Canonical small RNAs in plants,including micro RNAs and small interfering RNAs,are key triggers of RNA interference and regulate nearly every major biological process in plants.To establish systemic silencing,small RN...Canonical small RNAs in plants,including micro RNAs and small interfering RNAs,are key triggers of RNA interference and regulate nearly every major biological process in plants.To establish systemic silencing,small RNAs undergo both short-distance intracellular trafficking or intercellular communication and longdistance transport from one organ to another,even across parasites or pathogens.This enables the delivery of effector molecules throughout the plant,promoting the spread of gene silencing.Biologically,the spatiotemporal regulation of small RNAs results in gradient distributions within cells or along the direction of organogenesis.Furthermore,the spreading capacity of small RNAs,generated in somatic or nurse cells,can guide target gene silencing in germlines in plants.In this review,we summarize recent advances in understanding the regulation and functional roles of local trafficking and long-distance transport of plant small RNAs in developmental polarity,the maintenance of cell identity,and with a particular focus,the mechanisms of small RNA movement and delivery between companion cells and gametes in plants.Additionally,we discuss the methods and challenges of monitoring small RNA transport in vivo through live imaging,as well as the potential applications of small RNA transport and delivery in the development of RNA-based pesticides.展开更多
Liver-expressed antimicrobial peptide 2(LEAP2)is a key regulator of innate immune defense in teleosts,yet the molecular basis of its chemotactic function remains largely unidentified.Boleophthalmus pectinirostris MOSP...Liver-expressed antimicrobial peptide 2(LEAP2)is a key regulator of innate immune defense in teleosts,yet the molecular basis of its chemotactic function remains largely unidentified.Boleophthalmus pectinirostris MOSPD2(BpMOSPD2)was previously identified as a candidate receptor for BpLEAP2 in monocytes/macrophages(MO/MΦ).In the present study,BpLEAP2 stimulation was found to trigger a retromer-dependent intracellular trafficking program essential for BpMOSPD2-mediated chemotaxis.Exposure to BpLEAP2 significantly enhanced BpMO/MΦmigration and promoted the accumulation of BpMOSPD2 at the plasma membrane.Subcellular fractionation and immunofluorescence analyses revealed that BpMOSPD2 translocated from the endoplasmic reticulum(ER)to early endosomes upon BpLEAP2 stimulation,followed by redistribution to the cell surface.Blockade of ER export or knockdown of core retromer subunits(BpVPS35,BpVPS26,or BpVPS29)abolished membrane localization and attenuated BpLEAP2-induced migration.Co-immunoprecipitation combined with mass spectrometry confirmed direct binding between BpMOSPD2 and BpVPS35,while domain-mapping indicated that this interaction was not exclusively dependent on MSP or CRAL-TRIO domains.Depletion of individual retromer components led to retention of BpMOSPD2 in early endosomes,establishing the necessity of the retromer complex for receptor recycling.Functionally,disruption of this complex eliminated the pro-migratory activity of BpLEAP2 on BpMO/MΦ.These findings identify the retromer complex as a critical regulator of BpMOSPD2 trafficking and uncover a previously unrecognized mechanism through which BpLEAP2 promotes MO/MΦmigration in teleosts.展开更多
Objective To explore the possible differential trafficking properties of the dopamine D 1-like receptor subtypes, D 1 receptor and D5 receptor. Methods To visualize distributions of dopamine D 1-like receptor subtypes...Objective To explore the possible differential trafficking properties of the dopamine D 1-like receptor subtypes, D 1 receptor and D5 receptor. Methods To visualize distributions of dopamine D 1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein （GFP） were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine （ACh）. Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment. Results In resting conditions, D 1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D 1 receptors appeared in the cytosol, indicating that D 1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties. Conclusion Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.展开更多
Since the first electron micrograph of“lace-like structures”over 75 years ago,the endoplasmic reticulum(ER)is now viewed as a highly dynamic,constantly remodeling,continuous network of tubules and cisternae that pla...Since the first electron micrograph of“lace-like structures”over 75 years ago,the endoplasmic reticulum(ER)is now viewed as a highly dynamic,constantly remodeling,continuous network of tubules and cisternae that plays an important role in a broad range of cellular activities from calcium regulation to protein synthesis and trafficking.In neurons,the ER extends from the soma through the axon to presynaptic terminals,and throughout the dendritic arbor into as many as half of all postsynaptic dendritic spines at any given time(Falahati et al.,2022).展开更多
Plant viruses pose significant threats to agriculture,with many vectored by insect pests.The entry of viruses and their encoded proteins into the host nucleus is a critical step for promoting some viral replication an...Plant viruses pose significant threats to agriculture,with many vectored by insect pests.The entry of viruses and their encoded proteins into the host nucleus is a critical step for promoting some viral replication and enabling systemic infection.Laodelphax striatellus,also known as the small brown planthopper(SBPH),is an efficient vector for rice stripe virus(RSV),one of the most damaging viruses of rice.In this study,we demonstrate that RSV infection induces the expression of genes in both the classical and non-classical nuclear import pathways of SBPH.A gene belonging to the importinβfamily,importin 5(LsIPO5),was upregulated by 84%in SBPH midguts infected with RSV.The nuclear localization signal(NLS,^(168)YRSPSKKRHKYV^(179))is located within the nonstructural protein NS3 directly bound to LsIPO5,thereby facilitating NS3nuclear entry.Moreover,a RING-type E3 ligase(LsRING)in SBPH,which mediated the ubiquitination of NS3 in the insect vector,enhanced NS3 binding to LsIPO5 and facilitated NS3 perinuclear localization.Combined treatment of SBPH with both ds IPO5 and ds RING significantly reduced RSV loads,highlighting the importance of LsIPO5 and NS3 ubiquitination cooperation in facilitating viral replication.Our findings provide new insights into synergistic molecular mechanisms that govern RSV infection and suggest potential therapeutic targets to control viral transmission through their insect vectors.展开更多
Introduction:One of the main events that regulate a cell’s well-being is cell-to-cell communication.This intercellular mechanism of information transfer is often mediated by vesicular trafficking.Mitochondrial-derive...Introduction:One of the main events that regulate a cell’s well-being is cell-to-cell communication.This intercellular mechanism of information transfer is often mediated by vesicular trafficking.Mitochondrial-derived vesicles(MDVs)are an emerging subpopulation of extracellular vesicle(EV)first discovered in 2008 that allow mitochondria to communicate with their surroundings.展开更多
Phosphatidylethanolamine is a major phospholipid class abundant in the brain,particularly in the inner leaflet of the plasma and mitochondrial membranes.Although it is primarily synthesized from phosphatidylserine via...Phosphatidylethanolamine is a major phospholipid class abundant in the brain,particularly in the inner leaflet of the plasma and mitochondrial membranes.Although it is primarily synthesized from phosphatidylserine via decarboxylation in mitochondria or from ethanolamine via the cytidine diphosphate-ethanolamine pathway in the endoplasmic reticulum,phosphatidylethanolamine that resides in mitochondria is preferentially produced locally and is distinct and separate from the pool of phosphatidylethanolamine made in the endoplasmic reticulum.Mitochondria-derived phosphatidylethanolamine is not only essential for mitochondrial integrity but also is exported to other organelles to fulfill diverse cellular functions.Neurons are highly enriched with phosphatidylethanolamine,and the importance of phosphatidylethanolamine metabolism in neuronal health has recently been recognized following its reported links to Alzheimer’s disease,Parkinson’s disease,and hereditary spastic paraplegia,among other neurological disorders.Indeed,disturbances in mitochondrial function and phosphatidylethanolamine metabolism and the resulting neuronal dysfunction are the common features of individuals suffering from these diseases,highlighting the great importance of maintaining proper phosphatidylethanolamine homeostasis in neurons.In this review,we summarize the current knowledge of phosphatidylethanolamine metabolism and its role in neuronal function with a special emphasis on the phosphatidylethanolamine biosynthetic pathway in mitochondria.We then review findings on how phosphatidylethanolamine biosynthesis is affected in major neurodegenerative diseases.Finally,we highlight promising future research areas that will help advance the understanding of neuronal phosphatidylethanolamine mechanisms and identify phosphatidylethanolamine-targeted therapeutic strategies for combating such brain diseases.展开更多
荧光漂白恢复(fluorescence recovery after photobleaching,FRAP)技术作为定量分析细胞质膜流动性的关键手段之一,在细胞膜动力学研究领域具有广泛的应用价值。囊泡运输(包括胞吞作用与胞吐作用)作为细胞内外物质交换的核心生物学过程...荧光漂白恢复(fluorescence recovery after photobleaching,FRAP)技术作为定量分析细胞质膜流动性的关键手段之一,在细胞膜动力学研究领域具有广泛的应用价值。囊泡运输(包括胞吞作用与胞吐作用)作为细胞内外物质交换的核心生物学过程,其运行机制高度依赖于质膜的流动性。基于科教融合的教学理念,在“细胞生物学实验”研究生课程中融入FRAP技术实验模块,其核心目标在于引导学生探究“囊泡运输”与“细胞质膜流动性”的内在相关性。该实验教学体系不仅充分凸显了FRAP技术在活细胞动态监测中的独特优势,更通过“理论知识–实验操作–数据分析”的完整训练体系,强化了学生对膜流动性与囊泡运输等基础理论知识的理解;并通过引导学生探究膜异质性及微聚体空间组织特性等细胞生物学前沿科学问题,拓展了其学术视野,为后续开展膜蛋白相关研究奠定了方法学基础。展开更多
Inositol polyphosphate 5-phosphatases （5PTases） are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormon...Inositol polyphosphate 5-phosphatases （5PTases） are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormonerelated cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid （an auxin transport inhibitor） indicate that 5PTase13 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTasel3 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PIN1 and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTasel3 deficiency. These results suggest that 5PTasel3 may modulate auxin transport by regulating vesicle trafficking and thereby play a role in root gravitropism.展开更多
Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain,but it is unclear whether this mechanism actually mediates the spinal cord dorsal hom central sensitizati...Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain,but it is unclear whether this mechanism actually mediates the spinal cord dorsal hom central sensitization(a specific form of synaptic plasticity)that is associated with persistent pain.Recent studies have shown that peripheral inflammation drives changes in ct-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR)subunit trafficking in the dorsal horn and that such changes contribute to the hypersensitivity that underlies persistent pain.Here,we review current evi-dence to illustrate how spinal cord AMPARs participate in the dorsal hom central sensitization associated with persistent pain.Understanding these mechanisms may allow the development of novel therapeutic strategies for treating persistent pain.展开更多
Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for tr...Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.展开更多
The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocont...The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.展开更多
Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system...Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.展开更多
As a typical plant virus which has biocompatibility and high transfection efficiency,tobacco mosaic virus(TMV)has shown broad application potential in drug or gene delivery field.Elucidating its intracellular traffick...As a typical plant virus which has biocompatibility and high transfection efficiency,tobacco mosaic virus(TMV)has shown broad application potential in drug or gene delivery field.Elucidating its intracellular trafficking is of great importance in investigation of its cytotoxicity,targeting site,and delivery efficiency,and is advantageous to designing new TMV-based drug delivery systems with different targets.By taking advantage of the regulated pH value of different organelles in a mammalian cell,we exploit a pH detection strategy to investigate the intracellular trafficking pathway of TMV.Here,we report a single-wavelength excited ratiometric fluorescent pH probe.This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate(FITC)and pH-insensitive rhodamine B isothiocyanate(RBIRC)onto the inner surface of TMV.The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents.By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy,it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes.This work may provide important guidance on construction of TMV-based nano carriers.展开更多
Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell.These barriers are formed by assembly of lipids and protein...Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell.These barriers are formed by assembly of lipids and protein in aqueous environments.Lipids not only serve as building blocks for membrane construction,but also possess regulatory functions in cellular activities.These regulatory lipids are non-uniformly distributed in membrane systems;their temporal and spatial accumulation in specific membranes decodes environmental cues and changes cellular activity accordingly.Phosphoinositides(PIs)are phospholipids that exert regulatory effects.In recent years,research on PIs roles in regulating plant growth,development,and responses to environmental stress is increasing.Several reviews have been published on the composition of PIs,intermolecular transferring of PIs by lipid kinases(phosphatases)or PI-PLCs,subcellular localization,and specially their functions in plant developments.Herein,we review the crucial regulatory functions of PIs in plant stress responses,with a particular focus on PIs involved in membrane trafficking.展开更多
The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also ...The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also a well-known event for understanding molecular and cellular mechanisms in retrograde endosome-to-TGN trafficking.Although it has been well established in literature that the C-terminus of bovine CI-M6PR determines its retrograde trafficking,it remains unclear whether the luminal domain of the protein plays a role on these sorting events.In this study,we found that partial deletion of luminal domain of human CI-M6PR mistargeted the mutant protein to nonTGN compartments.Moreover,replacing the luminal domain of both bovine and human CI-M6PR with that from irrelevant membrane proteins such as CD8 or Tac also altered the TGN targeting of the chimeric proteins.On the other hand,only short sequence from HA fused with the transmembrane domain and C-terminus of the receptor,HA-hCIM6PR-tail,resulted in its preferential targeting to TGN as for the full length receptor,strongly suggesting that sorting of the receptor may be influenced by luminal sequence.Furthermore,using this luminal truncated form of HA-hCIM6 PR as a model cargo,we found that the trafficking of the chimeric protein was regulated by the retromer complex through interacting with SNX5.In conclusion,our study strongly suggested that the disrupted luminal domain from hCI-M6PR or other irrelevant membrane proteins interfere with the process of membrane trafficking and TGN targeting of CI-M6PR.展开更多
基金supported by the National Key Research and Development Program of China(2024YFF100303)the National Natural Science Foundation of China(32025005)。
文摘Canonical small RNAs in plants,including micro RNAs and small interfering RNAs,are key triggers of RNA interference and regulate nearly every major biological process in plants.To establish systemic silencing,small RNAs undergo both short-distance intracellular trafficking or intercellular communication and longdistance transport from one organ to another,even across parasites or pathogens.This enables the delivery of effector molecules throughout the plant,promoting the spread of gene silencing.Biologically,the spatiotemporal regulation of small RNAs results in gradient distributions within cells or along the direction of organogenesis.Furthermore,the spreading capacity of small RNAs,generated in somatic or nurse cells,can guide target gene silencing in germlines in plants.In this review,we summarize recent advances in understanding the regulation and functional roles of local trafficking and long-distance transport of plant small RNAs in developmental polarity,the maintenance of cell identity,and with a particular focus,the mechanisms of small RNA movement and delivery between companion cells and gametes in plants.Additionally,we discuss the methods and challenges of monitoring small RNA transport in vivo through live imaging,as well as the potential applications of small RNA transport and delivery in the development of RNA-based pesticides.
基金supported by the National Natural Science Foundation of China(32173004)Natural Science Foundation of Zhejiang Province(LZ23C190001)。
文摘Liver-expressed antimicrobial peptide 2(LEAP2)is a key regulator of innate immune defense in teleosts,yet the molecular basis of its chemotactic function remains largely unidentified.Boleophthalmus pectinirostris MOSPD2(BpMOSPD2)was previously identified as a candidate receptor for BpLEAP2 in monocytes/macrophages(MO/MΦ).In the present study,BpLEAP2 stimulation was found to trigger a retromer-dependent intracellular trafficking program essential for BpMOSPD2-mediated chemotaxis.Exposure to BpLEAP2 significantly enhanced BpMO/MΦmigration and promoted the accumulation of BpMOSPD2 at the plasma membrane.Subcellular fractionation and immunofluorescence analyses revealed that BpMOSPD2 translocated from the endoplasmic reticulum(ER)to early endosomes upon BpLEAP2 stimulation,followed by redistribution to the cell surface.Blockade of ER export or knockdown of core retromer subunits(BpVPS35,BpVPS26,or BpVPS29)abolished membrane localization and attenuated BpLEAP2-induced migration.Co-immunoprecipitation combined with mass spectrometry confirmed direct binding between BpMOSPD2 and BpVPS35,while domain-mapping indicated that this interaction was not exclusively dependent on MSP or CRAL-TRIO domains.Depletion of individual retromer components led to retention of BpMOSPD2 in early endosomes,establishing the necessity of the retromer complex for receptor recycling.Functionally,disruption of this complex eliminated the pro-migratory activity of BpLEAP2 on BpMO/MΦ.These findings identify the retromer complex as a critical regulator of BpMOSPD2 trafficking and uncover a previously unrecognized mechanism through which BpLEAP2 promotes MO/MΦmigration in teleosts.
文摘Objective To explore the possible differential trafficking properties of the dopamine D 1-like receptor subtypes, D 1 receptor and D5 receptor. Methods To visualize distributions of dopamine D 1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein （GFP） were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine （ACh）. Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment. Results In resting conditions, D 1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D 1 receptors appeared in the cytosol, indicating that D 1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties. Conclusion Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.
基金supported by AHA Career Development Award 938683 (to PJD)NIH grant R01MH123700 (to MLD)
文摘Since the first electron micrograph of“lace-like structures”over 75 years ago,the endoplasmic reticulum(ER)is now viewed as a highly dynamic,constantly remodeling,continuous network of tubules and cisternae that plays an important role in a broad range of cellular activities from calcium regulation to protein synthesis and trafficking.In neurons,the ER extends from the soma through the axon to presynaptic terminals,and throughout the dendritic arbor into as many as half of all postsynaptic dendritic spines at any given time(Falahati et al.,2022).
基金supported by the Natural Science Foundation of Jiangsu Province,China(BK20240902 and BK20240904)the National Natural Science Foundation of China(32272533)。
文摘Plant viruses pose significant threats to agriculture,with many vectored by insect pests.The entry of viruses and their encoded proteins into the host nucleus is a critical step for promoting some viral replication and enabling systemic infection.Laodelphax striatellus,also known as the small brown planthopper(SBPH),is an efficient vector for rice stripe virus(RSV),one of the most damaging viruses of rice.In this study,we demonstrate that RSV infection induces the expression of genes in both the classical and non-classical nuclear import pathways of SBPH.A gene belonging to the importinβfamily,importin 5(LsIPO5),was upregulated by 84%in SBPH midguts infected with RSV.The nuclear localization signal(NLS,^(168)YRSPSKKRHKYV^(179))is located within the nonstructural protein NS3 directly bound to LsIPO5,thereby facilitating NS3nuclear entry.Moreover,a RING-type E3 ligase(LsRING)in SBPH,which mediated the ubiquitination of NS3 in the insect vector,enhanced NS3 binding to LsIPO5 and facilitated NS3 perinuclear localization.Combined treatment of SBPH with both ds IPO5 and ds RING significantly reduced RSV loads,highlighting the importance of LsIPO5 and NS3 ubiquitination cooperation in facilitating viral replication.Our findings provide new insights into synergistic molecular mechanisms that govern RSV infection and suggest potential therapeutic targets to control viral transmission through their insect vectors.
基金supported by project Emerging Infectious Diseases One Health Basic and Translational Research Actions addressing Unmet Needs on Emerging Infectious Diseases,INF-ACT,Spoke 1 and Spoke 5,Project number PE00000007,CUP B53C20040570005(to PP and DN).
文摘Introduction:One of the main events that regulate a cell’s well-being is cell-to-cell communication.This intercellular mechanism of information transfer is often mediated by vesicular trafficking.Mitochondrial-derived vesicles(MDVs)are an emerging subpopulation of extracellular vesicle(EV)first discovered in 2008 that allow mitochondria to communicate with their surroundings.
基金supported by the National Institutes of Health(grant numbers R01NS089737,RF1NS130881,and R21AG089974,to QC).
文摘Phosphatidylethanolamine is a major phospholipid class abundant in the brain,particularly in the inner leaflet of the plasma and mitochondrial membranes.Although it is primarily synthesized from phosphatidylserine via decarboxylation in mitochondria or from ethanolamine via the cytidine diphosphate-ethanolamine pathway in the endoplasmic reticulum,phosphatidylethanolamine that resides in mitochondria is preferentially produced locally and is distinct and separate from the pool of phosphatidylethanolamine made in the endoplasmic reticulum.Mitochondria-derived phosphatidylethanolamine is not only essential for mitochondrial integrity but also is exported to other organelles to fulfill diverse cellular functions.Neurons are highly enriched with phosphatidylethanolamine,and the importance of phosphatidylethanolamine metabolism in neuronal health has recently been recognized following its reported links to Alzheimer’s disease,Parkinson’s disease,and hereditary spastic paraplegia,among other neurological disorders.Indeed,disturbances in mitochondrial function and phosphatidylethanolamine metabolism and the resulting neuronal dysfunction are the common features of individuals suffering from these diseases,highlighting the great importance of maintaining proper phosphatidylethanolamine homeostasis in neurons.In this review,we summarize the current knowledge of phosphatidylethanolamine metabolism and its role in neuronal function with a special emphasis on the phosphatidylethanolamine biosynthetic pathway in mitochondria.We then review findings on how phosphatidylethanolamine biosynthesis is affected in major neurodegenerative diseases.Finally,we highlight promising future research areas that will help advance the understanding of neuronal phosphatidylethanolamine mechanisms and identify phosphatidylethanolamine-targeted therapeutic strategies for combating such brain diseases.
文摘荧光漂白恢复(fluorescence recovery after photobleaching,FRAP)技术作为定量分析细胞质膜流动性的关键手段之一,在细胞膜动力学研究领域具有广泛的应用价值。囊泡运输(包括胞吞作用与胞吐作用)作为细胞内外物质交换的核心生物学过程,其运行机制高度依赖于质膜的流动性。基于科教融合的教学理念,在“细胞生物学实验”研究生课程中融入FRAP技术实验模块,其核心目标在于引导学生探究“囊泡运输”与“细胞质膜流动性”的内在相关性。该实验教学体系不仅充分凸显了FRAP技术在活细胞动态监测中的独特优势,更通过“理论知识–实验操作–数据分析”的完整训练体系,强化了学生对膜流动性与囊泡运输等基础理论知识的理解;并通过引导学生探究膜异质性及微聚体空间组织特性等细胞生物学前沿科学问题,拓展了其学术视野,为后续开展膜蛋白相关研究奠定了方法学基础。
文摘Inositol polyphosphate 5-phosphatases （5PTases） are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormonerelated cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid （an auxin transport inhibitor） indicate that 5PTase13 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTasel3 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PIN1 and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTasel3 deficiency. These results suggest that 5PTasel3 may modulate auxin transport by regulating vesicle trafficking and thereby play a role in root gravitropism.
基金supported by grants from the National Institutes of Health(NS058886 and NS072206)Rita Allen Foundation+1 种基金Mr.David Koch and the Patrick C.Walsh Prostate Cancer Research Fundthe Blaustein Pain Research Fund
文摘Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain,but it is unclear whether this mechanism actually mediates the spinal cord dorsal hom central sensitization(a specific form of synaptic plasticity)that is associated with persistent pain.Recent studies have shown that peripheral inflammation drives changes in ct-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR)subunit trafficking in the dorsal horn and that such changes contribute to the hypersensitivity that underlies persistent pain.Here,we review current evi-dence to illustrate how spinal cord AMPARs participate in the dorsal hom central sensitization associated with persistent pain.Understanding these mechanisms may allow the development of novel therapeutic strategies for treating persistent pain.
基金supported by grants from the NIH GM114660 to Y.Zhaothe Research Grants Council of Hong Kong (CUHK466011,465112,466613,CUHK2/CRF/11G,C401114R and Ao E/M-05/12)+2 种基金NSFC/RGC (N_CUHK406/12)NSFC (31270226 and 31470294)Shenzhen Peacock Project (KQTD201101) to L.Jiang
文摘Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.
基金Supported by The National Institutes of Health grants R01GM074876 (Caplan S and Naslavsky N),R01GM087455 (Caplan S),the Nebraska Dept. of Health (Naslavsky N)P20 RR018759 from the National Center
文摘The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.
基金Supported by The Swiss Science Foundation Grant,31003A_121963
文摘Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.
基金the National Key R&D Program of China(No.2018YFC1105300)the National Natural Science Foundation of China(Nos.51703230 and 21776021)+3 种基金the Bejjing Natural Science Foundation(No.7182110)the Cross Training Plan for High Level Talents in Beiingthe Youth Innovation Promotion Association of the Chinese Academy of Sciences(No.2017039)the Presidential Foundation of Technical Institute of Physics and Chemistry.
文摘As a typical plant virus which has biocompatibility and high transfection efficiency,tobacco mosaic virus(TMV)has shown broad application potential in drug or gene delivery field.Elucidating its intracellular trafficking is of great importance in investigation of its cytotoxicity,targeting site,and delivery efficiency,and is advantageous to designing new TMV-based drug delivery systems with different targets.By taking advantage of the regulated pH value of different organelles in a mammalian cell,we exploit a pH detection strategy to investigate the intracellular trafficking pathway of TMV.Here,we report a single-wavelength excited ratiometric fluorescent pH probe.This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate(FITC)and pH-insensitive rhodamine B isothiocyanate(RBIRC)onto the inner surface of TMV.The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents.By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy,it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes.This work may provide important guidance on construction of TMV-based nano carriers.
基金the National Natural Science Foundation of China (32100553, 32171956, and 31770294)Natural Science Foundation of Jiangsu Province (BK20200555)the Fundamental Research Funds for the Central Universities, and a start-up fund for advanced talents from Nanjing Agricultural University (680-804016 to F.L.)
文摘Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell.These barriers are formed by assembly of lipids and protein in aqueous environments.Lipids not only serve as building blocks for membrane construction,but also possess regulatory functions in cellular activities.These regulatory lipids are non-uniformly distributed in membrane systems;their temporal and spatial accumulation in specific membranes decodes environmental cues and changes cellular activity accordingly.Phosphoinositides(PIs)are phospholipids that exert regulatory effects.In recent years,research on PIs roles in regulating plant growth,development,and responses to environmental stress is increasing.Several reviews have been published on the composition of PIs,intermolecular transferring of PIs by lipid kinases(phosphatases)or PI-PLCs,subcellular localization,and specially their functions in plant developments.Herein,we review the crucial regulatory functions of PIs in plant stress responses,with a particular focus on PIs involved in membrane trafficking.
基金supported by the National Nature Science Foundation of China to Y.Liu(Grant No.31371436 and No.8157051134)Y.Huang(Grant No.81500678)the laboratory start-up grant from Nanjing Medical University to Y.Liu
文摘The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also a well-known event for understanding molecular and cellular mechanisms in retrograde endosome-to-TGN trafficking.Although it has been well established in literature that the C-terminus of bovine CI-M6PR determines its retrograde trafficking,it remains unclear whether the luminal domain of the protein plays a role on these sorting events.In this study,we found that partial deletion of luminal domain of human CI-M6PR mistargeted the mutant protein to nonTGN compartments.Moreover,replacing the luminal domain of both bovine and human CI-M6PR with that from irrelevant membrane proteins such as CD8 or Tac also altered the TGN targeting of the chimeric proteins.On the other hand,only short sequence from HA fused with the transmembrane domain and C-terminus of the receptor,HA-hCIM6PR-tail,resulted in its preferential targeting to TGN as for the full length receptor,strongly suggesting that sorting of the receptor may be influenced by luminal sequence.Furthermore,using this luminal truncated form of HA-hCIM6 PR as a model cargo,we found that the trafficking of the chimeric protein was regulated by the retromer complex through interacting with SNX5.In conclusion,our study strongly suggested that the disrupted luminal domain from hCI-M6PR or other irrelevant membrane proteins interfere with the process of membrane trafficking and TGN targeting of CI-M6PR.
