Early Endosomal Compartments Research Articles

Impaired endocytosis and accumulation in early endosomal compartments defines herpes simplex virus–mediated disruption of the nonclassical MHC class I–related molecule MR1

Presentation of metabolites by the Major Histocompatibility Complex Class-I-related protein 1 (MR1) molecule to Mucosal-Associated Invariant T (MAIT) cells is impaired during herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. This is surprising given these viruses do not directly synthesise MR1 ligands. We have previously identified several HSV proteins responsible for rapidly downregulating the intracellular pool of immature MR1, effectively inhibiting new surface antigen presentation, while pre-existing ligand-bound mature MR1 is surprisingly upregulated by HSV-1. Using flow cytometry, immunoblotting and high throughput fluorescence microscopy we demonstrate that the endocytosis of surface MR1 is impaired during HSV infection, and that internalised molecules accumulate in EEA1-labelled early endosomes, avoiding degradation. We establish that the short MR1 cytoplasmic tail is not required for HSV-1 mediated downregulation of immature molecules, however it may play a role in the retention of mature molecules on the surface and in early endosomes. We also determine that the HSV-1 US3 protein, the shorter US3.5 kinase and the full-length HSV-2 homolog, all predominantly target mature surface rather than total MR1 levels. We propose that the downregulation of intracellular and cell surface MR1 molecules by US3 and other HSV proteins is an immune-evasive countermeasure to minimise the effect of impaired MR1 endocytosis, which might otherwise render infected cells susceptible to MR1-mediated killing by MAIT cells.

RPG interacts with E3-ligase CERBERUS to mediate rhizobial infection in Lotus japonicus.

Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation.

Multifaceted Roles of Retromer in EGFR Trafficking and Signaling Activation.

Mammalian retromer complex contributes to multiple early endosome-associated trafficking pathways whose origins are dependent on which sorting nexin (SNX) they are complexed with. In an attempt to dissect out the contribution of individual retromer-SNX complexes, we examined the trafficking of EGFR in detail within a series of KO cell line models. We demonstrated that the depletion of retromer subunit Vps35 leads to decreased EGFR protein levels in resting cells with enhanced association of EGFR with lysosomal compartments. Compared to control cells, the addition of EGF to Vps35 KO cells resulted in a reduced rate of EGFR degradation; AKT activation and cell prolferation rates were elevated, while ERK activation remained relatively unchanged. These observations are consistent with a prolonged temporal association of EGFR within early endosomes due to the inefficiency of early endosome-associated protein trafficking pathways or organelle maturation due to retromer absence. We did not fully delineate the discrete contributions from retromer-associated SNXs to the phenotypes observed from retromer Vps35 depletion. While each of the knock-outs of SNX1/2, SNX3, or SNX27 promotes the enhanced association of EGFR with early endosomal compartments, only the decreased EGF-mediated EGFR degradation was observed in SNX1/2 dKO cells, while the enhanced AKT activation was only increased in SNX3 KO or SNX27 KO cells. Despite this, each of the knock-outs showed increased EGF-stimulated cell proliferation rates.

Smoothened (SMO) regulates insulin-like growth factor 1 receptor (IGF1R) levels and protein kinase B (AKT) localization and signaling

AbstractThe oncoprotein Smoothened (SMO), a Frizzled-class-G-protein-coupled receptor, is the central transducer of hedgehog (Hh) signaling. While canonical SMO signaling is best understood in the context of cilia, evidence suggests that SMO has other functions in cancer biology that are unrelated to canonical Hh signaling. Herein, we provided evidence that elevated levels of human SMO show a strong correlation with elevated levels of insulin-like growth factor 1 receptor (IGF1R) and reduced survival in diffuse large B-cell lymphoma (DLBCL). As an integral component of raft microdomains, SMO plays a fundamental role in maintaining the levels of IGF1R in lymphoma and breast cancer cells as well IGF1R-associated activation of protein kinase B (AKT). Silencing of SMO increases lysosomal degradation and favors a localization of IGF1R to late endosomal compartments instead of early endosomal compartments from which much of the receptor would normally recycle. In addition, loss of SMO interferes with the lipid raft localization and retention of the remaining IGF1R and AKT, thereby disrupting the primary signaling context for IGF1R/AKT. This activity of SMO is independent of its canonical signaling and represents a novel and clinically relevant contribution to signaling by the highly oncogenic IGF1R/AKT signaling axis.The smoothened (SMO) receptor maintains levels of insulin-like growth factor 1 receptor (IGF1R) in several types of cancer cells, and high protein levels of both receptors correlate with poor survival in diffuse large B-cell lymphoma (DLBCL) patients. Loss of SMO favors IGF1R degradation over recycling, and IGF1R-mediated AKT/PI3K signaling, but MAPK is not significantly impaired. The preferential disruption of AKT signaling correlates with a loss of IGF1R and AKT in raft microdomains.

A novel live-cell imaging assay reveals regulation of endosome maturation.

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

Abstract LB187: EGFR signaling and pharmacology in oncology revealed with an innovative RTK biosensor technology

Abstract EGFR is involved in key biological processes and its deregulation is associated with the development of many cancers. EGFR is implicated in tumor invasion, metastasis and angiogenesis. Moreover, intrinsic and acquired mutations of EGFR have been described to modify receptor signaling and be responsible for the appearance of drug resistance during treatment in a clinical setting. Development of tools providing new insight into RTK complex mechanisms is crucial to develop more effective RTK-targeting drugs. In this study, we present a live-cell ebBRET-based biosensor platform which includes 12 distinct biosensors for monitoring SH2-domain containing proteins-mediated downstream signaling of RTKs, hence following the activation of MAPK, Akt and PKC pathways. These biosensors are designed to measure receptor proximal events that are engaged upon receptor activation at the plasma membrane (PM) and early endosomal compartments (EE). Using EGFR and two of its ligands as a model system, we showed the capacity of these biosensors to differentiate unique signaling signatures of EGFR, at the PM or the EE, with EGF and Epiregulin ligands displaying differences of efficacy and potency. Indeed, EGF was more potent and efficacious than Epiregulin on all the studied pathways. Also, we detected activities at the EE with EGF stimulation but not with Epiregulin. Our data highlight the platform's capacity to follow the trafficking of RTK biosensors into different compartments and to reveal internalization selectivity and bias which can be observed with different ligands. Overcoming resistance has become an important challenge when developing new therapeutics for the treatment of cancer. We further demonstrated that EGFR deletions or single point mutations, found in Gliobastoma or NSCLC, have an impact on the constitutive activity of EGFR and the signaling profiles but also on inhibitor efficacies which could impact receptor trafficking from the PM to the EE. In addition, the sensitivity of the RTK platform allowed us to measure the signaling of endogenously expressed EGFR in pathophysiologically-relevant cell lines commonly used for their oncogenic properties, such as A431 human epidermoid carcinoma cells, N87 gastric carcinoma cells and T47D, MCF7 and SK-BR-3 breast adenocarcinoma cells. Finally, we illustrated, using BRET-based imaging, the recruitment of SH2 effectors at the PM or at the EE after a 10-minute or 60-minute incubation respectively with EGF, highlighting the translatability of the biosensor platform to microscopy. The ebBRET-based biosensor technology displayed new insights in RTK biology and revealed different modes of action, extensive RTK signal profiling, and trafficking of RTK effectors. It represents a powerful tool for the analysis of RTK mutations and for the identification of novel generation of TKIs and antibodies directed against RTKs. Citation Format: Florence Gross, Guilhem Dugast, Arturo Mancini, Hiroyuki Kobayashi, Michel Bouvier, Stephan Schann, Xavier Leroy, Laurent Sabbagh. EGFR signaling and pharmacology in oncology revealed with an innovative RTK biosensor technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB187.

Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells.

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross‐presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome‐like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C‐type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen‐containing compartments lacked co‐expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single‐cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co‐localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S‐independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross‐presentation by DCs. In summary, our data suggest that these antigen‐containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long‐lasting antigen cross‐presentation.

The Drosophila amyloid precursor protein homologue mediates neuronal survival and neuroglial interactions.

The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimer's disease (fAD), most research focuses on this aspect of APP biology. We investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway during development. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. We report that APPL loss of function (LOF) results in the dysregulation of endolysosomal function in neurons, with a notable enlargement of early endosomal compartments followed by neuronal cell death and the accumulation of dead neurons in the brain during a critical period at a young age. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, we show that the secreted extracellular domain of APPL interacts with glia and regulates the size of their endosomes, the expression of the Draper engulfment receptor, and the clearance of neuronal debris in an axotomy model. We propose that APP proteins represent a novel family of neuroglial signaling factors required for adult brain homeostasis.

Parasitoid wasp venom vesicles (venosomes) enter Drosophila melanogaster lamellocytes through a flotillin/lipid raft-dependent endocytic pathway

ABSTRACT Venosomes are extracellular vesicles found in the venom of Leptopilina endoparasitoids wasps, which transport and target virulence factors to impair the parasitoid egg encapsulation by the lamellocytes of their Drosophila melanogaster host larva. Using the co-immunolocalization of fluorescent L. boulardi venosomes and one of the putative-transported virulence factors, LbGAP, with known markers of cellular endocytosis, we show that venosomes endocytosis by lamellocytes is not a process dependent on clathrin or macropinocytosis and internalization seems to bypass the early endosomal compartment Rab5. After internalization, LbGAP colocalizes strongly with flotillin-1 and the GPI-anchored protein Atilla/L1 (a lamellocyte surface marker) suggesting that entry occurs via a flotillin/lipid raft-dependent pathway. Once internalized, venosomes reach all intracellular compartments, including late and recycling endosomes, lysosomes, and the endoplasmic reticulum network. Venosomes therefore enter their target cells by a specific mechanism and the virulence factors are widely distributed in the lamellocytes’ compartments to impair their functions.

Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Enlarged early endosomes have been visualized in Alzheimer’s disease (AD) and Down syndrome (DS) using conventional confocal microscopy at a resolution corresponding to endosomal size (hundreds of nm). In order to overtake the diffraction limit, we used super-resolution structured illumination microscopy (SR-SIM) and transmission electron microscopies (TEM) to analyze the early endosomal compartment in DS.By immunofluorescence and confocal microscopy, we confirmed that the volume of Early Endosome Antigen 1 (EEA1)-positive puncta was 13–19% larger in fibroblasts and iPSC-derived neurons from individuals with DS, and in basal forebrain cholinergic neurons (BFCN) of the Ts65Dn mice modelling DS. However, EEA1-positive structures imaged by TEM or SR-SIM after chemical fixation had a normal size but appeared clustered. In order to disentangle these discrepancies, we imaged optimally preserved High Pressure Freezing (HPF)-vitrified DS fibroblasts by TEM and found that early endosomes were 75% denser but remained normal-sized.RNA sequencing of DS and euploid fibroblasts revealed a subgroup of differentially-expressed genes related to cargo sorting at multivesicular bodies (MVBs). We thus studied the dynamics of endocytosis, recycling and MVB-dependent degradation in DS fibroblasts. We found no change in endocytosis, increased recycling and delayed degradation, suggesting a “traffic jam” in the endosomal compartment.Finally, we show that the phosphoinositide PI (3) P, involved in early endosome fusion, is decreased in DS fibroblasts, unveiling a new mechanism for endosomal dysfunctions in DS and a target for pharmacotherapy.

Galectin-8 Enhances T cell Response by Promotion of Antigen Internalization and Processing.

SummaryGalectin-8 (Gal-8) is a mammalian lectin endowed with immunostimulatory ability. In the present work, we demonstrate that Gal-8-glycan interactions on the surface of antigen-presenting cells (APCs) promote antigen binding and internalization, independently from antigen nature. Both Gal-8 and antigen were together internalized and localized in early endosomes. Interestingly, antigen processing by APCs was also accelerated in the presence of Gal-8 as a separate mechanism, distinct from the increased antigen internalization. Moreover, APCs pulsed together with antigen and Gal-8 were able to activate cognate CD4+ T cells more efficiently than those pulsed with antigen alone. This enhanced antigen presentation was still evident in the absence of costimulatory signals and APCs-derived soluble mediators. Therefore, our results provide evidence for as yet unrecognized mechanism by which Gal-8 stimulates the elicitation of the immune response in a lectin-dependent manner, by inducing antigen uptake and processing upon lattice formation at APCs surface.

Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation. IMPLICATIONS: Enlarged early endosomes play a Rab4-modulated role in regulation of EGFR activation in breast cancer cells.

Constitutive Differential Features of Type 2 Transglutaminase in Cells Derived from Celiac Patients and from Healthy Subjects.

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31–43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31–43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31–43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31–43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.

Correlative Light and Electron Microscopy Imaging of the Plant trans-Golgi Network.

The plant trans-Golgi network (TGN) is a multifunctional organelle derived from the Golgi. It consists of tubulovesicular compartments scattered in the cytosol. They produce secretory vesicles delivering proteins and polysaccharides to the cell wall. They also serve as early endosomal compartments, receiving endocytic cargos from the plasma membrane. This versatility is thought to originate from functional variations among individual TGN compartments. Correlative light and electron microscopy (CLEM) combines the imaging capability of light microscopy and electron microscopy (EM) to determine the location of macromolecules in EM images in the cellular context. It is possible to identify organelles associated with specific fluorescent markers and examine their membrane architectures at nanometer-level resolutions using CLEM. In this chapter, we will explain the CLEM method that our lab uses to investigate functional and structural heterogeneity among individual TGN compartments in plant cells.

Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis.

Endosomes play a major role in various cellular processes including cell-cell signaling, development and cellular responses to environment. Endosomes are dynamically organized into a complex set of endomembrane compartments themselves subcompartmentalized in distinct pools or subpopulations. It is increasingly evident that endosome dynamics and maturation is driven by local modification of lipid composition. The diversity of membrane lipids is impressive and their homeostasis often involves crosstalk between distinct lipid classes. Hence, biochemical characterization of endosomal membrane lipidome would clarify the maturation steps of endocytic routes. Immunopurification of intact endomembrane compartments has been employed in recent years to isolate early and late endosomal compartments and can even be used to separate subpopulations of early endosomes. In this section, we will describe the immunoprecipitation protocol to isolate endosomes with the aim to analyze the lipid content. We will detail a procedure to identify the total fatty acid and sterol content of isolated endosomes as a first line of lipid identification. Advantages and limitations of the method will be discussed as well as potential pitfalls and critical steps.

Screening Legionella effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.

PERK-Mediated Unfolded Protein Response Activation and Oxidative Stress in PARK20 Fibroblasts.

PARK20, an early onset autosomal recessive parkinsonism is due to mutations in the phosphatidylinositol-phosphatase Synaptojanin 1 (Synj1). We have recently shown that the early endosomal compartments are profoundly altered in PARK20 fibroblasts as well as the endosomal trafficking. Here, we report that PARK20 fibroblasts also display a drastic alteration of the architecture and function of the early secretory compartments. Our results show that the exit machinery from the Endoplasmic Reticulum (ER) and the ER-to-Golgi trafficking are markedly compromised in patient cells. As a consequence, PARK20 fibroblasts accumulate large amounts of cargo proteins within the ER, leading to the induction of ER stress. Interestingly, this stressful state is coupled to the activation of the PERK/eIF2α/ATF4/CHOP pathway of the Unfolded Protein Response (UPR). In addition, PARK20 fibroblasts reveal upregulation of oxidative stress markers and total ROS production with concomitant alteration of the morphology of the mitochondrial network. Interestingly, treatment of PARK20 cells with GSK2606414 (GSK), a specific inhibitor of PERK activity, restores the level of ROS, signaling a direct correlation between ER stress and the induction of oxidative stress in the PARK20 cells. All together, these findings suggest that dysfunction of early secretory pathway might contribute to the pathogenesis of the disease.

A Liposomal Platform for Delivery of a Protein Antigen to Langerin-Expressing Cells.

The skin is an attractive site for vaccination and harbors a dense network of Langerhans cells that are the prime target for antigen delivery approaches in the epidermis. While specific targeting of Langerhans cells has been shown to elicit the necessary T-cell response using antibody-based delivery approaches, the targeted administration of particulate antigens in the form of nanoparticle-based vaccine formulations has been challenging. We previously reported on a specific targeting ligand for human Langerin, a C-type lectin expressed on Langerhans cells. This ligand is presented on liposomes and renders them highly specific for the uptake by Langerhans cells. Here we show a detailed study of the uptake and intracellular routing of the particles in model cell lines by confocal and live cell imaging as well as flow cytometric assays. Liposomes are internalized into early endosomal compartments and accumulate in late endosomes and lysosomes, shortly followed by a release of the cargo. Furthermore, we show the encapsulation of protein antigens and their delivery to cell lines and primary human Langerhans cells. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.

Regulation of Somatostatin Receptor 2 Trafficking by C-Tail Motifs and the Retromer.

The Gi-coupled somatostatin receptor 2 (SST2) is a G protein-coupled receptor (GPCR) that mediates many of somatostatin's neuroendocrine actions. Upon stimulation, SST2 is rapidly internalized and transported to early endosomes before being recycled to the plasma membrane. However, little is known about the intracellular itinerary of SST2 after it moves to the early endosomal compartment or the cytoplasmic proteins that regulate its trafficking. As postsynaptic density protein/discs large 1/zonula occludens-1 (PDZ) domain interactions often regulate the trafficking and signaling potential of GPCRs, we examined the role of the SST2 PDZ ligand and additional C-terminal residues in controlling its intracellular trafficking. We determined that SST2 can recycle to the plasma membrane via multiple pathways, including a LAMP1/Rab7-positive late endosome to the trans-Golgi network (TGN) pathway. Trafficking from the late endosome to the TGN is often regulated by the retromer complex of endosomal coat proteins, and disrupting the retromer components sorting nexins 1/2 inhibits the budding of SST2 from late endosomes. Moreover, trafficking through the late endosomal/TGN pathway is dependent on an intact PDZ ligand and C-terminal tail, as truncating either the 3 or 10 C-terminal amino acids of SST2 alters the pathway through which it recycles to the plasma membrane. Moreover, addition of these amino acids to a heterologous receptor is sufficient to redirect it from a degradation pathway to a recycling itinerary. Our results demonstrate that endosomal trafficking of SST2 is dependent on numerous regulatory mechanisms controlled by its C terminus and the retromer machinery.

Ultrastructural Analysis of Vesicular Transport in Electrotransfection.

Emerging evidence from various studies indicates that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when it is used for electrotransfection. To provide morphological evidence of the process, we investigated ultrastructures in cells that were associated with the electrotransfected pDNA, using immunoelectron microscopy. The results demonstrate that four endocytic pathways are involved in the uptake of the pDNA, including caveolae- and clathrin-mediated endocytosis, macropinocytosis, and the clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endosomal compartment (CLIC/GEEC) pathway. Among them, macropinocytosis is the most common pathway utilized by cells having various pDNA uptake capacities, and the CLIC/GEEC pathway is observed primarily in human umbilical vein endothelial cells. Quantitatively, the endocytic pathways are more active in easy-to-transfect cells than in hard-to-transfect ones. Taken together, our data provide ultrastructural evidence showing that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA.