Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization

The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.     

AMP-Activated Protein Kinase Is Required for the Macropinocytic Internalization of Ebolavirus

Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute''s NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo. Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.     

Interaction of Cowpea Mosaic Virus (CPMV) Nanoparticles with Antigen Presenting Cells In Vitro and In Vivo

Background

Plant viruses such as Cowpea mosaic virus (CPMV) are increasingly being developed for applications in nanobiotechnology including vaccine development because of their potential for producing large quantities of antigenic material in plant hosts. In order to improve efficacy of viral nanoparticles in these types of roles, an investigation of the individual cell types that interact with the particles is critical. In particular, it is important to understand the interactions of a potential vaccine with antigen presenting cells (APCs) of the immune system. CPMV was previously shown to interact with vimentin displayed on cell surfaces to mediate cell entry, but the expression of surface vimentin on APCs has not been characterized.

Methodology

The binding and internalization of CPMV by several populations of APCs was investigated both in vitro and in vivo by flow cytometry and fluorescence confocal microscopy. The association of the particles with mouse gastrointestinal epithelium and Peyer''s patches was also examined by confocal microscopy. The expression of surface vimentin on APCs was also measured.

Conclusions

We found that CPMV is bound and internalized by subsets of several populations of APCs both in vitro and in vivo following intravenous, intraperitoneal, and oral administration, and also by cells isolated from the Peyer''s patch following gastrointestinal delivery. Surface vimentin was also expressed on APC populations that could internalize CPMV. These experiments demonstrate that APCs capture CPMV particles in vivo, and that further tuning the interaction with surface vimentin may facilitate increased uptake by APCs and priming of antibody responses. These studies also indicate that CPMV particles likely access the systemic circulation following oral delivery via the Peyer''s patch.     

Baculovirus GP64-mediated entry into mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.     

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.     

Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis

The rates of internalization and uncoating of 32P-labelled human immunodeficiency virus (HIV) in the human T lymphoid cell line CEM are consonant with a receptor-mediated endocytosis mechanism of entry. This interpretation was affirmed by electron microscopic observation of virions within endosomes. Virus binding and infectivity were inhibited to the same extent by pretreatment with OKT4A antibody, therefore, the CD4 receptor-dependent pathway of internalization appears to be the infectious route of entry. The pattern of internalization by the human monoblastoid cell line U937 proved to be more complex, involving rapid and efficient CD4-independent internalization. Electron microscopy revealed the presence of large intracellular vesicles, each containing several virions. Antibody against the CD4 receptor for virus efficiently blocked infection, but did not reduce significantly HIV binding or internalization in the U937 cell line. Consequently, U937 cells have a CD4-independent pathway of virus internalization that does not coincide with the route of entry for infectious HIV.     

EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Kaposi''s sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2''s tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.     

Trypanosoma cruzi uses macropinocytosis as an additional entry pathway into mammalian host cell

Several intracellular pathogens are internalized by host cells via multiple endocytic pathways. It is no different with Trypanosoma cruzi. Evidences indicate that T. cruzi entry may occur by endocytosis/phagocytosis or by an active manner. Although macropinocytosis is largely considered an endocytic process where cells internalize only large amounts of solutes, several pathogens use this pathway to enter into host cells. To investigate whether T. cruzi entry into peritoneal macrophages and LLC-MK2 epithelial cells can be also mediated through a macropinocytosis-like process, we used several experimental strategies presently available to characterize macropinocytosis such as the use of different inhibitors. These macropinocytosis' inhibitors blocked internalization of T. cruzi by host cells. To further support this, immunofluorescence microscopy and scanning electron microscopy techniques were used. Field emission scanning electron microscopy revealed that after treatment, parasites remained attached to the external side of host cell plasma membrane. Proteins such as Rabankyrin 5, tyrosine kinases, Pak1 and actin microfilaments, which participate in macropinosome formation, were localized at T. cruzi entry sites. We also observed co-localization between the parasite and an endocytic fluid phase marker. All together, these results indicate that T. cruzi is able to use multiple mechanisms of penetration into host cell, including macropinocytosis.     

Movement of plasma-membrane-associated clathrin spots along the microtubule cytoskeleton

The current understanding of the role of plasma- membrane-associated clathrin suggests that clathrin-coated pits form at the sites of activated receptors and then, following internalization, the clathrin coat is rapidly shed. Utilizing total internal reflection fluorescence microscopy (TIR-FM), we have documented linear lateral motion of cell-surface-associated dsRed-clathrin spots parallel to the plasma membrane. Clathrin spot motility was observed in multiple cell lines (MDCK, CHO, Cos-7 and HeLa). In MDCK cells dsRed-clathrin spots moved along linear pathways up to 4 μm in length with rates of approximately 0.8 μm/s. Spots did not generally undergo internalization during movement. The motion of these puncta was coincident with the microtubule cytoskeleton, and depolymerization of microtubules reduced spot motility over 10-fold. Over-expression of the microtubule-associated protein tau-EGFP decreased spot run length by 40% without affecting the rate of movement. Thus dsRed-clathrin puncta move along the microtubule cytoskeleton parallel to the cell surface.     

A role for ActA in epithelial cell invasion by Listeria monocytogenes

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes . An in frame Δ actA mutant was constructed in a hyperinvasive strain of prfA * genotype, in which all genes of the PrfA-dependent virulence regulon, including actA , are highly expressed in vitro . Loss of ActA production in prfA * bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the Δ actA prfA * mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA * bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro ) or prfA *Δ actA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA * strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.     

Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1-mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti-β1 integrin monoclonal antibody. In the mouse cell line GD25, a β1 null mutant, the αvβ3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to αvβ3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N-terminal fragments or the 120 kDa cell-binding fragment of Fn promote bacterial entry. Fn-coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn-coated beads are blocked by anti-β1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.     

Amiloride causes changes in the mechanical properties of hair cell bundles in the fish lateral line similar to those induced by dihydrostreptomycin

Amiloride is a known blocker of the mechano-electrical transduction current in sensory hair cells. Measurements of cupular motion in the lateral line organ of fish now show that amiloride concurrently changes the micromechanical properties of the hair cell bundles. The effects of amiloride on the mechanics and receptor potentials of the hair cells resemble those previously observed for the aminoglycoside drug dihydrostreptomycin (DHSM) and are similarly antagonized by Ca²⁺. We hypothesize that amiloride and DHSM act on hair cells in two correlated ways which manifest themselves in both the electrical and mechanical properties of the transduction process. One action is the reduction of the transduction current with a concurrent increase of the hair bundle stiffness. The other action is a shift of the hair cell''s operating point on a current–displacement curve, with a concomitant shift along the associated hair bundle stiffness–displacement curve. The latter action has the opposite effect to that of the first and thus may lead, at relatively low blocker concentrations, to both an increase of transduction current and a decrease in hair bundle stiffness.     

N-formyl peptide receptors in human neutrophils display distinct membrane distribution and lateral mobility when labeled with agonist and antagonist

Receptors for bacterial N-formyl peptides are instrumental for neutrophil chemotactic locomotion and activation at sites of infection. As regulatory mechanisms for signal transduction, both rapid coupling of the occupied receptor to cytoskeletal components, and receptor lateral redistribution, have been suggested (Jesaitis et al., 1986, 1989). To compare the distribution and lateral diffusion of the nonactivated and activated neutrophil N-formyl-peptide receptor, before internalization, we used a new fluorescent N-formyl-peptide receptor antagonist, tertbutyloxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH (Boc- FLFLF, 0.1-1 microM), and the fluorescent receptor agonist formyl-Nle- Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK, 0.1-1 microM). Fluorescent Boc-FLFLF did not elicit an oxidative burst in the neutrophil at 37 degrees C, as assessed by chemiluminescence and reduction of p-nitroblue tetrazolium chloride, but competed efficiently both with formyl-methionyl-leucyl- phenylalanine (fMLF) and fnLLFnLYK. It was not internalized, as evidenced by confocal microscopy and acid elution of surface bound ligand. The lateral mobility characteristics of the neutrophil fMLF receptor were investigated with the technique of FRAP. The diffusion coefficient (D) was similar for antagonist- and agonist-labeled receptors (D approximately 5 x 10(-10) cm2/s), but the fraction of mobile receptors was significantly lower in agonist- compared to antagonist-labeled cells, approximately 40% in contrast to approximately 60%. This reduction in receptor mobile fraction was slightly counteracted, albeit not significantly, by dihydrocytochalasin B (dhcB, 5 microM). To block internalization of agonist-labeled receptors, receptor mobility measurements were done at 14 degrees C. At this temperature, confocal microscopy revealed clustering of receptors in response to agonist binding, compared to a more uniform receptor distribution in antagonist-labeled cells. The pattern of agonist- induced receptor clustering was less apparent after dhcB treatment. To summarize, this work shows that activated N-formyl peptide receptors aggregate and immobilize in the plane of the neutrophil plasma membrane before internalization, a process that is affected, but not significantly reversed, by cytochalasin. The results are consistent with a model where arrested receptors are associated mainly with a cytochalasin-insensitive pool of cytoskeletal elements.     

The Ubiquitin/Proteasome System Mediates Entry and Endosomal Trafficking of Kaposi's Sarcoma-Associated Herpesvirus in Endothelial Cells

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi''s sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process.     

Membrane destabilizing properties of cell-penetrating peptides

Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R(7)W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thoren et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R(7)W. In this paper, the interactions of penetratin, R(7)W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R(7)W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.     

Extended exposure to substrate regulates the human equilibrative nucleoside transporter 1 (hENT1)

ABSTRACT

Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 μM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.     

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.