and an ATP-actin monomeric binding protein (profilin). In mammalian cells, critical components of the actin dynamics machinery such as the Arp2/3 complex, N-WASP, and the capping protein Eps8 are also transiently recruited to clathrin-positive endocytic sites, and their ablation impairs clathrin mediated endocytosis to various extents. Rab5 is a master regulator of endocytosis and endosomal dynamics. Rab5, however, is also involved in the control of actin dynamics. This dual function is underscored by a complex system of regulation, in which several activators of Rab5 (including the GEFs RIN1, Rabex-5, and Alsin) are also involved in the control of actin dynamics. RIN1 is an effector of Ras and binding to Ras enhances the Rab5 specific GEF activity of RIN1 that mediates EGFR mediated Rab5 activation via Ras in vivo. A more direct way of achieving Rab5 activation by surface receptors is through Rabex-5 that possesses two ubiquitin binding domains, which bind ubiquitinated proteins including EGFR.

Lipid rafts are plasma membrane microdomains enriched in cholesterol and sphingolipids that are involved in the lateral compartmentalization of molecules at the cell surface. Internalization of ligands and receptors by these domains occurs via a process defined as raft-dependent endocytosis. Caveolae are caveolin-1-enriched smooth invaginations of the plasma membrane that form a subdomain of lipid rafts. Endocytosis of rafts, including caveolar but also noncaveolar dynamin-dependent and dynamin-independent pathways, is characterized by its cholesterol sensitivity and clathrin-independence. In this review we will characterize lipid rafts and caveolae, their endocytosis and its regulation by the actin cytoskeleton, caveolin-1, dynamin, and cholesterol.

This chapter describes the various emerging evidence supporting the involvement of actin dynamics in endocytosis. It also briefly discusses the possible roles for microtubules during the initial and later phases of the endocytic process and describes the known molecular circuitries that link and integrate endocytosis and actin dynamics. Endocytosis of membrane receptors is governed by a complex structural machinery, which is in turn controlled by various accessory regulatory proteins. Links between endocytosis and the actin cytoskeleton have been revealed by a number of studies in yeast A variety of the so-called "end" or "dim" mutations, isolated in screening for endocytic defects. Various activators of Arp2/3, including Las17/Beelp, Abpl, and the type I myosin Myo3/5p have been reported to affect endocytosis to various extents. The role of microtubules in the endocytic process has been established mainly with the use of microtubule depolymerizing drugs, such as nocodazole. In cells kept in suspension, but not in cells grown on substrates, nocodazole was reported to reduce by 40% the initial rate of transferrin receptor internalization. This suggests that the overall structural organization of a cell might affect microtubule organization and dynamics, leading to alterations in membrane plasticity, and consequently affecting the ability of a receptor to be recruited to the invaginating clathrin-coated pit.

Publisher Summary The liver is an excellent organ for gene transfer in treating a wide variety of diseases that affect liver function. It is an ideal organ for a high amount of expression of therapeutic genes and efficient systemic distribution of the resulting therapeutic proteins secreted into the bloodstream. For strategies of liver-destined gene therapy, the liver sinusoid endothelium contains pores with a mean diameter of 100 nm, which allow small vectors to leave the blood circulation and reach the hepatocytes. The preparation of asialofetuin–liposomes targeted to hepatocytes can be made by covalent coupling of asialofetuin glycoprotein (ASF) onto the liposome surface, by the use of heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Changes in liposome charge can be made by inclusion on the liposome backbone of anionic or cationic lipids, cationic lipopeptides, and cationic polymers. Because the nuclear localization signal (NLS) is also a cationic polypeptide, it can interact directly with DNA by electrostatic interaction, increasing the nuclear availability of delivered DNA and thereby contributing to increase the efficacy or gene transfer.

Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

Publisher Summary This chapter describes the mechanism of macromolecular transport in newborn animals, focusing on the factors regulating gut closure. The neonatal mammalian small intestine has the capacity to transport macromolecules derived from breast milk. The transported macromolecules are biologically important in the neonate especially in hypogammaglobulinemic animals. Transport of macromolecules follows two different pathways: specific receptor mediated transcytosis and nonspecific transcytosis. Milk-born lactoferrin may have effects such as influence on iron metabolism, antibacterial action, mitogenic effects, and actions to potentiate the immunological system. The factors regulating gut closure are epidermal growth factor (EGF), insulin, secretin, prostaglandin, polyamines, and dietary fibre. The transfer of some macromolecules into the CSF via the blood circulation in suckling neonatal pigs is discussed in the chapter. The passage of macromolecules present in plasma into the brain is limited by the blood–brain barrier and blood cerebrospinal fluid barrier. It remains for future work to clarify if novel agents derived from milk contribute to the improvement of central nervous functions.

Summary At the start of the 1980s (Goldstein et al., 1979) it was known that receptor mediated endocytosis was a very efficient and specific means of taking proteins into cells. In addition, several high affinity receptors able to concentrate in coated pits, and recycle through the endocytic pathway, had been characterized (Steinman et al., 1983). At that time, the molecules regulating endocytosis, such as clathrin, adaptin and ATP-dependent proton pumps were beginning to be isolated. Since then, a rapid expansion of recombinant DNA technology, and the development of assays that reconstitute endocytosis in vitro , have allowed major advances in the understanding of endocytosis to be made. Recombinant DNA methods have revealed the primary amino acid sequences of many receptors that enter cells by endocytosis. Internalization or, sorting motifs, have been identified in the cytoplasmic tails of receptors. Internalization motifs bind to adaptin complexes, and adaptin seeds the assembly of clathrin coats on the cytoplasmic face of the plasma membrane. Taken together, these data provide a molecular basis for the clustering of receptors in coated pits. Structural rearrangements within the clathrin lattice then cause the invagination of the coated pit, and the formation of the coated vesicle. Clathrin coats are removed by an uncoating ATPase, and uncoated vesicles fuse with endosomes. The lumen of endosomes is acidic. This acid environment, which is maintained by an ATP-dependent proton pump, is essential for the physical dissociation of receptor-ligand complexes. Sorting of receptors and ligands is further enhanced by the movement of receptors into tubular endosomal elements that recycle receptors to the plasma membrane. Membrane fusion throughout the endocytic pathway requires ATP, GTP, Ca 2+ , and cytosolic proteins. Many of these cytosolic proteins are required for membrane fusion in general, and are used in vesicular transport steps throughout the cell. Specific cytosolic proteins, called small GTP-binding proteins, or rabs, however, regulate the specificity of membrane fusion. Rabs 4 and 5 bind GTP and act as molecular switches that regulate the formation and recycling of endocytic vesicles. The formation of coated vesicles, and their fusion with early compartments of the endocytic pathway, may also require dynamin. Fusion of endosomes with lysosomes may require the additional help of microtubules. A specialized form of endocytosis, called potocytosis, has been characterized recently. Ligands taken up by potocytosis bind to GPI-anchored receptors that concentrate in specialized domains of the plasma membrane called caveolae. Caveolae are coated with caveolin and invaginate to form a membrane compartment that is sealed off from the extracellular fluid. Caveolae do not pinch off from the cell surface and concentrate and process molecules within specialized domains of the plasma membrane. Caveolae may be used to take up low molecular weight ligands, such as vitamins, into cells, or for the localized generation of extracellular signaling molecules.

Publisher Summary This chapter discusses the receptor-mediated endocytosis. Receptor-mediated endocytosis is a mechanism used by cells for the internalization of selected plasma-membrane proteins. Many of these proteins are cell-surface receptors for extracellular macromolecules—for example, transferrin, low-density lipoprotein (LDL), insulin, asialoglycoprotein in hepatocytes, and antigen-bound immunoglobulin in B-lymphocytes. Endocytosis thus serves as a mechanism for the uptake of essential macromolecules and connects the outside world with the internal compartments of the cell. In the first step of the process, a ligand binds to a receptor on the plasma membrane. The receptor-ligand complexes become concentrated to the exclusion of other membrane proteins in specialized regions called coated pits. As the coated pit grows, it invaginates and detaches to form a coated vesicle. The rapid removal of the coat allows the vesicle to fuse with the endosome and further processing of receptor-ligand complex to proceed. Further, coated vesicles play additional roles within the cell. Coated vesicles are responsible for the export of lysosomal enzymes from the trans-Golgi network and have been implicated in the early events of regulated secretion. Under the electron microscope, the coated vesicle reveals a remarkable closed hexagonal and pentagonal lattice-like structure. The coated vesicle is composed of two discrete layers of coat proteins surrounding an inner layer of membrane-bound receptors.