Caveolin-dependent Endocytosis Research Articles

Absorption and transport mechanism of colloidal nanoparticles (CNPs) in lamb soup based on Caco-2 cell

Soup is an important presence in diet, but its absorption and transport mechanism by the human body remains unclear. In this study, Caco-2 intact cell and monolayer cell models were constructed to simulate small intestine absorption on colloidal nanoparticles (CNPs) isolated from lamb soup. The intracellular localization of CNPs was viewed by laser confocal microscopy (LSCM). CNPs uptake and release pathways were explored by differences in CNPs concentrations in 5 endocytosis inhibitor models and 4 exocytosis inhibitor models. Results indicated that CNPs endocytosis by Caco-2 cells was restrained by Nystatin and Cytochalasin D, with exocytosis being inhibited by Nocodazole and Monensin. Therefore, the major absorption pathways for CNPs were caveolin-dependent endocytosis, macropinocytosis and phagocytosis. The major transport pathways were microtubule-vesicle-mediated protein transport to the membrane and transportation between the Golgi apparatus and membrane. This study may provide theoretical support for the transport mechanism of soup products in the small intestine.

Caveolin-dependent endocytosis regulates arterial calcification

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB219 Introduction Patients with chronic kidney disease (CKD) have a significantly elevated cardiovascular risk, which is primarily due to premature vascular aging with arterial calcification - a substantial public health burden. Calcifying extracellular vesicles (EVs) are required for the onset and progression of calcification. Endocytosis and exocytosis are important for EV genesis and cargo loading, thus controlling EV release and propensities. Purpose We aim to investigate endocytosis, endosomal ripening, and exocytosis as critical mechanisms of EV-dependent mineralization. Methods and Results First, we characterized EVs from calcifying vascular smooth muscle cells (SMCs) compared to control SMCs. Western blot analysis revealed an increase in tissue nonspecific alkaline phosphatase (TNAP) among calcifying EVs but not in flotillin abundance. When compared to control EVs, calcifying EVs showed higher TNAP activity. Flow cytometry and a turbidity assay were then used to evaluate EV mineral cargo and aggregation potential. Compared to the control, EVs produced from SMCs cultivated under calcifying conditions had 5-fold more mineral-positive EVs (p=0.004) and a 3-fold higher nucleation potential (p<0.001). Next, endocytosis was blocked by targeting Dynamin-1 and –2, which inhibited caveolin- and clathrin-dependent endocytosis (Dynasore hydrate), or targeting clathrin-terminal domain, which inhibited solely clathrin-dependent endocytosis (Pitstop 2). Dynasore reduced matrix mineralization dose-dependently and lowered mineral-positive EVs by 18%, whereas Pitstop 2 had no effect, suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, Cav-1 silencing inhibited matrix mineralization and Cav-1 protein was more abundant in calcifying EVs than in control EVs. Dynasore and Pitstop-2 did not affect the EV number measured by nanoparticle tracking analysis. Inhibition of endosomal ripening by Rab7 inhibition did not affect SMC calcification. Exocytosis was inhibited by Rab27a inhibition, which reduced mineral deposition (-32%, p=0.017) without affecting EV mineral or EV quantity. Conclusion EVs released from calcifying SMCs showed a higher abundance of mineral and TNAP proteins, suggesting a role in mineral transport and deposition. Our data suggest that caveolin-dependent endocytosis is essential in EV-mediated SMC calcification. Mineral cargo in EVs is independent of Rab7 and Rab27a pathways.

Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

The Role of Vesicles in Transporting of Cholera Toxin

The review reports on the secretion pathways of the main virulence factor of Vibrio cholerae, cholera toxin, both through the two-stage Sec-dependent type 2 secretion system and with the help of vesicles of the outer membrane of V. cholerae. The ways of toxin transfer into the host organism, depending on its form, are discussed. The well-studied free soluble cholera toxin is secreted extracellularly and transmitted in a GM1-dependent manner through cholesterolrich lipid rafts. The transfer of cholera toxin associated with vesicles has advantages over free toxin, because substances inside the outer membrane vesicles are protected from external proteases and host antibodies by the membrane that forms the vesicle. Vesicular transporting of cholera toxin into the target cell occurs via clathrin-dependent, caveolin-dependent and lipid raft-dependent endocytosis. The specific transport route is determined by the structure of the vesicles. Clathrindependent endocytosis is described for V. cholerae strains cultivated at low osmolarity of the medium, whose outer membrane vesicles contain the cholera toxin subunit A inside. Lipid raft-dependent endocytosis is characteristic of vesicles in which cholera toxin is located on the surface. In addition, endocytosis of V. cholerae outer membrane vesicles through structures known as caveolae is presented.

Proteomic characterization of Opisthorchis felineus exosome-like vesicles and their uptake by human cholangiocytes

The epidemiologically important food-borne trematode Opisthorchis felineus infests the liver biliary tract of fish-eating mammals and causes disorders, including bile duct neoplasia. Many parasitic species release extracellular vesicles (EVs) that mediate host–parasite interaction. Currently, there is no information on O. felineus EVs.Using gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry, we aimed to characterize the proteome of EVs released by the adult O. felineus liver fluke. Differential abundance of proteins between whole adult worms and EVs was assessed by semiquantitative iBAQ (intensity-based absolute quantification). Imaging, flow cytometry, inhibitor assays, and colocalization assays were performed to monitor the uptake of the EVs by H69 human cholangiocytes.The proteomic analysis reliably identified 168 proteins (at least two peptides matched a protein). Among major proteins of EVs were ferritin, tetraspanin CD63, helminth defense molecule 1, globin 3, saposin B type domain-containing protein, 60S ribosomal protein, glutathione S-transferase GST28, tubulin, and thioredoxin peroxidase. Moreover, as compared to the whole adult worm, EVs proved to be enriched with tetraspanin CD63, saposin B, helminth defense molecule 1, and Golgi-associated plant pathogenesis-related protein 1 (GAPR1). We showed that EVs are internalized by human H69 cholangiocytes via clathrin-dependent endocytosis, whereas phagocytosis and caveolin-dependent endocytosis do not play a substantial role in this process.Our study describes for the first time proteomes and differential abundance of proteins in whole adult O. felineus worms and EVs released by this food-borne trematode. Studies elucidating the regulatory role of individual components of EVs of liver flukes should be continued to determine which components of EV cargo play the most important part in the pathogenesis of fluke infection and in a closely linked pathology: bile duct neoplasia. SignificanceThe food-borne trematode Opisthorchis felineus is a pathogen that causes hepatobiliary disorders in humans and animals. Our study describes for the first time the release of EVs by the liver fluke O. felineus, their microscopic and proteomic characterization, and internalization pathways by human cholangiocytes. Differential abundance of proteins between whole adult worms and EVs was assessed. EVs are enriched with canonical EV markers as well as parasite specific proteins, i.e. tetraspanin CD63, saposin B, helminth defense molecule 1, and others. Our findings will form the basis of the search for potential immunomodulatory candidates with therapeutic potential in the context of inflammatory diseases, as well as novel vaccine candidates.

Sulfation of Heparan and Chondroitin Sulfate Ligands Enables Cell-Specific Homing of Nanoprobes.

Demystifying the sulfation code of glycosaminoglycans (GAGs) to induce precise homing of nanoparticles in tumor cells or neurons influences the development of a potential drug- or gene-delivery system. However, GAGs, particularly heparan sulfate (HS) and chondroitin sulfate (CS), are structurally highly heterogeneous, and synthesizing well-defined HS/CS composed nanoparticles is challenging. Here, we decipher how specific sulfation patterns on HS and CS regulate receptor-mediated homing of nanoprobes in primary and secondary cells. We discovered that aggressive cancer cells such as MDA-MB-231 displayed a strong uptake of GAG-nanoprobes compared to mild or moderately aggressive cancer cells. However, there was no selectivity towards the GAG sequences, thus indicating the presence of more than one form of receptor-mediated uptake. However, U87 cells, olfactory bulb, and hippocampal primary neurons showed selective or preferential uptake of CS-E-coated nanoprobes compared to other GAG-nanoprobes. Furthermore, mechanistic studies revealed that the 4,6-O-disulfated-CS nanoprobe used the CD44 and caveolin-dependent endocytosis pathway for uptake. These results could lead to new opportunities to use GAG nanoprobes in nanomedicine.

Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells.

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Effect of molecular weight of polysaccharide on efficient plasmid DNA delivery by polyethylenimine‐polysaccharide‐Fe(III) complexes

AbstractPolyethylenimine (PEI) is considered as the gold standard for evaluating non‐viral gene vectors due to its proton sponge effect and high transfection efficiency. However, its cytotoxicity limits its application. Rice bran polysaccharide (RBP) is rich in nature and beneficial for preparation of biodegradable biomaterials. In this work, three PEI modified RBP‐Fe complexes (PIP‐1, PIP‐2, and PIP‐3) were prepared based on RBP with different molecular weight (Mw). Then the potential of PIP complexes for plasmid DNA (pDNA) delivery and the influence of Mw was studied. PIP complexes showed better safety and higher gene transfection efficiency than PEI. PIP‐1 was prepared by RBP with lower Mw and could formed PIP‐1/pDNA nanoparticle with lower particle size which exhibited higher gene transfection efficiency than other PIP complexes prepared by RBP with higher Mw. The results indicate that the PIP/pDNA complex exhibits a multi‐pathway cellular uptake mechanism. Clathrin dependent and caveolin dependent endocytosis and macropinocytosis were involved in the cellular uptake process of PIP/pDNA complex. It is hoped that this study can provide useful enlightenment for the research and development of gene vector based on polysaccharide.

Cerebral endothelial cell derived small extracellular vesicles improve cognitive function in aged diabetic rats.

Small extracellular vesicles (sEVs) mediate cell-cell communication by transferring their cargo biological materials into recipient cells. Diabetes mellitus (DM) induces cerebral vascular dysfunction and neurogenesis impairment, which are associated with cognitive decline and an increased risk of developing dementia. Whether the sEVs are involved in DM-induced cerebral vascular disease, is unknown. Therefore, we studied sEVs derived from cerebral endothelial cells (CEC-sEVs) of aged DM rats (DM-CEC-sEVs) and found that DM-CEC-sEVs robustly inhibited neural stem cell (NSC) generation of new neuroblasts and damaged cerebral endothelial function. Treatment of aged DM-rats with CEC-sEVs derived from adult healthy normal rats (N-CEC-sEVs) ameliorated cognitive deficits and improved cerebral vascular function and enhanced neurogenesis. Intravenously administered N-CEC-sEVs crossed the blood brain barrier and were internalized by neural stem cells in the neurogenic region, which were associated with augmentation of miR-1 and –146a and reduction of myeloid differentiation primary response gene 88 and thrombospondin 1 proteins. In addition, uptake of N-CEC-sEVs by the recipient cells was mediated by clathrin and caveolin dependent endocytosis signaling pathways. The present study provides ex vivo and in vivo evidence that DM-CEC-sEVs induce cerebral vascular dysfunction and neurogenesis impairment and that N-CEC-sEVs have a therapeutic effect on improvement of cognitive function by ameliorating dysfunction of cerebral vessels and increasing neurogenesis in aged DM rats, respectively.

Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells.

Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains—Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG—in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.

TFEB controls integrin-mediated endothelial cell adhesion by the regulation of cholesterol metabolism

The dynamic integrin-mediated adhesion of endothelial cells (ECs) to the surrounding ECM is fundamental for angiogenesis both in physiological and pathological conditions, such as embryonic development and cancer progression. The dynamics of EC-to-ECM adhesions relies on the regulation of the conformational activation and trafficking of integrins. Here, we reveal that oncogenic transcription factor EB (TFEB), a known regulator of lysosomal biogenesis and metabolism, also controls a transcriptional program that influences the turnover of ECM adhesions in ECs by regulating cholesterol metabolism. We show that TFEB favors ECM adhesion turnover by promoting the transcription of genes that drive the synthesis of cholesterol, which promotes the aggregation of caveolin-1, and the caveolin-dependent endocytosis of integrin β1. These findings suggest that TFEB might represent a novel target for the pharmacological control of pathological angiogenesis and bring new insights in the mechanism sustaining TFEB control of endocytosis.

Internalization of FITC-albumin in Human Adipose-derived Stem Cells: Involvement of Clathrin and Caveolin.

Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A1), clathrin-dependent endocytosis inhibitors (chloropromazine, phenylarsine oxide, and Pitstop2), and caveolin-dependent endocytosis inhibitors (nystatin and methyl-β-cyclodextrin). Furthermore, the knockdown of the clathrin heavy chain and caveolin-1 significantly reduced FITC-albumin uptake. These findings suggest that AdSCs take up albumin via endocytic pathways in which clathrin and caveolin are involved.

Poly(l-alanine-co-l-lysine)-g-Trehalose as a Biomimetic Cryoprotectant for Stem Cells.

Poly(l-alanine-co-l-lysine)-graft-trehalose (PAKT) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAKT. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAKT and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAKT solution, followed by cryopreservation at -196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0-1.0 and 0.0-15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAKT solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAKT was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAKT. 1H NMR and FTIR spectra suggested that the intracellular PEG-PAKTs interact with water and stabilize the cells during cryopreservation.

Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

A Disintegrin and Metalloproteinase with ThromboSpondin motif (ADAMTS) 5 functions as an anti-angiogenic and anti-cancer protein independent of its metalloproteinase activity. Both full-length ADAMTS5 and TS5-p45, the autocatalytically cleaved C-terminal 45 kDa truncate of ADAMTS5, inhibits angiogenesis, and induces endothelial cell (EC) apoptosis. However, how ADAMTS5 triggers EC apoptosis remains unclear. This work shows that caspase-8 (Cas-8) and caspase-9 (Cas-9) are involved in TS5-p45-induced EC apoptosis. We identify cell surface nucleolin (NCL) as a novel high-affinity receptor for TS5-p45 in ECs, mediating TS5-p45’s cell surface binding and pro-apoptotic function. We show that the central RNA-binding domain (RBD) of NCL is essential and sufficient for its binding to TS5-p45. Upon interacting with EC surface NCL, TS5-p45 is internalized through clathrin- and caveolin-dependent endocytosis and trafficked to the nucleus via late endosomes (LEs). We demonstrate that the nuclear trafficking of TS5-p45 is important for its pro-apoptotic activity as disruption of LE membrane integrity with an endosomolytic peptide suppressed both nuclear trafficking and pro-apoptotic activity of TS5-p45. Through cell surface biotinylation, we revealed that cell surface NCL shuttles extracellular TS5-p45 to the nucleus to mediate apoptosis. Furthermore, blocking the importin α1/ß1 receptor hindered the nuclear trafficking of TS5-p45, suggesting the involvement of the nuclear importing machinery for this nuclear translocation. RNA-seq identified many apoptosis-related genes that are differentially expressed at least two-fold in TS5-p45-treated ECs, with 10 of them qRT-PCR-validated and at least 5 of these genes potentially contributing to TS5-p45-NCL-induced apoptosis. Altogether, our work identifies NCL as a novel cell surface receptor for ADAMTS5 and demonstrates the critical role of NCL-mediated internalization and nuclear trafficking for ADAMTS5-induced EC apoptosis. These findings reveal novel mechanistic insights of the secreted metalloproteinase ADAMTS5 in angiogenesis inhibition.

Abstract TP144: Extracellular Vesicles Derived From Stroke Patient Clots Impair Cerebral Endothelial Cell Function

Introduction: Most patients undergoing thrombectomy fail for full reperfusion. The present study tested whether small extracellular vesicles (sEVs) from stroke patient clots impair cerebral endothelial cell (CEC) function, which may contribute to dysfunction of downstream CECs, leading to reduction of cerebral tissue perfusion. Methods: Per IRB-approved protocol, clots were collected from 60 patients (30 males) with acute ischemic stroke subjected to thrombectomy. Red and white clots were determined by immunochemistry. Clot-sEVs were isolated and characterized using ultracentrifugation, size distribution, and Western blot. The effects of clot-sEVs on human CEC (hCEC) permeability and proteins were measured using trans-endothelial assay and trans-endothelial electrical resistance (TEER) and Western blot, respectively. Nystatin, an inhibitor of caveolin-dependent endocytosis, was employed to study hCEC uptake of clot-sEVs. Results: Clot-sEVs had a mean size of 73±2nm. Cellular source analysis of clot-sEVs revealed that 22%, 30%, and 44% of sEVs from red clots and 28%, 28%, and 52% of sEVs from white clots were from neutrophils, endothelial cells, and platelets, respectively. Compared to sEVs derived from donor plasma (plasma-sEVs), treatment of hCECs with clot-sEVs significantly increased cell permeability (227% vs 100% of plasma-sEVs) and reduced TEER (66.6ω.cm 2 vs 87.3ω.cm 2 ). Clot-sEVs significantly increased pro-inflammatory and pro-coagulation proteins in the hCECs including ICAM1, VCAM1, TNF-a, HMBG1, tissue factor, and significantly reduced tight junction proteins ZO-1 and Claudin-5. The effects of white clot-sEVs on hCEC dysfunction were greater than red clot-sEVs. Nystatin significantly reduced hCEC uptake of clot-sEV from 232% to 142% and the clot-sEV-increased permeability. Moreover, Pearson correlation showed that the severity of clot-sEV impaired hCEC function was significantly correlated with final TICI scores of the individual patients (permeability vs TICI, r=-0.3348, p=0.02; TEER vs TICI, r=0,3666, p=0.01). Conclusion: Clot-sEVs contribute to hCEC dysfunction by augmenting pro-inflammatory and pro-coagulation proteins. Blocking uptake of clot-sEVs by recipient cells may improve patient outcomes.

Nucleosomes enter cells by clathrin- and caveolin-dependent endocytosis.

DNA damage and apoptosis lead to the release of free nucleosomes—the basic structural repeating units of chromatin—into the blood circulation system. We recently reported that free nucleosomes that enter the cytoplasm of mammalian cells trigger immune responses by activating cGMP-AMP synthase (cGAS). In the present study, we designed experiments to reveal the mechanism of nucleosome uptake by human cells. We showed that nucleosomes are first absorbed on the cell membrane through nonspecific electrostatic interactions between positively charged histone N-terminal tails and ligands on the cell surface, followed by internalization via clathrin- or caveolae-dependent endocytosis. After cellular internalization, endosomal escape occurs rapidly, and nucleosomes are released into the cytosol, maintaining structural integrity for an extended period. The efficient endocytosis of extracellular nucleosomes suggests that circulating nucleosomes may lead to cellular disorders as well as immunostimulation, and thus, the biological effects exerted by endocytic nucleosomes should be addressed in the future.

Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds.

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

Ratiometric Fluorescent DNA Nanostructure for Mitochondrial ATP Imaging in Living Cells Based on Hybridization Chain Reaction.

For intracellular molecular detection, the appropriate probes should include the abilities to enter target cells noninvasively, target specific sites, and then respond to the analytes reliably. Herein, a ratiometric fluorescent DNA nanostructure (RFDN) was designed for mitochondrial adenosine triphosphate (ATP) imaging in living cells. The DNA nanostructure was constructed by continuous hybridization of two hairpin DNA strands (HS1-Cy3 and HS2-Cy5) under the initiation of the trigger. HS1-Cy3 and HS2-Cy5 contained split aptamer fragments of ATP and are labeled with a fluorescent donor (Cy3) and acceptor (Cy5), respectively. The RFDN integrated multiple split aptamer fragments and increased the local concentration of sensing probes. The binding of ATP to aptamer fragments on the RFDN shortened the distance between Cy3 and Cy5, resulting in obvious ratiometric signals (fluorescence resonance energy transfer). The RFDN showed good biocompatibility and can be internalized into cells in a caveolin-dependent endocytosis pathway. The co-localization imaging results indicated that the DNA nanostructure could target the mitochondria via Cy3 and Cy5. Moreover, the confocal imaging results showed that the intracellular ATP changes stimulated by drugs in living cells could be indicated by the RFDN. In this way, the RFDN is expected to be a simple, flexible, and general platform for chemo/biosensing in living cells.

Internal cell-penetrating peptide-mediated internalization enables a chimeric lysin to target intracellular pathogens

Intracellular pathogens pose serious challenges to the public health worldwide. Lysin, peptidoglycan hydrolase from phage, is promising alternative to conventional antibiotics because of its high bactericidal activity and low risk of resistance. However, most proteinaceous lysins cannot penetrate the mammalian cell membrane because of size exclusion. Previously, we reported a broad-spectrum chimeric lysin, ClyR, with a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin and an SH-3b cell-wall binding domain from PlySs2 lysin. Herein, we further report that a novel internal cell-penetrating peptide (CPP) is predicted in the junction region of the two constitutive domains of ClyR, mediated by which ClyR can be internalized by epithelial cells through caveolin-dependent endocytosis to target intracellular pathogens. Residues K153, P154, R169, and R188 of the internal CPP were found to be essential for ClyR-mediated internalization and intracellular killing. RNA-seq analysis further showed that there are minor differences in transcript and metabolic profiles from epithelial cells exposed to 100 μg/ml ClyR for 24 h. Taken together, our findings demonstrate a novel mechanism of internalization by ClyR, providing new insights into the rational designing of the next-generation lysins to target both extracellular and intracellular pathogens.

Endocytic pathway inhibition attenuates extracellular vesicle-induced reduction of chemosensitivity to bortezomib in multiple myeloma cells

Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal cells (BMSCs) have been demonstrated as key factors in the progression and drug resistance of multiple myeloma (MM). EV uptake involves a variety of mechanisms which largely depend on the vesicle origin and recipient cell type. The aim of the present study was to identify the mechanisms involved in the uptake of BMSC-derived small EVs (sEVs) by MM cells, and to evaluate the anti-MM effect of targeting this process.Methods: Human BMSC-derived sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of chemical inhibitors and shRNA-mediated knockdown of endocytosis-associated genes on sEV uptake and cell apoptosis were analyzed by flow cytometry. The anti-MM effect of blocking sEV uptake was evaluated in vitro and in a xenograft MM mouse model.Results: sEVs derived from BMSC were taken up by MM cells in a time- and dose-dependent manner, and subsequently promoted MM cell cycling and reduced their chemosensitivity to bortezomib. Chemical endocytosis inhibitors targeting heparin sulphate proteoglycans, actin, tyrosine kinase, dynamin-2, sodium/proton exchangers, or phosphoinositide 3-kinases significantly reduced MM cell internalization of BMSC-derived sEVs. Moreover, shRNA-mediated knockdown of endocytosis-associated proteins, including caveolin-1, flotillin-1, clathrin heavy chain, and dynamin-2 in MM cells suppressed sEV uptake. Furthermore, an endocytosis inhibitor targeting dynamin-2 preferentially suppressed the uptake of sEV by primary MM cells ex vivo and enhanced the anti-MM effects of bortezomib in vitro and in a mouse model.Conclusion: Clathrin- and caveolin-dependent endocytosis and macropinocytosis are the predominant routes of sEV-mediated communication between BMSCs and MM cells, and inhibiting endocytosis attenuates sEV-induced reduction of chemosensitivity to bortezomib, and thus enhances its anti-MM properties.