目的:探讨磁性Fe_3O_4纳米颗粒对大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白表达的影响,阐明其作用机制。方法:将24只Wistar大鼠按体质量随机分成对照组和低、中、高剂量磁性Fe_3O_4纳米颗粒组,尾静脉注射不同剂量磁性Fe_...目的:探讨磁性Fe_3O_4纳米颗粒对大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白表达的影响,阐明其作用机制。方法:将24只Wistar大鼠按体质量随机分成对照组和低、中、高剂量磁性Fe_3O_4纳米颗粒组,尾静脉注射不同剂量磁性Fe_3O_4纳米颗粒24h后取脏器组织,Western blotting法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白的表达水平,荧光实时定量PCR法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain mRNA的表达水平。结果:与对照组比较,中和高剂量组大鼠肝脏和脾脏组织中Clathrin Heavy Chain蛋白和mRNA表达水平明显升高(P<0.05)。高剂量组大鼠肾脏组织中Clathrin Heavy Chain mRNA的表达水平与其他3组比较明显升高(P<0.05)。Caveolin-1蛋白表达水平在各剂量组之间比较差异无统计学意义(P>0.05);与对照组比较,低、中和高剂量组大鼠肝脏、肺脏和脾脏组织中Caveolin-1mRNA表达水平明显升高(P<0.05);各组肾脏组织中Caveolin-1 mRNA表达水平差异无统计学意义(P>0.05)。结论:磁性Fe_3O_4纳米颗粒能够诱导大鼠肝脏、肺脏、脾脏中Clathrin Heavy Chain蛋白表达增强,通过Clathrin Heavy Chain蛋白的内吞作用是磁性Fe_3O_4纳米颗粒进入大鼠肝脏、肺脏和脾脏细胞的途径之一。展开更多
Fc 伪 R，为 IgA 的 Fc 受体，为调停 IgA 的有免疫力的回答是必要的。以前的研究证明了 IgA 和 IgA 免疫者建筑群能是很快由 Fc 伪 R 的 endocytosed。然而，内在的机制仍然保持不清楚。这里，我们在 monocytic 房间线调查了 Fc 伪 R ...Fc 伪 R，为 IgA 的 Fc 受体，为调停 IgA 的有免疫力的回答是必要的。以前的研究证明了 IgA 和 IgA 免疫者建筑群能是很快由 Fc 伪 R 的 endocytosed。然而，内在的机制仍然保持不清楚。这里，我们在 monocytic 房间线调查了 Fc 伪 R 的 endocytic 小径， U937，那自然地快速的 Fc 伪 R 并且在 transfected 汉语仓鼠卵巢(CHO ) ， COS-7 和 Hela 房间。由使用不同 endocytic 小径的选择化学禁止者， Eps15 的主导否定的异种的 overexpression 并且 clathrin 击倒经由 RNA 干扰的重链(CHC ) ，我们证明 Fc 伪 R 的 endocytosis 通过一条调停 clathrin 的小径。endocytosed Fc 伪 R 走进 Rab5 积极、 Rab11 积极的内涵体。然而， Fc 伪 R 的 endocytosis 不能被 Rab5 的主导否定的异种堵住。我们也证明 Fc 伪 R 的 endocytosis 由 overexpressing 是 dynamin 依赖的 dynamin 的主导否定的异种。为 Fc 伪 R 的潜在的 endocytic 主题也被检验。出人意料地，我们发现 Fc 伪 R 的全部细胞质的域没为 Fc 伪 R 的 endocytic 进程被要求。我们断定 Fc 伪 R 的 endocytosis 是 clathrin-dependent, 并且 dynamin 依赖，但是没被 Rab5，和 endocytic 主题调整不位于 Fc 伪 R 的细胞质的领域。展开更多
Clathrin-mediated endocytosis(CME)is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins.Clathrin triskelia are composed of clathrin heavy chains(CHCs)and ligh...Clathrin-mediated endocytosis(CME)is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins.Clathrin triskelia are composed of clathrin heavy chains(CHCs)and light chains(CLCs),and the phytohormone auxin differentially regulates membraneassociated CLCs and CHCs,modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2(PIN2).However,the molecular mechanisms by which auxin regulates clathrin are still poorly understood.Transmembrane kinase(TMKs)family proteins are considered to contribute to auxin signaling and plant development;it remains unclear whether they are involved in PIN transport by CME.We assessed TMKs involvement in the regulation of clathrin by auxin,using genetic,pharmacological,and cytological approaches including live-cell imaging and immunofluorescence.In tmk1 mutant seedlings,auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis,leading to an impaired asymmetric distribution of PIN2 and therefore auxin.Furthermore,TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin.In summary,TMK1 is essential for auxin-regulated clathrin recruitment and CME.TMK1therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.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
The establishment of auxin maxima by PIN-FORMED 3 （PIN3）- and AUXIN RESISTANT l/LIKE AUX1 （LAX） 3 （AUX1/LAX3）-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, howeve...The establishment of auxin maxima by PIN-FORMED 3 （PIN3）- and AUXIN RESISTANT l/LIKE AUX1 （LAX） 3 （AUX1/LAX3）-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, however, the underlying regulatory mechanism has remained poorly understood. Here, we show that loss of function of clathrin light chain CLC2 and CLC3 genes enhanced auxin maxima and thereby hook curvature, alleviated the inhibitory effect of auxin overproduction on auxin maxima and hook curva- ture, and delayed blue light-stimulated auxin maxima reduction and hook opening. Moreover, pharmaco- logical experiments revealed that auxin maxima formation and hook curvature in clc2 clc3 were sensitive to auxin efflux inhibitors 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid but not to the auxin influx inhibitor 1-naphthoxyacetic acid. Live-cell imaging analysis further uncovered that loss of CLC2 and CLC3 function impaired PIN3 endocytosis and promoted its lateralization in the cortical cells but did not affect AUX1 localization. Taken together, these results suggest that clathrin regulates auxin maxima and thereby hook formation through modulating PIN3 localization and auxin efflux, providing a novel mechanism that integrates developmental signals and environmental cues to regulate plant skotomorphogenesis and photomorphogenesis.展开更多
GGGGCC repeat expansions in the C9 ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia(c9 ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9 ORF72 ...GGGGCC repeat expansions in the C9 ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia(c9 ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9 ORF72 produce five dipeptide repeat(DPR) proteins by an unconventional repeat-associated non-ATG(RAN)translation. Within the five DPR proteins, poly-PR and poly-GR that contain arginine are more toxic than the other DPRs(poly-GA, poly-GP, and poly-PA). Here, we demonstrated that poly-PR peptides transferred into cells by endocytosis in a clathrin-dependent manner, leading to endoplasmic reticulum stress and cell death. In SH-SY5 Y cells and primary cortical neurons, poly-PR activated JUN amino-terminal kinase(JNK) and increased the levels of p53 and Bax. The uptake of poly-PR peptides by cells was significantly inhibited by knockdown of clathrin or by chlorpromazine, an inhibitor that blocks clathrin-mediated endocytosis. Inhibition of clathrin-dependent endocytosis by chlorpromazine significantly blocked the transfer of poly-PR peptides into cells, and attenuated poly-PRinduced JNK activation and cell death. Our data revealed that the uptake of poly-PR undergoes clathrin-dependentendocytosis and blockade of this process prevents the toxic effects of synthetic poly-PR peptides.展开更多
The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- m...The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- motic conditions disrupt the equilibrium of the plasma membrane： hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root mer- istem cells. Acute hyperosmotic treatments （ionic and nonionic） enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presum- ably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin- mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.展开更多
Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell.The endocytic pathway consists of distinct membrane compartments;internalized molecules are de...Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell.The endocytic pathway consists of distinct membrane compartments;internalized molecules are delivered to early endosomes,and some of them are recycled back to the surface,whereas other molecules are sent to late endosomes and lysosomes for degradation.However,little is known about how mitochondria are involved in the endocytic pathway.Here,we report that FM dyes, membrane-impermeant fluorescent lipid probes,can traffic to mitochondria directly from the plasma membrane by clathrinmediated endocytosis.FM dye entry into mitochondria uses microtubule-dependent active transport,but the mechanism is different from the classical endocytic pathway.Hence,this study reveals a previously unrealized lipid trafficking pathway from the plasma membrane to mitochondria.展开更多
Clathrin heavy chain(Chc)is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but ap-pears to have physiological roles also at the organisma...Clathrin heavy chain(Chc)is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but ap-pears to have physiological roles also at the organismal level.Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells.How-ever,the physiological functions of Chc in insects remain poorly understood.Here,we identified a Chc ortholog from a Locusta migratoria transcriptome database.RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs.In this develop-mental stage,LmChc showed the highest expression in the ovary followed by hemolymph,testis,hindgut,midgut,and foregut.In isolated hemocytes,we detected the Chc protein in patches at the plasma membrane.To examine the role of LmChc in L.migratoria during development,RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs.Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult.In addition,silencing of LmChc resulted in abnormal development of the ovaries,the size of which was significantly smaller than that in controls.Taken together,our results suggest that LmChc is a vital gene in L.migratoria that plays an important role in growth,development,and reproduction.LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.展开更多
PIN-FORMED(PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion,...PIN-FORMED(PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers.Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.展开更多
OBJECTIVE:To explore the neuroprotective mechanisms of Tongluo Huatan capsule(THC)in a rat model of vascular dementia(VD).METHODS:A rat model of VD was established by repeated clamping of bilateral common carotid arte...OBJECTIVE:To explore the neuroprotective mechanisms of Tongluo Huatan capsule(THC)in a rat model of vascular dementia(VD).METHODS:A rat model of VD was established by repeated clamping of bilateral common carotid arteries with the intraperitoneal injection of sodium nitroprusside solution.VD rats were administered THC,memantine hydrochloride,or distilled water daily for 14 d after operation.Learning and memory abilities were assessed using the step-down passive avoidance test,novel object recognition(NOR)test,and Morris water maze(MWM)test.Pathological changes in the hippocampus were observed through hematoxylin and eosin and Nissl staining.The expression levels of clathrin,RAB5 B,andN-methyl-D-aspartic acid receptor 1(NMDAR1)were measured by immunohistochemistry staining,real-time quantitative polymerase chain reaction and Western blot.RESULTS:Rats in VD group showed impaired learning and memory abilities(step-down passive avoidance,NOR,and MWM)and abnormalities in neuronal morphology(light microscopy)in the hippocampus.The m RNA or protein expression levels of clathrin and RAB5 B were decreased,and NMDAR1 was increased in hippocampal tissues(P<0.05).Administration of THC promoted the learning and memory abilities and the morphological structure of hippocampal neurons in VD rats.Besides,THC enhanced m RNA or protein expression levels of clathrin and RAB5 B,and decreased NMDAR1(P<0.05).CONCLUSION:THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.展开更多
文摘目的:探讨磁性Fe_3O_4纳米颗粒对大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白表达的影响,阐明其作用机制。方法:将24只Wistar大鼠按体质量随机分成对照组和低、中、高剂量磁性Fe_3O_4纳米颗粒组,尾静脉注射不同剂量磁性Fe_3O_4纳米颗粒24h后取脏器组织,Western blotting法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白的表达水平,荧光实时定量PCR法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain mRNA的表达水平。结果:与对照组比较,中和高剂量组大鼠肝脏和脾脏组织中Clathrin Heavy Chain蛋白和mRNA表达水平明显升高(P<0.05)。高剂量组大鼠肾脏组织中Clathrin Heavy Chain mRNA的表达水平与其他3组比较明显升高(P<0.05)。Caveolin-1蛋白表达水平在各剂量组之间比较差异无统计学意义(P>0.05);与对照组比较,低、中和高剂量组大鼠肝脏、肺脏和脾脏组织中Caveolin-1mRNA表达水平明显升高(P<0.05);各组肾脏组织中Caveolin-1 mRNA表达水平差异无统计学意义(P>0.05)。结论:磁性Fe_3O_4纳米颗粒能够诱导大鼠肝脏、肺脏、脾脏中Clathrin Heavy Chain蛋白表达增强,通过Clathrin Heavy Chain蛋白的内吞作用是磁性Fe_3O_4纳米颗粒进入大鼠肝脏、肺脏和脾脏细胞的途径之一。
文摘Fc 伪 R，为 IgA 的 Fc 受体，为调停 IgA 的有免疫力的回答是必要的。以前的研究证明了 IgA 和 IgA 免疫者建筑群能是很快由 Fc 伪 R 的 endocytosed。然而，内在的机制仍然保持不清楚。这里，我们在 monocytic 房间线调查了 Fc 伪 R 的 endocytic 小径， U937，那自然地快速的 Fc 伪 R 并且在 transfected 汉语仓鼠卵巢(CHO ) ， COS-7 和 Hela 房间。由使用不同 endocytic 小径的选择化学禁止者， Eps15 的主导否定的异种的 overexpression 并且 clathrin 击倒经由 RNA 干扰的重链(CHC ) ，我们证明 Fc 伪 R 的 endocytosis 通过一条调停 clathrin 的小径。endocytosed Fc 伪 R 走进 Rab5 积极、 Rab11 积极的内涵体。然而， Fc 伪 R 的 endocytosis 不能被 Rab5 的主导否定的异种堵住。我们也证明 Fc 伪 R 的 endocytosis 由 overexpressing 是 dynamin 依赖的 dynamin 的主导否定的异种。为 Fc 伪 R 的潜在的 endocytic 主题也被检验。出人意料地，我们发现 Fc 伪 R 的全部细胞质的域没为 Fc 伪 R 的 endocytic 进程被要求。我们断定 Fc 伪 R 的 endocytosis 是 clathrin-dependent, 并且 dynamin 依赖，但是没被 Rab5，和 endocytic 主题调整不位于 Fc 伪 R 的细胞质的领域。
基金supported by grants to Jianwei Pan from the National Natural Science Foundation of China (91754104,31820103008,and 31670283)grants to Chao Wang from the Youth Program of National Natural Science Foundation of China (31801193)grants to Haijun Wu from the Startup Foundation for Introducing Talent of Lanzhou University (No.561120206)。
文摘Clathrin-mediated endocytosis(CME)is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins.Clathrin triskelia are composed of clathrin heavy chains(CHCs)and light chains(CLCs),and the phytohormone auxin differentially regulates membraneassociated CLCs and CHCs,modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2(PIN2).However,the molecular mechanisms by which auxin regulates clathrin are still poorly understood.Transmembrane kinase(TMKs)family proteins are considered to contribute to auxin signaling and plant development;it remains unclear whether they are involved in PIN transport by CME.We assessed TMKs involvement in the regulation of clathrin by auxin,using genetic,pharmacological,and cytological approaches including live-cell imaging and immunofluorescence.In tmk1 mutant seedlings,auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis,leading to an impaired asymmetric distribution of PIN2 and therefore auxin.Furthermore,TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin.In summary,TMK1 is essential for auxin-regulated clathrin recruitment and CME.TMK1therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.
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.
文摘The establishment of auxin maxima by PIN-FORMED 3 （PIN3）- and AUXIN RESISTANT l/LIKE AUX1 （LAX） 3 （AUX1/LAX3）-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, however, the underlying regulatory mechanism has remained poorly understood. Here, we show that loss of function of clathrin light chain CLC2 and CLC3 genes enhanced auxin maxima and thereby hook curvature, alleviated the inhibitory effect of auxin overproduction on auxin maxima and hook curva- ture, and delayed blue light-stimulated auxin maxima reduction and hook opening. Moreover, pharmaco- logical experiments revealed that auxin maxima formation and hook curvature in clc2 clc3 were sensitive to auxin efflux inhibitors 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid but not to the auxin influx inhibitor 1-naphthoxyacetic acid. Live-cell imaging analysis further uncovered that loss of CLC2 and CLC3 function impaired PIN3 endocytosis and promoted its lateralization in the cortical cells but did not affect AUX1 localization. Taken together, these results suggest that clathrin regulates auxin maxima and thereby hook formation through modulating PIN3 localization and auxin efflux, providing a novel mechanism that integrates developmental signals and environmental cues to regulate plant skotomorphogenesis and photomorphogenesis.
基金supported by the National Natural Science Foundation of China (81761148024 and 31871023)the National Key Scientific R&D Program of China (2016YFC1306000)+1 种基金Suzhou Clinical Research Center of Neurological Disease (Szzx201503)a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China
文摘GGGGCC repeat expansions in the C9 ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia(c9 ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9 ORF72 produce five dipeptide repeat(DPR) proteins by an unconventional repeat-associated non-ATG(RAN)translation. Within the five DPR proteins, poly-PR and poly-GR that contain arginine are more toxic than the other DPRs(poly-GA, poly-GP, and poly-PA). Here, we demonstrated that poly-PR peptides transferred into cells by endocytosis in a clathrin-dependent manner, leading to endoplasmic reticulum stress and cell death. In SH-SY5 Y cells and primary cortical neurons, poly-PR activated JUN amino-terminal kinase(JNK) and increased the levels of p53 and Bax. The uptake of poly-PR peptides by cells was significantly inhibited by knockdown of clathrin or by chlorpromazine, an inhibitor that blocks clathrin-mediated endocytosis. Inhibition of clathrin-dependent endocytosis by chlorpromazine significantly blocked the transfer of poly-PR peptides into cells, and attenuated poly-PRinduced JNK activation and cell death. Our data revealed that the uptake of poly-PR undergoes clathrin-dependentendocytosis and blockade of this process prevents the toxic effects of synthetic poly-PR peptides.
文摘The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- motic conditions disrupt the equilibrium of the plasma membrane： hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root mer- istem cells. Acute hyperosmotic treatments （ionic and nonionic） enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presum- ably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin- mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.
基金the National Key Research and Development Program of China (2017YFA0104704 and 2016YFA0501000)the National Natural Science Foundation of China (31071251,31490592,and 31872773)+1 种基金Basic Research Program of Education Department of Jiangsu Province (17KJA180009)the Six TaLent Peaks Project in jiangsu Province (2017-SWYY-056).
文摘Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell.The endocytic pathway consists of distinct membrane compartments;internalized molecules are delivered to early endosomes,and some of them are recycled back to the surface,whereas other molecules are sent to late endosomes and lysosomes for degradation.However,little is known about how mitochondria are involved in the endocytic pathway.Here,we report that FM dyes, membrane-impermeant fluorescent lipid probes,can traffic to mitochondria directly from the plasma membrane by clathrinmediated endocytosis.FM dye entry into mitochondria uses microtubule-dependent active transport,but the mechanism is different from the classical endocytic pathway.Hence,this study reveals a previously unrealized lipid trafficking pathway from the plasma membrane to mitochondria.
基金supported by theNational Natural Science Foundation of China(NSFC31730074).
文摘Clathrin heavy chain(Chc)is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but ap-pears to have physiological roles also at the organismal level.Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells.How-ever,the physiological functions of Chc in insects remain poorly understood.Here,we identified a Chc ortholog from a Locusta migratoria transcriptome database.RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs.In this develop-mental stage,LmChc showed the highest expression in the ovary followed by hemolymph,testis,hindgut,midgut,and foregut.In isolated hemocytes,we detected the Chc protein in patches at the plasma membrane.To examine the role of LmChc in L.migratoria during development,RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs.Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult.In addition,silencing of LmChc resulted in abnormal development of the ovaries,the size of which was significantly smaller than that in controls.Taken together,our results suggest that LmChc is a vital gene in L.migratoria that plays an important role in growth,development,and reproduction.LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.
基金This work was supported by the National Natural Science Foundation of China(Nos.31801193,31820103008,91754104,and 31670283)the Fundamental Research Funds for the Central Universities(Nos.lzujbky-2018-28 and lzujbky-2020-it13)。
文摘PIN-FORMED(PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers.Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.
基金Supported by the National Natural Science Foundation:The Research of Shenzhi Jiannao formula Regulation on Clathrin-mediated NMDA Receptors Endocytosis of Vascular Dementia(No.81673910)。
文摘OBJECTIVE:To explore the neuroprotective mechanisms of Tongluo Huatan capsule(THC)in a rat model of vascular dementia(VD).METHODS:A rat model of VD was established by repeated clamping of bilateral common carotid arteries with the intraperitoneal injection of sodium nitroprusside solution.VD rats were administered THC,memantine hydrochloride,or distilled water daily for 14 d after operation.Learning and memory abilities were assessed using the step-down passive avoidance test,novel object recognition(NOR)test,and Morris water maze(MWM)test.Pathological changes in the hippocampus were observed through hematoxylin and eosin and Nissl staining.The expression levels of clathrin,RAB5 B,andN-methyl-D-aspartic acid receptor 1(NMDAR1)were measured by immunohistochemistry staining,real-time quantitative polymerase chain reaction and Western blot.RESULTS:Rats in VD group showed impaired learning and memory abilities(step-down passive avoidance,NOR,and MWM)and abnormalities in neuronal morphology(light microscopy)in the hippocampus.The m RNA or protein expression levels of clathrin and RAB5 B were decreased,and NMDAR1 was increased in hippocampal tissues(P<0.05).Administration of THC promoted the learning and memory abilities and the morphological structure of hippocampal neurons in VD rats.Besides,THC enhanced m RNA or protein expression levels of clathrin and RAB5 B,and decreased NMDAR1(P<0.05).CONCLUSION:THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.
