Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well a...Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUTl-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane （DRM） fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiq- uitin system （SUS） to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane- associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUTl-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was ob- served between the StSUTl-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUTl-interacting proteins, a protein disulfide isomerase （PDI）, interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.展开更多
Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip gro...Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic fila- ments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP （fluorescence recovery after photobleaching） technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted ＂V＂ cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.展开更多
The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we rep...The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we report that a formin family protein,AtFH5,is localized to the vesicles and rotates ahead of Lifeact-mEGFPlabeled actin filaments during pollen germination.The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-1 abeled vesicles and the polymerization of actin filaments:vesicle-localized AtFH5 promotes actin assembly,and the polymerization and elongation of actin filaments,in turn,is essential for the mobility of AtFH5-1 abeled vesicles in pollen grains.Taken together,our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.展开更多
Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hyp...Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid （NPA）, an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membraneselective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBPl-overexpressing seedlings are less sensitive to brefeldin A （BFA） treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.展开更多
The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli.
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
We report in this study the identification of a natural product-like antagonist(1a) of Vps34 as a potent autophagy modulator via structure-based virtual screening. Aurone derivative 1a strongly inhibited Vps34 activit...We report in this study the identification of a natural product-like antagonist(1a) of Vps34 as a potent autophagy modulator via structure-based virtual screening. Aurone derivative 1a strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, 1a prevents autophagy in human cells induced either by starvation or by an mT OR inhibitor. In silico modeling and kinetic data revealed that 1a could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy in vivo and without inducing heart or liver damage in mice. 1a could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases.展开更多
在真核生物细胞中,各细胞器间物质和信息的交流是细胞生命活动的基本保证,而囊泡转运是细胞器之间物质和信息交流的主要方式。大多数的囊泡融合过程是由可溶性的N-乙基马来酰亚胺敏感因子连接物复合体(Soluble N-ethyl-maleimide-sensit...在真核生物细胞中,各细胞器间物质和信息的交流是细胞生命活动的基本保证,而囊泡转运是细胞器之间物质和信息交流的主要方式。大多数的囊泡融合过程是由可溶性的N-乙基马来酰亚胺敏感因子连接物复合体(Soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors,SNAREs)介导的,物种间的SNAREs具有高度保守的特性。与其他真核生物相比,植物的基因组编码更多的SNAREs。研究证明,植物的SNAREs是一个多功能的蛋白家族,在植物的许多生理过程中都有着重要的作用。本文对植物SNAREs作用的分子机理及生物学功能的最新研究进展做一概述。展开更多
Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors(SNAREs)complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells.SNARE assemb...Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors(SNAREs)complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells.SNARE assembly involves four different SNARE motifs,Qa,Qb,Qc,and R,providedby three orfour SNARE proteins.YKT6 is an atypical R-SNARE that lacks a transmembrane domain and is involved in multiple vesicle-target membrane fusions.Although YKT6 is evolutionarily conserved and essential,its function and regulation in different phyla seem distinct.Arabidopsis YKT61,the yeast and metazoan YKT6 homologue,is essential for gametophytic development,plays a critical role in sporophytic cells,and me-diates multiple vesicle-target membrane fusion.However,its molecular regulation is unclear.We report here that YKT61 is S-acylated.Abolishing its S-acylation by a C195S mutation dissociates YKT61 from endomembrane structures and causes its functional loss.Although interacting with various SNARE pro-teins,YKT61functions not as a canonical R-SNAREbut coordinates with otherR-SNAREs to participate in theformationof SNAREcomplexes.Phylum-specific molecular regulation of YKT6 may be evolvedto allow more efficient SNARE assembly in different eukaryotic cells.展开更多
Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vi...Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo. Organelles are major players in various cellular functions; however, the influence of nano- topography on organelles has not yet been elucidated. In the present study, a micropit-nanotube topography （MNT） was fabricated on the titanium surface, and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells. Communication between organelles, identified by organelle-specific GTPase expression, was evaluated by quantitative polymerase chain reaction and western blotting. Transmission electron microscopy was performed to evaluate the organelle structure. There were no significant differences in organelle distribution or number between the MNT and flat surface. However, organelle-specific GTPases on the MNT were dramatically downregulated. In addition, obvious endoplasmic reticulum lumen dilation was observed on the MNT surface, and the unfolded protein response （UPR） was also initiated. Regarding the relationships among organelle trafficking, UPR, and osteogenic differentiation, our findings may provide important insights into the signal transduction induced by nanotopography.展开更多
文摘Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUTl-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane （DRM） fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiq- uitin system （SUS） to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane- associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUTl-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was ob- served between the StSUTl-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUTl-interacting proteins, a protein disulfide isomerase （PDI）, interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.
基金This work was supported by grants from the Ministry of Science and Technology of China （2013CB945100） and the National Natural Science Foundation of China （31671390 and 31471266）. X.Q. was supported by post-doctoral fellowships from Tsinghua-Peking Joint Center for Life Sciences and the China Postdoctoral Science Foundation （grant no. 2015M571028）.
文摘Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic fila- ments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP （fluorescence recovery after photobleaching） technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted ＂V＂ cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.
基金grants from the National Natural Science Foundation of China (31570323 and 31770206).
文摘The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we report that a formin family protein,AtFH5,is localized to the vesicles and rotates ahead of Lifeact-mEGFPlabeled actin filaments during pollen germination.The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-1 abeled vesicles and the polymerization of actin filaments:vesicle-localized AtFH5 promotes actin assembly,and the polymerization and elongation of actin filaments,in turn,is essential for the mobility of AtFH5-1 abeled vesicles in pollen grains.Taken together,our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.
基金This work was supported by the National Natural Science Foundation of China （No. 90717001, 30721061, 30425029） and Science and Technology Commission of Shanghai Municipality （08XD14049）.We thank Jian Xu （Utrecht University, Netherlands） for providing Arabidopsis seeds containing DR5-GUS and PIN2-EGFP expression cassettes. No conflict of interest declared,
文摘Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid （NPA）, an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membraneselective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBPl-overexpressing seedlings are less sensitive to brefeldin A （BFA） treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.
基金supported by UC Davis start up fundsa Hellman fellowship to G.D. N.W. was supported by a Plant Sciences GSR and the CREATE-IGERT NSF DGE-0653984 grant
文摘The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli.
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 Hong Kong Baptist University (FRG2/ 16–17/007, FRG2/17–18/003, China)the Health and Medical Research Fund (HMRF/14150561, China)+9 种基金the Research Grants Council (HKBU/12301115, China)the National Natural Science Foundation of China (21575121 and 21775131, China)the Hong Kong Baptist University Century Club Sponsorship Scheme 2018 (China)the Interdisciplinary Research Matching Scheme (RC-IRMS/16–17/03, China)Interdisciplinary Research Clusters Matching Scheme (RC-IRCs/17–18/03, China)Innovation and Technology Fund (ITS/260/16FX, China), Matching Proof of Concept Fund (MPCF-001–2017/18, China)Collaborative Research Fund (C5026-16G, China), SKLEBA and HKBU Strategic Development Fund (SKLP_1718_P04, China)the Science and Technology Development Fund, Macao SAR (0072/ 2018/A2, China)the University of Macao (MYRG2016-00151ICMS-QRCM and MYRG2018-00187-ICMS, China)a Discovery Project Grant (DP160101682, Australia) from the Australian Research Council
文摘We report in this study the identification of a natural product-like antagonist(1a) of Vps34 as a potent autophagy modulator via structure-based virtual screening. Aurone derivative 1a strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, 1a prevents autophagy in human cells induced either by starvation or by an mT OR inhibitor. In silico modeling and kinetic data revealed that 1a could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy in vivo and without inducing heart or liver damage in mice. 1a could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases.
文摘在真核生物细胞中,各细胞器间物质和信息的交流是细胞生命活动的基本保证,而囊泡转运是细胞器之间物质和信息交流的主要方式。大多数的囊泡融合过程是由可溶性的N-乙基马来酰亚胺敏感因子连接物复合体(Soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors,SNAREs)介导的,物种间的SNAREs具有高度保守的特性。与其他真核生物相比,植物的基因组编码更多的SNAREs。研究证明,植物的SNAREs是一个多功能的蛋白家族,在植物的许多生理过程中都有着重要的作用。本文对植物SNAREs作用的分子机理及生物学功能的最新研究进展做一概述。
基金This work is supported by National Natural Science Foundation of China(31970332).
文摘Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors(SNAREs)complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells.SNARE assembly involves four different SNARE motifs,Qa,Qb,Qc,and R,providedby three orfour SNARE proteins.YKT6 is an atypical R-SNARE that lacks a transmembrane domain and is involved in multiple vesicle-target membrane fusions.Although YKT6 is evolutionarily conserved and essential,its function and regulation in different phyla seem distinct.Arabidopsis YKT61,the yeast and metazoan YKT6 homologue,is essential for gametophytic development,plays a critical role in sporophytic cells,and me-diates multiple vesicle-target membrane fusion.However,its molecular regulation is unclear.We report here that YKT61 is S-acylated.Abolishing its S-acylation by a C195S mutation dissociates YKT61 from endomembrane structures and causes its functional loss.Although interacting with various SNARE pro-teins,YKT61functions not as a canonical R-SNAREbut coordinates with otherR-SNAREs to participate in theformationof SNAREcomplexes.Phylum-specific molecular regulation of YKT6 may be evolvedto allow more efficient SNARE assembly in different eukaryotic cells.
基金This work was granted by the National Natural Science Foundation of China （Nos. 81470785 and 81530051） and Program for Changjiang Scholars and Innovative Research Team in University （No. IRT13051）. We appreciate the grant from Department of Oral and Maxillofacial Surgery, School of Stomatology, the Fourth Military Medical University. The authors also thank the help from Department of Toxicology, the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, Shaanxi Provincial Key Lab of Free Radical Biology and Medicine, School of Public Health.
文摘Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo. Organelles are major players in various cellular functions; however, the influence of nano- topography on organelles has not yet been elucidated. In the present study, a micropit-nanotube topography （MNT） was fabricated on the titanium surface, and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells. Communication between organelles, identified by organelle-specific GTPase expression, was evaluated by quantitative polymerase chain reaction and western blotting. Transmission electron microscopy was performed to evaluate the organelle structure. There were no significant differences in organelle distribution or number between the MNT and flat surface. However, organelle-specific GTPases on the MNT were dramatically downregulated. In addition, obvious endoplasmic reticulum lumen dilation was observed on the MNT surface, and the unfolded protein response （UPR） was also initiated. Regarding the relationships among organelle trafficking, UPR, and osteogenic differentiation, our findings may provide important insights into the signal transduction induced by nanotopography.
