Dynamin-dependent Pathway Research Articles

ELMO interacts with DOCK180 to enhance coelomocyte phagocytosis in Vibrio splendidus challenged Apostichopus japonicus

Engulfment and cell motility protein (ELMO) was identified as a novel, evolutionarily conserved upstream regulator of the RAC signaling pathway in mammals, which was essential for diverse physiological and pathological processes of cells, such as phagocytosis and cell migration. However, the functions and the underlying mechanisms of ELMO1 are generally poorly understood in invertebrates. In this study, we found that the endocytosis and GTP activity were significantly enriched in AjELMO1-interfered coelomocytes of Apostichopus japonicus by RNA-seq. Moreover, overexpression or interference of AjELMO1 significantly altered the coelomocyte phagocytosis levels induced by Vibrio splendidus AJ01, leading to an increase and decrease in the intracellular AJ01 burden by flow cytometry and bacteriophage plate counting in vivo and in vitro. Further functional analysis with different endocytosis inhibitors treatment revealed that AjELMO1 mediated AJ01 internalization via the actin-dependent endocytic pathways through the changes of F-actin/G-actin, but not the clathrin-, macropinocytosisi-, and dynamin-dependent endocytic pathways. The dedicator of cytokinesis protein 1 (AjDOCK180) was identified as an interacting protein of AjELMO1 using co-IP and GST-Pull down followed by mass spectrometry identification. Further function analysis also revealed that AjDOCK180 promotes coelomocyte phagocytosis through the actin endocytosis pathway via the changes in the phagocytosis levels, the intracellular AJ01 burden, and the ratio of F-actin/G-actin in coelomocytes. Additionally, we found that the phagocytic activity in sea cucumber coelomocytes promoted by AjELMO1 can be blocked by interference with AjDOCK180. Altogether, our data demonstrate that AjELMO1-AjDOCK180 is involved in resisting AJ01 infection via medicating coelomocyte phagocytosis through the actin-dependent pathway.

Uptake Pathway of Styrene Maleic Acid Copolymer-Coated Lipid Emulsions Under Acidic Tumor Microenvironment

The purpose of this study was to elucidate and compare styrene maleic acid copolymer (SMA)-coated lipid emulsions (SMA emulsions) uptake pathway in vascular endothelial cells and surrounding cancer cells under not only neutral but also acidic pH, which is often observed in tumor microenvironment. DiI-labeled SMA emulsions were prepared using 1-palmitoyl-2-oleoyl-sn‑glycero-3-phosphocholine and triolein. In murine melanoma B16-BL6 (B16) cells and human umbilical vein endothelial cells (HUVEC), DiI-labeled SMA emulsions uptake under near-neutral (pH 7.4) and acidic (pH 6.0) conditions was determined by fluorescent analysis. SMA emulsions were taken up more efficiently into HUVEC than B16 cells under acidic condition in a temperature-dependent manner. Uptake study using endocytosis inhibitors showed that SMA emulsions were taken up by macropinocytosis and clathrin-mediated endocytosis in B16 cells. In HUVEC, however, they were taken up by clathrin- and caveolae-independent, but dynamin-dependent pathway. SMA emulsions would be internalized efficiently into vascular endothelial cells as well as cancer cells under acidic microenvironment via different endocytosis pathways. SMA emulsions could be a promising drug delivery carrier for anti-angiogenic drugs.

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells.

Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Hydroxyl-Rich Hydrophilic Endocytosis-Promoting Peptide with No Positive Charge.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

Ligand-bound integrin αvβ6 internalisation and trafficking.

The integrin αvβ6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvβ6. As αvβ6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvβ6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvβ6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvβ6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvβ6-dependent migration (αvβ6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvβ6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvβ6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvβ6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvβ6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvβ6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvβ6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvβ6-specific therapeutics and potentially the types of therapeutic load.

Internalization of Polymeric Bacterial Peptidoglycan Occurs through Either Actin or Dynamin Dependent Pathways.

Peptidoglycan (PGN), a polymeric glycan macromolecule, is a major constituent of the bacterial cell wall and a conserved pathogen-associated molecular pattern (PAMP) that triggers immune responses through cytosolic sensors. Immune cells encounter both PGN polymers and hydrolyzed muropeptides during infections, and primary human innate immune cells respond better to polymeric PGN than the minimal bioactive subunit muramyl dipeptide (MDP). While MDP is internalized through macropinocytosis and/or clathrin-mediated endocytosis, the internalization of particulate polymeric PGN is unresolved. We show here that PGN macromolecules isolated from Bacillus anthracis display a broad range of sizes, making them amenable for multiple internalization pathways. Pharmacologic inhibition indicates that PGN primarily, but not exclusively, is internalized by actin-dependent endocytosis. An alternate clathrin-independent but dynamin dependent pathway supports 20–30% of PGN uptake. In primary monocytes, this alternate pathway does not require activities of RhoA, Cdc42 or Arf6 small GTPases. Selective inhibition of PGN uptake shows that phagolysosomal trafficking, processing and downstream immune responses are drastically affected by actin depolymerization, while dynamin inhibition has a smaller effect. Overall, we show that polymeric PGN internalization occurs through two endocytic pathways with distinct potentials to trigger immune responses.

Synthesis, Self-Assembly and In Vitro Cellular Uptake Kinetics of Nanosized Drug Carriers Based on Aggregates of Amphiphilic Oligomers of N-Vinyl-2-pyrrolidone.

Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier–membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.

Europium sulfide nanoprobes predict antiretroviral drug delivery into HIV-1 cell and tissue reservoirs.

Background: Delivery of long-acting nanoformulated antiretroviral drugs (ARVs) to human immunodeficiency virus type one cell and tissue reservoirs underlies next generation antiretroviral therapeutics. Nanotheranostics, comprised of trackable nanoparticle adjuncts, can facilitate ARV delivery through real-time drug tracking made possible through bioimaging platforms.Methods: To model HIV-1 therapeutic delivery, europium sulfide (EuS) nanoprobes were developed, characterized and then deployed to cells, tissues, and rodents. Tests were performed with nanoformulated rilpivirine (NRPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used clinically to suppress or prevent HIV-1 infection. First, CD4+ T cells and monocyte-derived macrophages were EuS-treated with and without endocytic blockers to identify nanoprobe uptake into cells. Second, Balb/c mice were co-dosed with NRPV and EuS or lutetium177-doped EuS (177LuEuS) theranostic nanoparticles to assess NRPV biodistribution via mass spectrometry. Third, single photon emission computed tomography (SPECT-CT) and magnetic resonance imaging (MRI) bioimaging were used to determine nanotheranostic and NRPV anatomic redistribution over time.Results: EuS nanoprobes and NRPV entered cells through dynamin-dependent pathways. SPECT-CT and MRI identified biodistribution patterns within the reticuloendothelial system for EuS that was coordinate with NRPV trafficking.Conclusions: EuS nanoprobes parallel the uptake and biodistribution of NRPV. These data support their use in modeling NRPV delivery to improve treatment strategies.

PSIII-36 The endocytic pathway plays a major role in the regulation of tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells

Abstract Postweaning pigs are subjected to nutrient deprivation during which intestinal epithelial cells undergo increased turnover. To preserve intestinal function, intestinal epithelial cells must activate adaptive mechanisms that allow them to cope with starvation-induced stress; most importantly, the preservation of intestinal barrier function. The objective of this study was to investigate the underlying mechanisms involved in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were subjected to total nutrient starvation in Krebs-Ringer bicarbonate (KRB) buffer for 0, 3, 6, 12 and 24 h. Abundance of tight junction proteins was determined by RT-PCR, western blotting and immunofluorescence. Compared with control group (0 h), the protein expression of claudin 1, claudin 3 and claudin 4 protein was downregulated up to 6 h of starvation and then increased thereafter (P < 0.01). However, there was no change in the protein level of occludin and ZO-1. To determine the contribution of the lysosome and the ubiquitin proteasome pathways to regulation of tight junction protein abundance, the lysosome (Bafilomycin A1) and the proteasome (MG132) inhibitors were used in nutrient starved cells. Results showed the degradation of claudin 1, 3 and 4 up to 6 h of starvation was through the lysosomal pathway. Surprisingly, re-synthesis of claudins 4 and claudin 3 after prolonged starvation (12 and 24 h) was prevented when cells were treated with bafilomycin A1 and MG132, respectively. The autophagy-lysosome pathway inhibitors (Wortmannin and MHY1485) and endosome-lysosome pathway inhibitors (Dynasore and Pitstop 2) were further used to determine the specific roles of these pathways. In summary, the degradation of claudin 3 and claudin 4 during short-term starvation (up to 6 h) was through the dynamin-dependent endocytic pathway. However, re-synthesis of these proteins after prolonged starvation relies on both the lysosome and proteasome pathways.

G protein-regulated endocytic trafficking of adenylyl cyclase type 9.

GPCRs are increasingly recognized to initiate signaling via heterotrimeric G proteins as they move through the endocytic network, but little is known about how relevant G protein effectors are localized. Here we report selective trafficking of adenylyl cyclase type 9 (AC9) from the plasma membrane to endosomes while adenylyl cyclase type 1 (AC1) remains in the plasma membrane, and stimulation of AC9 trafficking by ligand-induced activation of Gs-coupled GPCRs. AC9 transits a similar, dynamin-dependent early endocytic pathway as ligand-activated GPCRs. However, unlike GPCR traffic control which requires β-arrestin but not Gs, AC9 traffic control requires Gs but not β-arrestin. We also show that AC9, but not AC1, mediates cAMP production stimulated by endogenous receptor activation in endosomes. These results reveal dynamic and isoform-specific trafficking of adenylyl cyclase in the endocytic network, and a discrete role of a heterotrimeric G protein in regulating the subcellular distribution of a relevant effector.

Orchestration of human macrophage NLRP3 inflammasome activation by Staphylococcus aureus extracellular vesicles

Release of extracellular vesicles (EVs) is a common feature among eukaryotes, archaea, and bacteria. However, the biogenesis and downstream biological effects of EVs released from gram-positive bacteria remain poorly characterized. Here, we report that EVs purified from a community-associated methicillin-resistant Staphylococcus aureus strain were internalized into human macrophages in vitro and that this process was blocked by inhibition of the dynamin-dependent endocytic pathway. Human macrophages responded to S. aureus EVs by TLR2 signaling and activation of NLRP3 inflammasomes through K+ efflux, leading to the recruitment of ASC and activation of caspase-1. Cleavage of pro-interleukin (IL)-1β, pro-IL-18, and gasdermin-D by activated caspase-1 resulted in the cellular release of the mature cytokines IL-1β and IL-18 and induction of pyroptosis. Consistent with this result, a dose-dependent cytokine response was detected in the extracellular fluids of mice challenged intraperitoneally with S. aureus EVs. Pore-forming toxins associated with S. aureus EVs were critical for NLRP3-dependent caspase-1 activation of human macrophages, but not for TLR2 signaling. In contrast, EV-associated lipoproteins not only mediated TLR2 signaling to initiate the priming step of NLRP3 activation but also modulated EV biogenesis and the toxin content of EVs, resulting in alterations in IL-1β, IL-18, and caspase-1 activity. Collectively, our study describes mechanisms by which S. aureus EVs induce inflammasome activation and reveals an unexpected role of staphylococcal lipoproteins in EV biogenesis. EVs may serve as a novel secretory pathway for S. aureus to transport protected cargo in a concentrated form to host cells during infections to modulate cellular functions.

The Plasticity of Mesenchymal Stem Cells in Regulating Surface HLA-I

SummaryA low surface expression level of human leukocyte antigen class I (HLA-I) ensures that the mesenchymal stem cells (MSCs) escape from the allogeneic recipients' immunological surveillance. Here, we discovered that both transcriptional and synthesis levels of HLA-I in MSCs increased continuously after interferon (IFN)-γ treatment, whereas interestingly, their surface HLA-I expression was downregulated after reaching an HLA-I surface expression peak. Microarray data indicated that the post-transcriptional process plays an important role in the downregulation of surface HLA-I. Further studies identified that IFN-γ-treated MSCs accelerated HLA-I endocytosis through a clathrin-independent dynamin-dependent endocytosis pathway. Furthermore, cells that have self-downregulated surface HLA-I expression elicit a weaker immune response than they previously could. Thus uncovering the plasticity of MSCs in the regulation of HLA-I surface expression would reveal insights into the membrane transportation events leading to the maintenance of low surface HLA-I expression, providing more evidence for selecting and optimizing low-immunogenic MSCs to improve the therapeutic efficiency.

Improving the production of recombinant human bone morphogenetic protein-4 in Chinese hamster ovary cell cultures by inhibition of undesirable endocytosis.

Endocytic regulation serves a critical role in modulating the extracellular level of signaling molecules, such as bone morphogenetic proteins (BMPs). Unfortunately, endocytosis may result in poor yields of recombinant human BMP-4 (rhBMP-4) from Chinese hamster ovary (CHO) cell cultures. When rhBMP-4 was incubated with CHO cells, rhBMP-4 was actively internalized into cells. Cell surface bound heparan sulfate proteoglycans (HSPGs) served as the major receptors for rhBMP-4 internalization. Removal of cell surface heparan sulfate (HS) by heparinases or reduction of HSPG synthesis by knockdown of xylosyltransferase2 (xylt2) in CHO cells decreased internalization of rhBMP-4. In addition, treatment with endocytosis inhibitors (chlorpromazine, genistein, and dynasore) identified a clathrin- and dynamin-dependent endocytic pathway as the major route for rhBMP-4 internalization. To enhance product yield by minimizing rhBMP-4 internalization in recombinant CHO (rCHO) cell cultures, we have tested various strategies to reduce HSPG synthesis (knockdown of xylt2 and sodium chlorate treatment) or inhibit the binding of rhBMP-4 to cell-surface-bound HSPGs (supplementation with heparin or dextran sulfate [DS]). Among these approaches, DS, which is a linear anionic sulfated polysaccharide with similarity to HS chains, was the most effective in enhancing rhBMP-4 production in rCHO cell cultures. Compared with the control cultures, DS addition to the culture medium (1.0 g/L) resulted in 1.4-fold and 2.3-fold increases in maximum rhBMP-4 concentration in batch and fed-batch cultures, respectively. Taken together, the addition of DS, an effective competitor of HSPGs, improved rhBMP-4 production in rCHO cell cultures through blockage of rhBMP-4 internalization.

Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

.BackgroundMacrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages.MethodsTHP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays.ResultsTHP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages.ConclusionOur results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible.

PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells.

Prostaglandin E2 (PGE2) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1, PTGS2, MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE2-induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression.

Phenotypically Screened Carbon Nanoparticles for Enhanced Combinatorial Therapy in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is a highly aggressive type of breast cancer with high resistance to current standard therapies. We demonstrate that phenotypically stratified carbon nanoparticle is highly effective in delivering a novel combinatorial triple drug formulation for synergistic regression of TNBC in vitro and in vivo. The combinatorial formulation is comprised of repurposed inhibitors of STAT3 (nifuroxazide), topoisomerase-II-activation-pathway (amonafide) and NFκb (pentoxifylline). Synergistic effect of drug combination was established in a panel of TNBC-lines comprising mesenchymal-stem-like, mesenchymal and basal-like cells along with non-TNBC-cells. The delivery of combinatorial drug formulation was achieved using a phenotypically screened carbon nanoparticles for TNBC cell lines. Results indicated a remarkable five-fold improvement (IC50-6.75µM) from the parent drugs with a combinatorial index <1 in majority of the TNBC cells. Multi-compartmental carbon nanoparticles were then parametrically assessed based on size, charge (positive/negative/neutral) and chemistry (functionalities) to study their likelihood of crossing endocytic barriers from phenotypical standpoint in TNBC lines. Interestingly, a combination of clathrin mediated, energy and dynamin dependent pathways were predominant for sulfonated nanoparticles, whereas pristine and phospholipid particles followed all the investigated endocytic pathways. An exactitude 'omics' approach helps to predict that phospholipid encapsulated-particles will predominantly accumulate in TNBC comprising the drug-'cocktail'. We investigated the protein expression effects inducing synergistic effect and simultaneously suppressing drug resistance through distinct mechanisms of action.

Modified low density lipoprotein (mLDL) uptake by B cells alters B cell phenotype and impacts atherosclerosis.

Abstract Atherosclerosis is a multifaceted chronic inflammatory disease characterized by the accumulation of modified lipoproteins and immune cells in the aortic wall and vascular dysfunction. The uptake of modified low density lipoproteins (mLDL) by MFs plays an important role in atherosclerosis through the modulation of cholesterol metabolism and MF activation. To date, the impact of mLDL on B cell phenotype and functions is unaddressed. We show that B cells can uptake acetylated and oxidized LDL (acLDL and oxLDL, respectively) via a dynamin-dependent pathway in a concentration, time, and temperature-dependent manner. Assessment of the transcriptional profiles of in vivo acLDL+ B cells and acLDL- B cells by RNAseq suggests that acLDL+ B cells assume an anergic like phenotype. Anergy plays an important role in keeping autoreactive B cells in a state of unresponsiveness and the loss of anergy contributes to the development of autoimmune diseases. Currently, nothing is known about the role and regulation of B cell anergy in atherosclerosis. We show here for the first time that GRAIL, an E3 ubiquitin ligase associated with T cell tolerance, is expressed in B cells. To determine whether B cell-specific GRAIL expression plays a role in atherosclerosis, we performed adoptive transfer of GRAIL-deficient or GRAIL-sufficient B cells into B-cell-deficient mice on apolipoprotein-E deficient background (uM−/− Apoe−/−) mice and examined atherogenesis after 8 weeks of western diet feeding. B cell-specific GRAIL-deficiency supported plaque burden and orchestrated inflammation in the aortic wall. These data suggest that there is a specific mechanism by which B cells recognize mLDL and regulate their anergy-dependent response to mLDL in atherosclerosis.

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis.

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus Tracking.

Entry is the first critical step for the infection of influenza A virus and of great significance for the research and development of antiflu drugs. Influenza A virus depends on exploitation of cellular endocytosis to enter its host cells, and its entry behaviors in distinct routes still need further investigation. With the aid of a single-virus tracking technique and quantum dots, we have realized real-time and multicolor visualization of the endocytic process of individual viruses and comprehensive dissection of two distinct dynamin-dependent endocytic pathways of influenza A virus, either dependent on clathrin or not. Based on the sequential progression of protein recruitment and viral motility, we have revealed the asynchronization in the recruitments of clathrin and dynamin during clathrin-dependent entry of the virus, with a large population of events for short-lived recruitments of these two proteins being abortive. In addition, the differentiated durations of dynamin recruitment and responses to inhibitors in these two routes have evidenced somewhat different roles of dynamin. Besides promoting membrane fission in both entry routes, dynamin also participates in the maturation of a clathrin-coated pit in the clathrin-dependent route. Collectively, the current study displays a dynamic and precise image of the entry process of influenza A virus and elucidates the mechanisms of distinct entry routes. This quantum dot-based single-virus tracking technique is proven to be well-suited for investigating the choreographed interactions between virus and cellular proteins.

Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

Endocytosis of ligand-receptor complexes regulates signal transduction during development. In particular, clathrin and dynamin-dependent endocytosis has been well studied in the context of patterning of the Drosophila wing disc, wherein apically secreted Wingless (Wg) encounters its receptor, DFrizzled2 (DFz2), resulting in a distinctive dorso-ventral pattern of signaling outputs. Here, we directly track the endocytosis of Wg and DFz2 in the wing disc and demonstrate that Wg is endocytosed from the apical surface devoid of DFz2 via a dynamin-independent CLIC/GEEC pathway, regulated by Arf1, Garz, and class I PI3K. Subsequently, Wg containing CLIC/GEEC endosomes fuse with DFz2-containing vesicles derived from the clathrin and dynamin-dependent endocytic pathway, which results in a low pH-dependent transfer of Wg to DFz2 within the merged and acidified endosome to initiate Wg signaling. The employment of two distinct endocytic pathways exemplifies a mechanism wherein cells in tissues leverage multiple endocytic pathways to spatially regulate signaling.