Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the d...Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.展开更多
Nanomedicines employ multiple endocytic pathways to enter cells.Their following fate is interesting,but it is not sufficient understood currently.This review introduces the endocytic pathways,presents new technologies...Nanomedicines employ multiple endocytic pathways to enter cells.Their following fate is interesting,but it is not sufficient understood currently.This review introduces the endocytic pathways,presents new technologies to confirm the specific endocytic pathways and discusses factors for pathway selection.In addition,some intriguing implication about nanomedicine design based on endocytosis will also be discussed at the end.This review may provide new thoughts for the design of novel multifunctional nanomedicines.展开更多
In this study, a three-dimensional mathematical model was used to study the contribution of clathrins during the process of cellular uptake of spherical nanoparticles under different membrane tensions. The clathrin-co...In this study, a three-dimensional mathematical model was used to study the contribution of clathrins during the process of cellular uptake of spherical nanoparticles under different membrane tensions. The clathrin-coated pit (CCP) that forms around the inward budding of the cell membrane was modeled as a vesicle with bending rigidity. An optimization algorithm was proposed for minimizing the total energy of the system, which comprises the deforming nanoparticle, receptor-ligand bonds, cell membrane, and CCP, in which way, the profile of the system is acquired. The results showed that the CCP enable full wrapping of the nanoparticles at various membrane tensions. When the cell membrane tension increases, the total deformation energy also increases, but the ratio of CCP bending to the minimum value of the total energy of the system decreases. The results also showed that the diameter of the endocytic vesicles determined by the competition between the stretching of the cell membrane and confinement of the coated pits are much larger than the nanoparticles, which is quit different as the results in passive endocytosis that is not facilitated by the CCPs. The present results indicate that variations of tension on cell membranes constitutes a biophysical marker for understanding the size distribution of CCPs observed in experiments. The present results also suggest that the early abortion of endocytosis is related to that the receptor-ligand bonds cannot generate adequate force to wrap the nanoparticles into the cell membrane before the clathrins respond to support the endocytic vesicles. Correspondingly, late abortion may relate to the inability of CCPs to confine the nanoparticles until the occurrence of the necking stage of endocytosis.展开更多
Epidermal growth factor receptor(EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non...Epidermal growth factor receptor(EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer(NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors(TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligandindependent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLCassociated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligasemediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenicprocesses. Therefore, in this review, we will discuss the links between mutant EGFR signaling and endocytic properties, and introduce potential mechanisms by which altered endocytic properties of mutant EGFRs may alter signaling and vice versa as well as their implications for NSCLC therapy.展开更多
In this work,a three-dimensional axisymmetric model with nanoparticle,receptor-ligand bonds and cell membrane as a system was used to study the quasi-static receptor-mediated endocytosis process of spherical nanoparti...In this work,a three-dimensional axisymmetric model with nanoparticle,receptor-ligand bonds and cell membrane as a system was used to study the quasi-static receptor-mediated endocytosis process of spherical nanoparticles in drug delivery.The minimization of the system energy function was carried out numerically,and the deformations of nanoparticle,receptor-ligand bonds and cell membrane were predicted.Results show that passive endocytosis may fail due to the rupture of receptor-ligand bonds during the wrapping process,and the size and rigidity of nanoparticles affect the total deformation energy and the terminal wrapping stage.Our results suggest that,in addition to the energy requirement,the success of passive endocytosis also depends on the maximum strength of the receptor-ligand bonds.展开更多
Mesenchymal stem cells(MSCs)closely interact with the immune system,and they are known to secrete inflammatory cytokines in response to stress stimuli.The biological function of MSC-derived inflammatory cytokines rema...Mesenchymal stem cells(MSCs)closely interact with the immune system,and they are known to secrete inflammatory cytokines in response to stress stimuli.The biological function of MSC-derived inflammatory cytokines remains elusive.Here,we reveal that even under physiological conditions,MSCs produce and release a low level of tumor necrosis factor alpha(TNFα),which is unexpectedly required for preserving the self-renewal and differentiation of MSCs via autocrine/paracrine signaling.Furthermore,TNFαcritically maintains MSC function in vivo during bone homeostasis.Mechanistically,we unexpectedly discovered that physiological levels of TNFαsafeguard MSC homeostasis in a receptor-independent manner through mechanical force-driven endocytosis and that endocytosed TNFαbinds to mammalian target of rapamycin(mTOR)complex 2 and restricts mTOR signaling.Importantly,inhibition of mTOR signaling by rapamycin serves as an effective osteoanabolic therapeutic strategy to protect against TNFαdeficiency and mechanical unloading.Collectively,these findings unravel the physiological framework of the dynamic TNFαshuttlebased mTOR equilibrium that governs MSC and bone homeostasis.展开更多
In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis a...In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis and transport of the ConA were observed展开更多
Pulsed electric field has been used widely as a nonviral approach to improving gene delivery in basic and translational research[1-2].The technique has been called electrotransfection(ET),electroporation,electrogene t...Pulsed electric field has been used widely as a nonviral approach to improving gene delivery in basic and translational research[1-2].The technique has been called electrotransfection(ET),electroporation,electrogene transfer,and gene electroinjection in the literature [1,3].It has a great potential to improve clinical treatment of diseases through delivery of vaccines and therapeutic genes,genome and epigenome editing,and generation of human induced pluripotent stem cells for tissue engineering[1-3].During ET,extracellular transport of plasmid DNA(pDNA)relies on electrophoresis,which is critical for applications in vivo.However,mechanisms of intracellular transport remain to be understood.The lack of understanding has hindered the translation of ET technology to the clinic.It is well known that pulsed electric field can generate transient hydrophilic pores in the plasma membrane(i.e.,electroporation)that permit membrane-impermeant molecules to enter cells.Although the pores have yet to be visualized directly under a microscope,the electric field-induced membrane permeabilization has been demonstrated through experimental measurements of electrical conductance of synthetic lipid membranes and plasma membranes,direct observation of fluorescent markers crossing the membranes facing both cathode and anode,and numerical simulations of the membrane permeabilization[1,3].Results from the simulations have predicted that the cutoff size of the pores is on the order of a few hundred nanometers,and the lifetime of the pores that are larger than 100 nm is on the order of 10 msec.Although these data provide a solid evidence of the membrane permeabilization,recent studies have demonstrated that the generation of the pores is insufficient for ET[1,4].The reasons are as follows.First,the lifetime of the pores is several orders of magnitude shorter than the time scale for pDNA uptake,which is on the order of 10 min.Second,complex formation between pDNA and plasma membrane is a necessary condition for successful gene transfer.Third,inhibition of clathrin mediated endocytosis or Rac-1 dependent micropinocytosis can reduce the amount of pDNA internalized by cells [1].Finally,we demonstrate that few pDNA molecules can be observed in the cytosol that are not associated with the intracellular vesicles[5],suggesting that pDNA uptake is mediated by endocytosis.In addition to the internalization,ET requires the pDNA in the cytoplasm to reach the nucleus.To understand mechanisms of intracellular trafficking of pDNA,we have examined time-dependent pDNA distributions in cells,quantitatively determined percentages of pDNA molecules associated with different endocytic compartments using transmission electron microscopy(TEM),and investigated different approaches to facilitate cytoplasmic transport and nuclear entry of pDNA.Our data have shown that electrotransfected pDNA is located in different vesicular ultrastructures at or near the plasma membrane at10 min post application of electric pulses[5].In the hard-to-transfect cells(e.g.,4T1),pDNA penetration from the cell surface is less active,and the total number of vesicular structures associated with pDNA is low,compared to those in the easyto-transfect cells(e.g.,COS7).Our data have also shown that macropinocytosis is the most common pathway shared by all types of cells.To investigate how improve pDNA transport in cells,we have photochemically treated cells to non-specifically induce pDNA escape from intracellular vesicles,or blocked endosome and autophagic vacuole maturation through treatment of cells with Bafilomycin Al,an inhibitor of vacuolar H+ATPase.Our data demonstrate that both treatments can lead to reduction of ET efficiency although the treatment for inducing endosomal escape can enhance poly-L-lysine mediated gene delivery.These data suggest that the vesicles play an important role in protecting the naked pDNA during intracellular trafficking.The nuclear envelope is another major barrier to ET.To facilitate the nuclear entry,we have examined three different approaches.One is to synchronize the nuclear envelope breakdown(NEBD)prior to ET;the second approach is to pre-treat cells with a nuclear pore dilating agent(i.e.,trans-1,2-cyclohexanediol);and the third one is to incorporate a nuclear targeting sequence(NTS)(i.e.,SV40)into the pDNA.Our data have shown that the synchronization of the NEBD can significantly improve the ET efficiency without compromising the cell viability.The nuclear pore dilation can improve the ET as well but the dilating agent is cytotoxic.The incorporation of NTS into pDNA can improve the gene delivery efficiency but the improvement is cell-type dependent,suggesting that the NTS has to be screened and optimized for the cells of interest.In summary,the transient pores in the plasma membrane induced by the electric pulses will enable cellular uptake of membrane-impermeant molecules up to the size of small proteins.Larger molecules(e.g.,pDNA)have to be internalized via endocytic processes triggered by the pulsed electric field.Within the cells,pDNA transport is mediated by vesicles and can be blocked by non-specific escape from vesicles or inhibition of vesicle maturation.The nuclear entry of pDNA can be enhanced,without compromising cell viability,through the use of the NTS or the synchronization of the NEBD.展开更多
Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria...Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria.Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections.In a previous study,we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance.The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.Results:In the present study,we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model.The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection.This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis.Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4,Src signalling,or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria,weakened the ability of the monocytes to eliminate the bacteria,and increased the pH of the endosomes.Conclusion:Together,our results reveal the effects of TLR4 on the control of E.coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model,providing theoretical support for breeding disease resistance in the future.Furthermore,Src and caveolin 1(CAV1)could be potentially valuable targets for the control of infectious diseases.展开更多
The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjus...The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjusts the plasmalemma tension,organizes membrane protein microdomains,remodels the cell surface and drives vesicle motion in order to fine-tune exocytosis,endocytosis and recycling of secretory vesicles.In this review,we discuss how these mechanisms work in secretory cells.展开更多
Human salivary histatin 1(Hst1)exhibits a series of cell-activating properties,such as promoting cell spreading,migration,and metabolic activity.We recently have shown that fluorescently labeled Hst1(F-Hst1)targets an...Human salivary histatin 1(Hst1)exhibits a series of cell-activating properties,such as promoting cell spreading,migration,and metabolic activity.We recently have shown that fluorescently labeled Hst1(F-Hst1)targets and activates mitochondria,presenting an important molecular mechanism.However,its regulating signaling pathways remain to be elucidated.We investigated the influence of specific inhibitors of G protein-coupled receptors(GPCR),endocytosis pathways,extracellular signal-regulated kinases1/2(ERK1/2)signaling,p38 signaling,mitochondrial respiration and Na+/K+-ATPase activity on the uptake,mitochondria-targeting and-activating properties of F-Hst1.We performed a si RNA knockdown(KD)to assess the effect of Sigma-2 receptor(S2R)/Transmembrane Protein 97(TMEM97)—a recently identified target protein of Hst1.We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1.Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1.The inhibitors of GPCR,ERK1/2,phagocytosis,and clathrin-mediated endocytosis(CME)as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake,which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity.Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1.We further showed the intracellular trafficking and targeting process of F-Hst1,in which early endosome plays an important role.Overall,phagocytosis,CME,GPCR,ERK signaling,and S2R/TMEM97 are involved in the internalization of Hst1,while only CME and S2R/TMEM97 are critical for its subcellular targeting.The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.展开更多
Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal c...Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.展开更多
AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cho...AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.展开更多
Aim:Extinction of aversive memories associated with drug withdrawal has been proposed as a therapeutic strategy for the treatment of drug addiction.However,the mechanisms underlying extinction of such memory are poorl...Aim:Extinction of aversive memories associated with drug withdrawal has been proposed as a therapeutic strategy for the treatment of drug addiction.However,the mechanisms underlying extinction of such memory are poorly understood.This study was,therefore,undertaken to investigate the role of Rho GTPase Rac1-mediated GABAAR endocytosis in the vmPFC in extinction of aversive memories associated with drug withdrawal.Methods:conditioned place aversion(CPA)was used as a model for measurement of the aversive memories of opiate withdrawal.Extinction experiments were performed as described in our previous study(Wang et al.,2012).Results:we found that extinction of CPA required activation of Rac1 in the vmPFC in a brain-derived neurotrophic factor(BDNF)-dependent manner,which triggers actin polymerization via Pak1-cofilin signaling pathway,leading to synaptic localization of activity-regulated cytoskeleton-associated protein(Arc)in the vmPFC.The synaptic Arc further determines GABAA receptor(GABAAR)endocytosis that is necessary and sufficient for vmPFC long-term potentiation and CPA extinction.Thus,extinction of an aversive memory associated with drug withdrawal is intriguingly controlled by Rac1-dependent GABAAR endocytosis in the vmPFC,thereby suggesting therapeutic targets to promote extinction of the unwanted memory.Conclusion:BDNF dependent Rac1 GTPase activation in the vmPFC contributes to aversive memory extinction by Arc-mediated GABAA receptor endocytosis.展开更多
Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1...Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1L1)is a major membrane protein responsible for cholesterol absorption,in which the physiological role of vesicular endocytosis is still controversial,and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo.Here,we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model,and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption.The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets.The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine,and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol.Of note,the vesicles colocalize with the early endosomal marker early endosome antigen 1(EEA1)and the filipin-stained free cholesterol.Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles.Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process.NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.展开更多
Many types of plastic products,including polystyrene,have long been used in commercial and industrial applications.Microplastics and nanoplastics,plastic particles derived from these plastic products,are emerging as e...Many types of plastic products,including polystyrene,have long been used in commercial and industrial applications.Microplastics and nanoplastics,plastic particles derived from these plastic products,are emerging as environmental pollutants that can pose health risks to a wide variety of living organisms,including humans.However,it is not well understood how microplastics and nanoplastics affect cellular functions and induce stress responses.Humans can be exposed to polystyrene-microplastics and polystyrene-nanoplastics through ingestion,inhalation,or skin contact.Most ingested plastics are excreted from the body,but inhaled plastics may accumulate in the lungs and can even reach the brain via the nose-to-brain route.Small-sized polystyrene-nanoplastics can enter cells by endocytosis,accumulate in the cytoplasm,and cause various cellular stresses,such as inflammation with increased pro-inflammatory cytokine production,oxidative stress with generation of reactive oxygen species,and mitochondrial dysfunction.They induce autophagy activation and autophagosome formation,but autophagic flux may be impaired due to lysosomal dysfunction.Unless permanently exposed to polystyrene-nanoplastics,they can be removed from cells by exocytosis and subsequently restore cellular function.However,neurons are very susceptible to this type of stress,thus even acute exposure can lead to neurodegeneration without recovery.This review focuses specifically on recent advances in research on polystyrene-nanoplastic-induced cytotoxicity and neurotoxicity.Furthermore,in this review,based on mechanistic studies of polystyrene-nanoplastics at the cellular level other than neurons,future directions for overcoming the negative effects of polystyrene-nanoplastics on neurons were suggested.展开更多
Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different...Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.展开更多
Parkinson's disease is characterized by the selective degeneration of dopamine neurons in the nigrostriatal pathway and dopamine deficiency in the striatum.The precise reasons behind the specific degeneration of t...Parkinson's disease is characterized by the selective degeneration of dopamine neurons in the nigrostriatal pathway and dopamine deficiency in the striatum.The precise reasons behind the specific degeneration of these dopamine neurons remain largely elusive.Genetic investigations have identified over 20 causative PARK genes and 90 genomic risk loci associated with both familial and sporadic Parkinson's disease.Notably,several of these genes are linked to the synaptic vesicle recycling process,particularly the clathrinmediated endocytosis pathway.This suggests that impaired synaptic vesicle recycling might represent an early feature of Parkinson's disease,followed by axonal degeneration and the eventual loss of dopamine cell bodies in the midbrain via a"dying back"mechanism.Recently,several new animal and cellular models with Parkinson's disease-linked mutations affecting the endocytic pathway have been created and extensively characterized.These models faithfully recapitulate certain Parkinson's disease-like features at the animal,circuit,and cellular levels,and exhibit defects in synaptic membrane trafficking,further supporting the findings from human genetics and clinical studies.In this review,we will first summarize the cellular and molecular findings from the models of two Parkinson's disease-linked clathrin uncoating proteins:auxilin(DNAJC6/PARK19)and synaptojanin 1(SYNJ1/PARK20).The mouse models carrying these two PARK gene mutations phenocopy each other with specific dopamine terminal pathology and display a potent synergistic effect.Subsequently,we will delve into the involvement of several clathrin-mediated endocytosis-related proteins(GAK,endophilin A1,SAC2/INPP5 F,synaptotagmin-11),identified as Parkinson's disease risk factors through genome-wide association studies,in Parkinson's disease pathogenesis.We will also explore the direct or indirect roles of some common Parkinson's disease-linked proteins(alpha-synuclein(PARK1/4),Parkin(PARK2),and LRRK2(PARK8))in synaptic endocytic trafficking.Additionally,we will discuss the emerging novel functions of these endocytic proteins in downstream membrane traffic pathways,particularly autophagy.Given that synaptic dysfunction is considered as an early event in Parkinson's disease,a deeper understanding of the cellular mechanisms underlying synaptic vesicle endocytic trafficking may unveil novel to rgets for early diagnosis and the development of interventional therapies for Parkinson's disease.Future research should aim to elucidate why generalized synaptic endocytic dysfunction leads to the selective degeneration of nigrostriatal dopamine neurons in Parkinson's disease.展开更多
Although bulk endocytosis has been found in a number of neuronal and endocrine cells,the molecular mechanism and physiological function of bulk endocytosis remain elusive.In pancreatic beta cells,we have observed bulk...Although bulk endocytosis has been found in a number of neuronal and endocrine cells,the molecular mechanism and physiological function of bulk endocytosis remain elusive.In pancreatic beta cells,we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization.Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca^(2+) entry and suppressed by the inhibition of dynamin function.Moreover,defects in bulklike endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice,which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability.展开更多
Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind terminal sialic...Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind terminal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloroquine, can rescue H5N1-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocytosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research.展开更多
基金supported by the National Basic Research Program of China(Grant No.:2015CB553701)the National Science and Technology Major Project,China(Grant No.:2019ZX09732001).
文摘Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.
文摘Nanomedicines employ multiple endocytic pathways to enter cells.Their following fate is interesting,but it is not sufficient understood currently.This review introduces the endocytic pathways,presents new technologies to confirm the specific endocytic pathways and discusses factors for pathway selection.In addition,some intriguing implication about nanomedicine design based on endocytosis will also be discussed at the end.This review may provide new thoughts for the design of novel multifunctional nanomedicines.
基金the National Natural Science Foundation of China (Grant 11872040)the Natural Science and Engineering Research Council of Canada.
文摘In this study, a three-dimensional mathematical model was used to study the contribution of clathrins during the process of cellular uptake of spherical nanoparticles under different membrane tensions. The clathrin-coated pit (CCP) that forms around the inward budding of the cell membrane was modeled as a vesicle with bending rigidity. An optimization algorithm was proposed for minimizing the total energy of the system, which comprises the deforming nanoparticle, receptor-ligand bonds, cell membrane, and CCP, in which way, the profile of the system is acquired. The results showed that the CCP enable full wrapping of the nanoparticles at various membrane tensions. When the cell membrane tension increases, the total deformation energy also increases, but the ratio of CCP bending to the minimum value of the total energy of the system decreases. The results also showed that the diameter of the endocytic vesicles determined by the competition between the stretching of the cell membrane and confinement of the coated pits are much larger than the nanoparticles, which is quit different as the results in passive endocytosis that is not facilitated by the CCPs. The present results indicate that variations of tension on cell membranes constitutes a biophysical marker for understanding the size distribution of CCPs observed in experiments. The present results also suggest that the early abortion of endocytosis is related to that the receptor-ligand bonds cannot generate adequate force to wrap the nanoparticles into the cell membrane before the clathrins respond to support the endocytic vesicles. Correspondingly, late abortion may relate to the inability of CCPs to confine the nanoparticles until the occurrence of the necking stage of endocytosis.
基金Supported by the NIH grant to Band H,No.CA99163,CA87986,CA105489 and CA116552a Department of Defense grant to Band H,No.W81WH-11-1-0167+4 种基金the NIH grant to Band V,No.CA96844 and CA144027Department of Defense grant to Band V,No.W81XWH-07-1-0351 and W81XWH-11-1-0171the Nebraska Department of Health and Human Services LB-506 grant to Band H,No.2014-01the NCI Core Support Grant to the UNMC Buffett Cancer CenterBielecki TA was a predoctoral trainee under the NCI Institutional Cancer Biology Training Grant,No.CA009476
文摘Epidermal growth factor receptor(EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer(NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors(TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligandindependent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLCassociated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligasemediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenicprocesses. Therefore, in this review, we will discuss the links between mutant EGFR signaling and endocytic properties, and introduce potential mechanisms by which altered endocytic properties of mutant EGFRs may alter signaling and vice versa as well as their implications for NSCLC therapy.
基金This work was supported by the National Natural Science Foundations of China(11372191,11232010)the Natural Science and Engineering Research Council of Canada.
文摘In this work,a three-dimensional axisymmetric model with nanoparticle,receptor-ligand bonds and cell membrane as a system was used to study the quasi-static receptor-mediated endocytosis process of spherical nanoparticles in drug delivery.The minimization of the system energy function was carried out numerically,and the deformations of nanoparticle,receptor-ligand bonds and cell membrane were predicted.Results show that passive endocytosis may fail due to the rupture of receptor-ligand bonds during the wrapping process,and the size and rigidity of nanoparticles affect the total deformation energy and the terminal wrapping stage.Our results suggest that,in addition to the energy requirement,the success of passive endocytosis also depends on the maximum strength of the receptor-ligand bonds.
基金This work was supported by grants from the National Institute of Dental and Craniofacial Research,National Institutes of Health,Department of Health and Human Services(K99E025915 to C.C.)a Schoenleber Pilot Research Grant(to S.S.)from the University of Pennsylvania School of Dental Medicine,the Guangdong Financial Fund for High-Caliber Hospital Construction,the Postdoctoral Innovative Talents Support Program of China(BX20190380 to B.S.)the General Program of the China Postdoctoral Science Foundation(2019M663986 to B.S.).
文摘Mesenchymal stem cells(MSCs)closely interact with the immune system,and they are known to secrete inflammatory cytokines in response to stress stimuli.The biological function of MSC-derived inflammatory cytokines remains elusive.Here,we reveal that even under physiological conditions,MSCs produce and release a low level of tumor necrosis factor alpha(TNFα),which is unexpectedly required for preserving the self-renewal and differentiation of MSCs via autocrine/paracrine signaling.Furthermore,TNFαcritically maintains MSC function in vivo during bone homeostasis.Mechanistically,we unexpectedly discovered that physiological levels of TNFαsafeguard MSC homeostasis in a receptor-independent manner through mechanical force-driven endocytosis and that endocytosed TNFαbinds to mammalian target of rapamycin(mTOR)complex 2 and restricts mTOR signaling.Importantly,inhibition of mTOR signaling by rapamycin serves as an effective osteoanabolic therapeutic strategy to protect against TNFαdeficiency and mechanical unloading.Collectively,these findings unravel the physiological framework of the dynamic TNFαshuttlebased mTOR equilibrium that governs MSC and bone homeostasis.
文摘In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis and transport of the ConA were observed
基金supported by grants from National Institutes of Health ( GM098520 and GM130830)National Science Foundation ( CBET-1264186)
文摘Pulsed electric field has been used widely as a nonviral approach to improving gene delivery in basic and translational research[1-2].The technique has been called electrotransfection(ET),electroporation,electrogene transfer,and gene electroinjection in the literature [1,3].It has a great potential to improve clinical treatment of diseases through delivery of vaccines and therapeutic genes,genome and epigenome editing,and generation of human induced pluripotent stem cells for tissue engineering[1-3].During ET,extracellular transport of plasmid DNA(pDNA)relies on electrophoresis,which is critical for applications in vivo.However,mechanisms of intracellular transport remain to be understood.The lack of understanding has hindered the translation of ET technology to the clinic.It is well known that pulsed electric field can generate transient hydrophilic pores in the plasma membrane(i.e.,electroporation)that permit membrane-impermeant molecules to enter cells.Although the pores have yet to be visualized directly under a microscope,the electric field-induced membrane permeabilization has been demonstrated through experimental measurements of electrical conductance of synthetic lipid membranes and plasma membranes,direct observation of fluorescent markers crossing the membranes facing both cathode and anode,and numerical simulations of the membrane permeabilization[1,3].Results from the simulations have predicted that the cutoff size of the pores is on the order of a few hundred nanometers,and the lifetime of the pores that are larger than 100 nm is on the order of 10 msec.Although these data provide a solid evidence of the membrane permeabilization,recent studies have demonstrated that the generation of the pores is insufficient for ET[1,4].The reasons are as follows.First,the lifetime of the pores is several orders of magnitude shorter than the time scale for pDNA uptake,which is on the order of 10 min.Second,complex formation between pDNA and plasma membrane is a necessary condition for successful gene transfer.Third,inhibition of clathrin mediated endocytosis or Rac-1 dependent micropinocytosis can reduce the amount of pDNA internalized by cells [1].Finally,we demonstrate that few pDNA molecules can be observed in the cytosol that are not associated with the intracellular vesicles[5],suggesting that pDNA uptake is mediated by endocytosis.In addition to the internalization,ET requires the pDNA in the cytoplasm to reach the nucleus.To understand mechanisms of intracellular trafficking of pDNA,we have examined time-dependent pDNA distributions in cells,quantitatively determined percentages of pDNA molecules associated with different endocytic compartments using transmission electron microscopy(TEM),and investigated different approaches to facilitate cytoplasmic transport and nuclear entry of pDNA.Our data have shown that electrotransfected pDNA is located in different vesicular ultrastructures at or near the plasma membrane at10 min post application of electric pulses[5].In the hard-to-transfect cells(e.g.,4T1),pDNA penetration from the cell surface is less active,and the total number of vesicular structures associated with pDNA is low,compared to those in the easyto-transfect cells(e.g.,COS7).Our data have also shown that macropinocytosis is the most common pathway shared by all types of cells.To investigate how improve pDNA transport in cells,we have photochemically treated cells to non-specifically induce pDNA escape from intracellular vesicles,or blocked endosome and autophagic vacuole maturation through treatment of cells with Bafilomycin Al,an inhibitor of vacuolar H+ATPase.Our data demonstrate that both treatments can lead to reduction of ET efficiency although the treatment for inducing endosomal escape can enhance poly-L-lysine mediated gene delivery.These data suggest that the vesicles play an important role in protecting the naked pDNA during intracellular trafficking.The nuclear envelope is another major barrier to ET.To facilitate the nuclear entry,we have examined three different approaches.One is to synchronize the nuclear envelope breakdown(NEBD)prior to ET;the second approach is to pre-treat cells with a nuclear pore dilating agent(i.e.,trans-1,2-cyclohexanediol);and the third one is to incorporate a nuclear targeting sequence(NTS)(i.e.,SV40)into the pDNA.Our data have shown that the synchronization of the NEBD can significantly improve the ET efficiency without compromising the cell viability.The nuclear pore dilation can improve the ET as well but the dilating agent is cytotoxic.The incorporation of NTS into pDNA can improve the gene delivery efficiency but the improvement is cell-type dependent,suggesting that the NTS has to be screened and optimized for the cells of interest.In summary,the transient pores in the plasma membrane induced by the electric pulses will enable cellular uptake of membrane-impermeant molecules up to the size of small proteins.Larger molecules(e.g.,pDNA)have to be internalized via endocytic processes triggered by the pulsed electric field.Within the cells,pDNA transport is mediated by vesicles and can be blocked by non-specific escape from vesicles or inhibition of vesicle maturation.The nuclear entry of pDNA can be enhanced,without compromising cell viability,through the use of the NTS or the synchronization of the NEBD.
基金supported by National Science and Technology Major Project of China(2016ZX08008-003).
文摘Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria.Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections.In a previous study,we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance.The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.Results:In the present study,we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model.The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection.This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis.Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4,Src signalling,or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria,weakened the ability of the monocytes to eliminate the bacteria,and increased the pH of the endosomes.Conclusion:Together,our results reveal the effects of TLR4 on the control of E.coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model,providing theoretical support for breeding disease resistance in the future.Furthermore,Src and caveolin 1(CAV1)could be potentially valuable targets for the control of infectious diseases.
基金This work was supported by the Grants PICT 2764-2016,PICT 02849-2018 and PICT 02041-2019 from the Agencia Nacional de Promoción de la Investigación,el Desarrollo Tecnológico y la Innovación(Argentina)ICN09_022 from ICM-ANID(Chile).
文摘The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjusts the plasmalemma tension,organizes membrane protein microdomains,remodels the cell surface and drives vesicle motion in order to fine-tune exocytosis,endocytosis and recycling of secretory vesicles.In this review,we discuss how these mechanisms work in secretory cells.
基金funded by Eurostars project,grant number E!12764。
文摘Human salivary histatin 1(Hst1)exhibits a series of cell-activating properties,such as promoting cell spreading,migration,and metabolic activity.We recently have shown that fluorescently labeled Hst1(F-Hst1)targets and activates mitochondria,presenting an important molecular mechanism.However,its regulating signaling pathways remain to be elucidated.We investigated the influence of specific inhibitors of G protein-coupled receptors(GPCR),endocytosis pathways,extracellular signal-regulated kinases1/2(ERK1/2)signaling,p38 signaling,mitochondrial respiration and Na+/K+-ATPase activity on the uptake,mitochondria-targeting and-activating properties of F-Hst1.We performed a si RNA knockdown(KD)to assess the effect of Sigma-2 receptor(S2R)/Transmembrane Protein 97(TMEM97)—a recently identified target protein of Hst1.We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1.Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1.The inhibitors of GPCR,ERK1/2,phagocytosis,and clathrin-mediated endocytosis(CME)as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake,which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity.Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1.We further showed the intracellular trafficking and targeting process of F-Hst1,in which early endosome plays an important role.Overall,phagocytosis,CME,GPCR,ERK signaling,and S2R/TMEM97 are involved in the internalization of Hst1,while only CME and S2R/TMEM97 are critical for its subcellular targeting.The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.
文摘Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.
基金Supported by the Austrian Science Fund,No.P20116-B13 and No.P22838-B13
文摘AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.
文摘Aim:Extinction of aversive memories associated with drug withdrawal has been proposed as a therapeutic strategy for the treatment of drug addiction.However,the mechanisms underlying extinction of such memory are poorly understood.This study was,therefore,undertaken to investigate the role of Rho GTPase Rac1-mediated GABAAR endocytosis in the vmPFC in extinction of aversive memories associated with drug withdrawal.Methods:conditioned place aversion(CPA)was used as a model for measurement of the aversive memories of opiate withdrawal.Extinction experiments were performed as described in our previous study(Wang et al.,2012).Results:we found that extinction of CPA required activation of Rac1 in the vmPFC in a brain-derived neurotrophic factor(BDNF)-dependent manner,which triggers actin polymerization via Pak1-cofilin signaling pathway,leading to synaptic localization of activity-regulated cytoskeleton-associated protein(Arc)in the vmPFC.The synaptic Arc further determines GABAA receptor(GABAAR)endocytosis that is necessary and sufficient for vmPFC long-term potentiation and CPA extinction.Thus,extinction of an aversive memory associated with drug withdrawal is intriguingly controlled by Rac1-dependent GABAAR endocytosis in the vmPFC,thereby suggesting therapeutic targets to promote extinction of the unwanted memory.Conclusion:BDNF dependent Rac1 GTPase activation in the vmPFC contributes to aversive memory extinction by Arc-mediated GABAA receptor endocytosis.
基金This work was supported by grants from the National Key R&D Program and the National Natural Science Foundation of China(2019YFA0802503,91857203,2018YFA0800602)Collaborative Innovation Program of Shanghai Municipal Health Commission(2020CXJQ01).
文摘Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1L1)is a major membrane protein responsible for cholesterol absorption,in which the physiological role of vesicular endocytosis is still controversial,and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo.Here,we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model,and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption.The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets.The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine,and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol.Of note,the vesicles colocalize with the early endosomal marker early endosome antigen 1(EEA1)and the filipin-stained free cholesterol.Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles.Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process.NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.
基金supported by the Basic Study and Interdisciplinary R&D Foundation of the University of Seoul(2019)grants,Nos.201910021035202006251003(both to KYR and JC)。
文摘Many types of plastic products,including polystyrene,have long been used in commercial and industrial applications.Microplastics and nanoplastics,plastic particles derived from these plastic products,are emerging as environmental pollutants that can pose health risks to a wide variety of living organisms,including humans.However,it is not well understood how microplastics and nanoplastics affect cellular functions and induce stress responses.Humans can be exposed to polystyrene-microplastics and polystyrene-nanoplastics through ingestion,inhalation,or skin contact.Most ingested plastics are excreted from the body,but inhaled plastics may accumulate in the lungs and can even reach the brain via the nose-to-brain route.Small-sized polystyrene-nanoplastics can enter cells by endocytosis,accumulate in the cytoplasm,and cause various cellular stresses,such as inflammation with increased pro-inflammatory cytokine production,oxidative stress with generation of reactive oxygen species,and mitochondrial dysfunction.They induce autophagy activation and autophagosome formation,but autophagic flux may be impaired due to lysosomal dysfunction.Unless permanently exposed to polystyrene-nanoplastics,they can be removed from cells by exocytosis and subsequently restore cellular function.However,neurons are very susceptible to this type of stress,thus even acute exposure can lead to neurodegeneration without recovery.This review focuses specifically on recent advances in research on polystyrene-nanoplastic-induced cytotoxicity and neurotoxicity.Furthermore,in this review,based on mechanistic studies of polystyrene-nanoplastics at the cellular level other than neurons,future directions for overcoming the negative effects of polystyrene-nanoplastics on neurons were suggested.
基金supported by the National Natural Science Foundation of China(U22A20520)the Innovation Team Project of Modern Agricultural Industrial Technology System of Guangdong Province,China(2023KJ119)the Natural Science Foundation Program of Guangdong Province,China(2023A1515012206)。
文摘Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.
文摘Parkinson's disease is characterized by the selective degeneration of dopamine neurons in the nigrostriatal pathway and dopamine deficiency in the striatum.The precise reasons behind the specific degeneration of these dopamine neurons remain largely elusive.Genetic investigations have identified over 20 causative PARK genes and 90 genomic risk loci associated with both familial and sporadic Parkinson's disease.Notably,several of these genes are linked to the synaptic vesicle recycling process,particularly the clathrinmediated endocytosis pathway.This suggests that impaired synaptic vesicle recycling might represent an early feature of Parkinson's disease,followed by axonal degeneration and the eventual loss of dopamine cell bodies in the midbrain via a"dying back"mechanism.Recently,several new animal and cellular models with Parkinson's disease-linked mutations affecting the endocytic pathway have been created and extensively characterized.These models faithfully recapitulate certain Parkinson's disease-like features at the animal,circuit,and cellular levels,and exhibit defects in synaptic membrane trafficking,further supporting the findings from human genetics and clinical studies.In this review,we will first summarize the cellular and molecular findings from the models of two Parkinson's disease-linked clathrin uncoating proteins:auxilin(DNAJC6/PARK19)and synaptojanin 1(SYNJ1/PARK20).The mouse models carrying these two PARK gene mutations phenocopy each other with specific dopamine terminal pathology and display a potent synergistic effect.Subsequently,we will delve into the involvement of several clathrin-mediated endocytosis-related proteins(GAK,endophilin A1,SAC2/INPP5 F,synaptotagmin-11),identified as Parkinson's disease risk factors through genome-wide association studies,in Parkinson's disease pathogenesis.We will also explore the direct or indirect roles of some common Parkinson's disease-linked proteins(alpha-synuclein(PARK1/4),Parkin(PARK2),and LRRK2(PARK8))in synaptic endocytic trafficking.Additionally,we will discuss the emerging novel functions of these endocytic proteins in downstream membrane traffic pathways,particularly autophagy.Given that synaptic dysfunction is considered as an early event in Parkinson's disease,a deeper understanding of the cellular mechanisms underlying synaptic vesicle endocytic trafficking may unveil novel to rgets for early diagnosis and the development of interventional therapies for Parkinson's disease.Future research should aim to elucidate why generalized synaptic endocytic dysfunction leads to the selective degeneration of nigrostriatal dopamine neurons in Parkinson's disease.
基金supported by a grant from the National Natural Science Foundation of China(Grant No.30871225)a grant from Beijing Municipal Science and Technology Commission(Grant No.7121008)+1 种基金a grant from the Ministry of Science and Technology(Grant No.SQ2011SF11B01041)the fund from The Key Construction Program of the National“985”Project from the Department of Education of China to Peking University.
文摘Although bulk endocytosis has been found in a number of neuronal and endocrine cells,the molecular mechanism and physiological function of bulk endocytosis remain elusive.In pancreatic beta cells,we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization.Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca^(2+) entry and suppressed by the inhibition of dynamin function.Moreover,defects in bulklike endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice,which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability.
基金Supported by the National Natural Science Foundation of China (Grant No. 30623009)National Basic Research Program of China (Grant No. 2005CB523000)
文摘Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind terminal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloroquine, can rescue H5N1-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocytosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research.
