Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Obje...Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Objectives: To establish an in vitro infection model of M. pachydermatis-exposed RK13 cells in order to evaluate cell morphological changes as well as changes in the structure of actin filaments. Methods: Cultures of RK13 cells were infected with M. pachydermatis, alterations caused by the yeast were evaluated by optical and fluorescence microscopy. Results: M. pachydermatis adheres itself to the cell and produces the formation of multiple agglomerates that cause changes in cell morphology, formation of cell aggregates in overlays, presence of syncytia and destruction of cell culture structure. The damaged cells presented changes in the actin filaments consisting of thickening of the cell cortex and loss of stress fibers. On the other hand, the formation of perinuclear actin rings in the yeasts was observed. Conclusions: An in vitro infection model was established with M. pachydermatis and alterations in cell morphology were observed consisting of changes in the structure of the actin filaments, overgrowth of the cells and the presence of syncytia.展开更多
The phragmoplast,a structure crucial for the completion of cytokinesis in plant cells,is composed of antiparallel microtubules(MTs)and actin filaments(AFs).However,how the parallel structure of phragmoplast MTs and AF...The phragmoplast,a structure crucial for the completion of cytokinesis in plant cells,is composed of antiparallel microtubules(MTs)and actin filaments(AFs).However,how the parallel structure of phragmoplast MTs and AFs is maintained,especially during centrifugal phragmoplast expansion,remains elusive.Here,we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein(AtMAC).When AtMAC was deleted,the phragmoplast showed disintegrity during centrifugal expansion,and the resulting phragmoplast fragmentation led to incomplete cell plates.Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis.Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro,and the truncated AtMAC protein,N-CC1,was the key domain controlling the ability of AtMAC.Further analysis showed that N-CC1(51–154)is the key domain for binding MTs,and N-CC1(51–125)for binding AFs.In conclusion,AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion,which is required for complete cytokinesis.展开更多
ABP41 is identified as a novel member of plant villin/gelsolin/fragmin superfamily proteins from lily pollen, which binds stoichiometrically to actin filaments and severs them in vitro. To further understand its in-vi...ABP41 is identified as a novel member of plant villin/gelsolin/fragmin superfamily proteins from lily pollen, which binds stoichiometrically to actin filaments and severs them in vitro. To further understand its in-vivo function and the potential molecular mechanisms, biochemical analysis, fluorescence microscopic observation and microinjection assays were performed. Different biochemical measurements showed that ABP41 maintained actin filaments in forms of short F-actin in vitro. Microinjection of ABP41 into pollen tubes could fragment the pre-existing actin filaments, inhibit the velocity of cytoplasmic streaming, and shorten the length of the clear zone of pollen tube. In addition, it was found that the endogenous ABP41 expressing level was dynamically corresponding to the short actin filament structure in pollen at different stages of pollen germination. Our results suggest that ABP41 is involved in the regulation of actin dynamics during the pollen germination process via maintenance of short dynamic actin filaments.展开更多
Actin filaments (AFs) in un-fixed pollen tubes of Amaryllis vittata Ait were visualized after TRITC-phalloidin staining with DMSO as a permeabilising agent. Typically, strands or hundles of microfilaments (Mfs) were d...Actin filaments (AFs) in un-fixed pollen tubes of Amaryllis vittata Ait were visualized after TRITC-phalloidin staining with DMSO as a permeabilising agent. Typically, strands or hundles of microfilaments (Mfs) were distributed in the extreme tip as well as pollen tubes in a form of network.Fluorescent granules or circles of various sizes were frequently found that continued with the filamentous structures. In addition, a more brightly stained structure, possibly Mf organizing center, was observed. Treatment of pollen tubes with cytochalasin D(CD)for increasing time intervals (5-40 minutes) caused gradual reduction of strands until flurescent granules filled up the pollen tubes. Mcanwhile, cytoplasmie streaming was inhibited completely. Though closely associated with vegetative nuclei (VN) and generative cells (GC), AFs were not found in the cytoplasm of GC.Mg++concentration greatly affected the isolated Mfs.展开更多
Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin c...Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.展开更多
A new and computationally efficient version of the immersed boundary method,which is combined with the coarse-graining method,is introduced for modeling inextensible filaments immersed in low-Reynolds number flows.Thi...A new and computationally efficient version of the immersed boundary method,which is combined with the coarse-graining method,is introduced for modeling inextensible filaments immersed in low-Reynolds number flows.This is used to represent actin biopolymers,which are constituent elements of the cytoskeleton,a complex network-like structure that plays a fundamental role in shape morphology.An extension of the traditional immersed boundary method to include a stochastic stress tensor is also proposed in order to model the thermal fluctuations in the fluid at smaller scales.By way of validation,the response of a single,massless,inextensible semiflexible filament immersed in a thermally fluctuating fluid is obtained using the suggested numerical scheme and the resulting time-averaged contraction of the filament is compared to the theoretical value obtained from the worm-like chain model.展开更多
BACKGROUND: In experimental acute pancreatitis, a large amount of reactive oxygen species are produced, and in turn cytoskeletal changes may be induced in pancreatic tissue. These changes contribute to an imbalance of...BACKGROUND: In experimental acute pancreatitis, a large amount of reactive oxygen species are produced, and in turn cytoskeletal changes may be induced in pancreatic tissue. These changes contribute to an imbalance of digestive enzyme segregation, transport, exocytosis and activation, resulting in cell injury. In this study, we assessed the effects of chondroitin sulfate (CS) on attenuation of oxidative damage and protection of F-actin in rats with acute necrotizing pancreatitis (ANP). METHODS: Ninety male Wistar rats were divided randomly into three groups. Group A was infused with 5% sodium taurocholate; group B was treated with CS; and group C served as control. Rats from the three groups were killed at 1, 3 or 8 hours. The levels were measured of malonyl dialdehyde (MDA), total superoxide dismutase (SOD), glutathione synthetase (GSH), serum amylase (SAM) and adenosine triphosphate (ATP). F-actin immunostained with rhodamine-phalloidin was analyzed using a confocal laser scanning system and the content of F-actin protein was determined. RESULTS: The levels of SAM increased in groups A and B, whereas the levels of GSH, SOD and ATP in group A decreased markedly during pancreatitis, and MDA increased significantly. The levels of GSH, SOD and ATP in group B were higher than those in group A, but the level of MDA was lower than in group A. At the same time, ANP resulted in early disruption of the cytoskeleton with dramatic changes and a loss of F-actin. Administration of CS moderated the damage to the actin cytoskeleton. CONCLUSIONS: Retrograde infusion of sodium taurocholate via the pancreatic duct may produce pancreatic necrosis and a marked increase in serum amylase activity, induce a severe depletion of ATP level, prime lipid peroxidation, and damage F-actin. Treatment with CS can ameliorate pancreatic cell conditions, limit cell membrane peroxidation, protect F-actin, and attenuate pancreatitis.展开更多
Objective:It has been documented that ezrin/radixin/moesin(ERM)phosphorylation by the p38 mitogen-activated protein kinase(MAPK),Rho/ROCK,and protein kinase C(PKC)pathways leads to filamentous actin(F-actin)reorganiza...Objective:It has been documented that ezrin/radixin/moesin(ERM)phosphorylation by the p38 mitogen-activated protein kinase(MAPK),Rho/ROCK,and protein kinase C(PKC)pathways leads to filamentous actin(F-actin)reorganization and microvascular endothelial cell hyperpermeability.In this study,we investigated the effects of Xijiao Dihuang Decoction combined with Yinqiao Powder(XDY)on influenza virus(IV)-induced F-actin restructuring and ERM phosphorylation regulated by the Rho/Rho kinase 1(ROCK),p38 MAPK,and PKC signaling pathways in pulmonary microvascular endothelial cells(PMVECs).Methods:Serum containing XDY(XDY-CS;13.8 g/kg)was acquired using standard protocols for serum pharmacology.Primary PMVECs were obtained from male Wistar rats and cultured.After adsorption of IV A(multiplicity of infection,0.01)for 1 h,medium with 20%XDY-CS was added to the PMVECs.The distributions of F-actin and phosphorylated ERM were determined by confocal microscopy,and F-actin expression was measured by flow cytometry.The expression levels of ROCK1,phosphorylated myosin phosphatase target-subunit(p-MYPT),phosphorylated MAPK kinase,phosphorylated p38(p-p38),phosphorylated PKC(p-PKC),and phosphorylated ERM(p-ERM)were determined by western blotting.Results:F-actin reorganization in IV-infected PMVECs was reversed by XDY-CS treatment,which was accompanied by reduced p-ERM production.The p-ERM protein accumulated at plasma membrane of PMVECs infected with IV,which was also inhibited by XDY-CS treatment.展开更多
文摘Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Objectives: To establish an in vitro infection model of M. pachydermatis-exposed RK13 cells in order to evaluate cell morphological changes as well as changes in the structure of actin filaments. Methods: Cultures of RK13 cells were infected with M. pachydermatis, alterations caused by the yeast were evaluated by optical and fluorescence microscopy. Results: M. pachydermatis adheres itself to the cell and produces the formation of multiple agglomerates that cause changes in cell morphology, formation of cell aggregates in overlays, presence of syncytia and destruction of cell culture structure. The damaged cells presented changes in the actin filaments consisting of thickening of the cell cortex and loss of stress fibers. On the other hand, the formation of perinuclear actin rings in the yeasts was observed. Conclusions: An in vitro infection model was established with M. pachydermatis and alterations in cell morphology were observed consisting of changes in the structure of the actin filaments, overgrowth of the cells and the presence of syncytia.
基金supported by grants from the National Natural Science Foundation of China(92254303 and 32170335)to Haiyun Ren and(32200272)to Pingzhou Duthe start-up fund of Beijing Normal University at Zhuhai(310432102)to Pingzhou Du。
文摘The phragmoplast,a structure crucial for the completion of cytokinesis in plant cells,is composed of antiparallel microtubules(MTs)and actin filaments(AFs).However,how the parallel structure of phragmoplast MTs and AFs is maintained,especially during centrifugal phragmoplast expansion,remains elusive.Here,we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein(AtMAC).When AtMAC was deleted,the phragmoplast showed disintegrity during centrifugal expansion,and the resulting phragmoplast fragmentation led to incomplete cell plates.Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis.Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro,and the truncated AtMAC protein,N-CC1,was the key domain controlling the ability of AtMAC.Further analysis showed that N-CC1(51–154)is the key domain for binding MTs,and N-CC1(51–125)for binding AFs.In conclusion,AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion,which is required for complete cytokinesis.
基金This work was supported by the National Basic Research Program of China （grant no. 2007CB108700, 2006CB100100） and the National Natural Science Foundation of China （30630005, 30470176）to H.R. No conflict of interest declared.
文摘ABP41 is identified as a novel member of plant villin/gelsolin/fragmin superfamily proteins from lily pollen, which binds stoichiometrically to actin filaments and severs them in vitro. To further understand its in-vivo function and the potential molecular mechanisms, biochemical analysis, fluorescence microscopic observation and microinjection assays were performed. Different biochemical measurements showed that ABP41 maintained actin filaments in forms of short F-actin in vitro. Microinjection of ABP41 into pollen tubes could fragment the pre-existing actin filaments, inhibit the velocity of cytoplasmic streaming, and shorten the length of the clear zone of pollen tube. In addition, it was found that the endogenous ABP41 expressing level was dynamically corresponding to the short actin filament structure in pollen at different stages of pollen germination. Our results suggest that ABP41 is involved in the regulation of actin dynamics during the pollen germination process via maintenance of short dynamic actin filaments.
文摘Actin filaments (AFs) in un-fixed pollen tubes of Amaryllis vittata Ait were visualized after TRITC-phalloidin staining with DMSO as a permeabilising agent. Typically, strands or hundles of microfilaments (Mfs) were distributed in the extreme tip as well as pollen tubes in a form of network.Fluorescent granules or circles of various sizes were frequently found that continued with the filamentous structures. In addition, a more brightly stained structure, possibly Mf organizing center, was observed. Treatment of pollen tubes with cytochalasin D(CD)for increasing time intervals (5-40 minutes) caused gradual reduction of strands until flurescent granules filled up the pollen tubes. Mcanwhile, cytoplasmie streaming was inhibited completely. Though closely associated with vegetative nuclei (VN) and generative cells (GC), AFs were not found in the cytoplasm of GC.Mg++concentration greatly affected the isolated Mfs.
基金National Science Foundation of Fujian Province of China(No.B0610031)Science and Technology Program of Put-Jan Municipality of China(No.2006N17)
文摘Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.
文摘A new and computationally efficient version of the immersed boundary method,which is combined with the coarse-graining method,is introduced for modeling inextensible filaments immersed in low-Reynolds number flows.This is used to represent actin biopolymers,which are constituent elements of the cytoskeleton,a complex network-like structure that plays a fundamental role in shape morphology.An extension of the traditional immersed boundary method to include a stochastic stress tensor is also proposed in order to model the thermal fluctuations in the fluid at smaller scales.By way of validation,the response of a single,massless,inextensible semiflexible filament immersed in a thermally fluctuating fluid is obtained using the suggested numerical scheme and the resulting time-averaged contraction of the filament is compared to the theoretical value obtained from the worm-like chain model.
文摘BACKGROUND: In experimental acute pancreatitis, a large amount of reactive oxygen species are produced, and in turn cytoskeletal changes may be induced in pancreatic tissue. These changes contribute to an imbalance of digestive enzyme segregation, transport, exocytosis and activation, resulting in cell injury. In this study, we assessed the effects of chondroitin sulfate (CS) on attenuation of oxidative damage and protection of F-actin in rats with acute necrotizing pancreatitis (ANP). METHODS: Ninety male Wistar rats were divided randomly into three groups. Group A was infused with 5% sodium taurocholate; group B was treated with CS; and group C served as control. Rats from the three groups were killed at 1, 3 or 8 hours. The levels were measured of malonyl dialdehyde (MDA), total superoxide dismutase (SOD), glutathione synthetase (GSH), serum amylase (SAM) and adenosine triphosphate (ATP). F-actin immunostained with rhodamine-phalloidin was analyzed using a confocal laser scanning system and the content of F-actin protein was determined. RESULTS: The levels of SAM increased in groups A and B, whereas the levels of GSH, SOD and ATP in group A decreased markedly during pancreatitis, and MDA increased significantly. The levels of GSH, SOD and ATP in group B were higher than those in group A, but the level of MDA was lower than in group A. At the same time, ANP resulted in early disruption of the cytoskeleton with dramatic changes and a loss of F-actin. Administration of CS moderated the damage to the actin cytoskeleton. CONCLUSIONS: Retrograde infusion of sodium taurocholate via the pancreatic duct may produce pancreatic necrosis and a marked increase in serum amylase activity, induce a severe depletion of ATP level, prime lipid peroxidation, and damage F-actin. Treatment with CS can ameliorate pancreatic cell conditions, limit cell membrane peroxidation, protect F-actin, and attenuate pancreatitis.
基金This work was supported by funding from the National Natural Science Foundation of China(Grant Nos.81473520 and 81102697).
文摘Objective:It has been documented that ezrin/radixin/moesin(ERM)phosphorylation by the p38 mitogen-activated protein kinase(MAPK),Rho/ROCK,and protein kinase C(PKC)pathways leads to filamentous actin(F-actin)reorganization and microvascular endothelial cell hyperpermeability.In this study,we investigated the effects of Xijiao Dihuang Decoction combined with Yinqiao Powder(XDY)on influenza virus(IV)-induced F-actin restructuring and ERM phosphorylation regulated by the Rho/Rho kinase 1(ROCK),p38 MAPK,and PKC signaling pathways in pulmonary microvascular endothelial cells(PMVECs).Methods:Serum containing XDY(XDY-CS;13.8 g/kg)was acquired using standard protocols for serum pharmacology.Primary PMVECs were obtained from male Wistar rats and cultured.After adsorption of IV A(multiplicity of infection,0.01)for 1 h,medium with 20%XDY-CS was added to the PMVECs.The distributions of F-actin and phosphorylated ERM were determined by confocal microscopy,and F-actin expression was measured by flow cytometry.The expression levels of ROCK1,phosphorylated myosin phosphatase target-subunit(p-MYPT),phosphorylated MAPK kinase,phosphorylated p38(p-p38),phosphorylated PKC(p-PKC),and phosphorylated ERM(p-ERM)were determined by western blotting.Results:F-actin reorganization in IV-infected PMVECs was reversed by XDY-CS treatment,which was accompanied by reduced p-ERM production.The p-ERM protein accumulated at plasma membrane of PMVECs infected with IV,which was also inhibited by XDY-CS treatment.
