AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological character...AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine sple展开更多
Extracellular vesicles(EVs)are small membranous particles that can mediate cell-to-cell communication and which are divided into at least three categories according to their subcellular origin and size:exosomes,microv...Extracellular vesicles(EVs)are small membranous particles that can mediate cell-to-cell communication and which are divided into at least three categories according to their subcellular origin and size:exosomes,microvesicles,and apoptotic bodies.Exosomes are the smallest(30–150 nm)of these EVs,and play an important role in EV-mediated cell-to-cell interactions,by transferring proteins,nucleic acids and,lipids from their parental cells to adjacent or distant cells to alter their phenotypes.Most exosome studies in the past two decades have focused on their nucleic acid composition and their transfer ofmRNAs and microRNAs to neighboring cells.However,exosomes also carry specific membrane proteins that can identify the physiological and pathological states of their parental cells or indicate their preferential target cells or tissues.Exosome membrane protein expression can also be directly employed or modified to allow exosomes to serve as drug delivery systems and therapeutic platforms,including in targeted therapy approaches.This review will briefly summarize information on exosome membrane proteins components and their role in exosome–cell interactions,including proteins associated with specific cell-interactions and diseases,and the potential for using exosome membrane proteins in therapeutic targeting approaches.展开更多
Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formati...Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid （Y-2-H） assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular florescence complementation （BIFC） microscopy, sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants （PRsec6） displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokine- sis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machin- ery or directly regulate membrane fusion during cell plate formation in plants.展开更多
Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha...Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha-dystroglycan(α-DG)to its endosomal receptor lysosome-associated membrane protein 1(LAMP1).A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch.To test the hypothesis that other non-conserved residues also contribute to receptor switch,we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1.Four residues,L84,K88,L107,and H170,were identified as critical for receptor switch.Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus(LCMV)residue(L84 N,K88E,L10F,and H170S)reduced the binding affinity of LASV GP1 for LAMP1.Moreover,all mutations caused decreases in glycoprotein precursor(GPC)-mediated membrane fusion at both pH 4.5 and 5.2.The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types,while L107F and H170S had only mild effects on infectivity.Using biolayer light interferometry assay,we found that all four mutants had decreased binding affinity to LAMP1,in the order of binding affinity being L84 N>L107F>K88E>H170S.The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.展开更多
The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with...The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment.In particular,proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes.In this review,the potential of enhanced molecular dynamics to address cell-biology problems is discussed.The cases of membrane deformation,membrane fusion,and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables.Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented.Finally,novel strategies merging cell biology and physics are considered as future lines of research.展开更多
Exosomes are membrane-bound nanoscale extracellular vesicles,which produced by almost all organisms.Due to the excellent biocompatibility,long circulation time as well as low immunogenicity,exosomes as naturally-deriv...Exosomes are membrane-bound nanoscale extracellular vesicles,which produced by almost all organisms.Due to the excellent biocompatibility,long circulation time as well as low immunogenicity,exosomes as naturally-derived drug delivery carriers have experienced explosive growth over the past decades.However,issues such as insufficient loading efficiency,heterogeneous delivery efficiency,uncontrollable targeting ability,and low production limit their wide application.Recently,the emerging exosome-liposome fusion strategy has become a potential approach to solve such issues.Thus,this review mainly focuses on the currently developed exosome-liposome fusion strategy and their application in drug delivery as well as disease treatment.This review aims to shed light on the advantages of fusion strategy in drug delivery and provides a better understanding for more rational design.The current challenge and future perspective regarding their clinical translation and application will also be discussed.展开更多
Influenza A virus(IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membran...Influenza A virus(IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor(M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR,the nuclear accumulation of viral nucleoprotein(NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking,or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin(HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes,thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR–HA interaction in the fusion of viral and late endosomal membranes during IAV replication.展开更多
Viral envelope fusion with the host plasma membrane(PM)for genome release is a hallmark step in the life cycle of many enveloped viruses.This process is regulated by a complex network of biomolecules on the PM,but rob...Viral envelope fusion with the host plasma membrane(PM)for genome release is a hallmark step in the life cycle of many enveloped viruses.This process is regulated by a complex network of biomolecules on the PM,but robust tools to precisely elucidate the dynamic mechanisms of virus-PM fusion events are still lacking.Here,we developed a quantitative single-virus tracking approach based on highly efficient dual-color labelling of viruses and batch trajectory analysis to achieve the spatiotemporal quantification of fusion events.This approach allows us to comprehensively analyze the membrane fusion mechanism utilized by pseudotyped severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)at the singlevirus level and precisely elucidate how the relevant biomolecules synergistically regulate the fusion process.Our results revealed that SARS-CoV-2 may promote the formation of supersaturated clusters of cholesterol to facilitate the initiation of the membrane fusion process and accelerate the viral genome release.展开更多
基金Supported jby the Natural Science Foundation of Guangdong Province China,No.980180
文摘AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine sple
基金The work was partially supported by research funding provided by the National Institutes of Health(Grants No.U01CA214254,R01HD090927,R01AI122932,R01AI113725,and R21Al126361-01),and Arizona Biomedical Research Commission(ABRC)young investigator award.
文摘Extracellular vesicles(EVs)are small membranous particles that can mediate cell-to-cell communication and which are divided into at least three categories according to their subcellular origin and size:exosomes,microvesicles,and apoptotic bodies.Exosomes are the smallest(30–150 nm)of these EVs,and play an important role in EV-mediated cell-to-cell interactions,by transferring proteins,nucleic acids and,lipids from their parental cells to adjacent or distant cells to alter their phenotypes.Most exosome studies in the past two decades have focused on their nucleic acid composition and their transfer ofmRNAs and microRNAs to neighboring cells.However,exosomes also carry specific membrane proteins that can identify the physiological and pathological states of their parental cells or indicate their preferential target cells or tissues.Exosome membrane protein expression can also be directly employed or modified to allow exosomes to serve as drug delivery systems and therapeutic platforms,including in targeted therapy approaches.This review will briefly summarize information on exosome membrane proteins components and their role in exosome–cell interactions,including proteins associated with specific cell-interactions and diseases,and the potential for using exosome membrane proteins in therapeutic targeting approaches.
基金grants from the National Natural Science Foundation of China，a Research Fund for the Doctoral Program of Higher Education
文摘Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid （Y-2-H） assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular florescence complementation （BIFC） microscopy, sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants （PRsec6） displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokine- sis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machin- ery or directly regulate membrane fusion during cell plate formation in plants.
基金supported by the National Key Research and Development Program of China(2023YFC2605504,2022YFC2303300)the National Natural Sciences Foundation of China(82172273 and 31670165)+3 种基金the Open Research Fund Program of the State Key Laboratory of Virology of China(2023JZZD-01)the Health research project of Shaanxi Province(2022D040)the Science and Technology Planning Project of Shaanxi Provincial Education Department(22JK0545)the Natural Science Basic Research Program of Shaanxi(2024JC-YBQN-0922).
文摘Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha-dystroglycan(α-DG)to its endosomal receptor lysosome-associated membrane protein 1(LAMP1).A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch.To test the hypothesis that other non-conserved residues also contribute to receptor switch,we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1.Four residues,L84,K88,L107,and H170,were identified as critical for receptor switch.Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus(LCMV)residue(L84 N,K88E,L10F,and H170S)reduced the binding affinity of LASV GP1 for LAMP1.Moreover,all mutations caused decreases in glycoprotein precursor(GPC)-mediated membrane fusion at both pH 4.5 and 5.2.The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types,while L107F and H170S had only mild effects on infectivity.Using biolayer light interferometry assay,we found that all four mutants had decreased binding affinity to LAMP1,in the order of binding affinity being L84 N>L107F>K88E>H170S.The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.
基金Grants from CONICET(PIP-0409CO)ANPCyT(PICT2020-1897)are gratefully acknowledged。
文摘The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment.In particular,proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes.In this review,the potential of enhanced molecular dynamics to address cell-biology problems is discussed.The cases of membrane deformation,membrane fusion,and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables.Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented.Finally,novel strategies merging cell biology and physics are considered as future lines of research.
基金supported by the National Key Research&Development Program of China(Nos.2021YFC2302400,2021YFA1201000,2021YFE0106900)the National Natural Science Foundation of China(Nos.32171394,32101148,82202338)+2 种基金the Beijing Nova Program(Interdisciplinary Cooperation Project)from Beijing Municipal Science&Technology Commission(No.20220484207)the Beijing Natural Science Foundation(No.L222128)the Fundamental Research Funds for the Central Universities(No.2022CX01013).
文摘Exosomes are membrane-bound nanoscale extracellular vesicles,which produced by almost all organisms.Due to the excellent biocompatibility,long circulation time as well as low immunogenicity,exosomes as naturally-derived drug delivery carriers have experienced explosive growth over the past decades.However,issues such as insufficient loading efficiency,heterogeneous delivery efficiency,uncontrollable targeting ability,and low production limit their wide application.Recently,the emerging exosome-liposome fusion strategy has become a potential approach to solve such issues.Thus,this review mainly focuses on the currently developed exosome-liposome fusion strategy and their application in drug delivery as well as disease treatment.This review aims to shed light on the advantages of fusion strategy in drug delivery and provides a better understanding for more rational design.The current challenge and future perspective regarding their clinical translation and application will also be discussed.
基金supported by the National Natural Science Foundation of China(32192453,32172847)the National Key Research and Development Program of China(2021YFD1800204)+1 种基金the Laboratory of Lingnan Modern Agriculture Project(NT2021007)the earmarked fund for CARS-41。
文摘Influenza A virus(IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor(M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR,the nuclear accumulation of viral nucleoprotein(NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking,or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin(HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes,thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR–HA interaction in the fusion of viral and late endosomal membranes during IAV replication.
基金supported by the National Natural Science Foundation of China(22293032,22293030,and 91859123)the National Key Research and Development Program of China(2019YFA0210500)+1 种基金the Fundamental Research Funds for the Central Universities of China(63211023)the financial support from Haihe Laboratory of Sustainable Chemical Transformations.
文摘Viral envelope fusion with the host plasma membrane(PM)for genome release is a hallmark step in the life cycle of many enveloped viruses.This process is regulated by a complex network of biomolecules on the PM,but robust tools to precisely elucidate the dynamic mechanisms of virus-PM fusion events are still lacking.Here,we developed a quantitative single-virus tracking approach based on highly efficient dual-color labelling of viruses and batch trajectory analysis to achieve the spatiotemporal quantification of fusion events.This approach allows us to comprehensively analyze the membrane fusion mechanism utilized by pseudotyped severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)at the singlevirus level and precisely elucidate how the relevant biomolecules synergistically regulate the fusion process.Our results revealed that SARS-CoV-2 may promote the formation of supersaturated clusters of cholesterol to facilitate the initiation of the membrane fusion process and accelerate the viral genome release.
