Objective To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level. Methods Immature mouse oocytes were cultured in vitro with a known dynein ATPase activ...Objective To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level. Methods Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level. Results In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 mmol/L SOV and control groups (P<0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 mmol/L. In incon- tinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 mmol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P<0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed. Conclusion Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or MⅠto anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.展开更多
We propose a dynamic mechanism for the processive motility of dynein on microtubules (MTs). The force generated for the motion of dynein is purely mechanical in origin. When a dynein monomer binds to a MT, the AAA rin...We propose a dynamic mechanism for the processive motility of dynein on microtubules (MTs). The force generated for the motion of dynein is purely mechanical in origin. When a dynein monomer binds to a MT, the AAA ring of dynein might fit into one of the trenches on the outer surface of the MT, with the linker domain leaning on the ratchet-shaped protofilament. At room temperature, the dynein molecule exhibits random thermal motion on the outer surface of the MT. The collision between the asymmetric ratchet teeth and the linker exerts a reactive impulsive force on the dynein molecule. The probability of producing an impulse with a longitudinal component pointing to either end of the MT depends on the instantaneous motion of dynein, the shape of the linker, and the mass distribution of the dynein with/without a load. In the dynamic mechanism, dynein monomers can move independently and processively toward either end of the MT. Many observations of the motility of dynein can be reproduced in a simulation system.展开更多
Kinesin 和达因在是为追踪的活跃细胞器和房间分割负责的分子的马达的二个重要的班。他们能一起工作带货物，沿着以一个协调方法的微导管移动。我们使用蒙特卡罗方法模仿这个协调运动的动力学。基于四个必要假设，我们的模拟复制一些特...Kinesin 和达因在是为追踪的活跃细胞器和房间分割负责的分子的马达的二个重要的班。他们能一起工作带货物，沿着以一个协调方法的微导管移动。我们使用蒙特卡罗方法模仿这个协调运动的动力学。基于四个必要假设，我们的模拟复制一些特征在 vivo 实验最近。货物的快移动速度被模仿，速度分发被介绍。展开更多
Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that drive ciliary beat have been extensively studied i...Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that drive ciliary beat have been extensively studied in unicellular Chlamydomonas, to what extent such knowledge can be applied to vertebrate is poorly known. In Chlamydomonas, Dynein-f controls flagellar waveforms but is dispensable for beating. The flagellar assembly of its heavy chains (HCs) requires its intermediate chain (IC) IC140 but not IC138. Here we show that, unlike its Chlamydomonas counte『part, vertebrate Dynein-f is essential for ciliary beat. We confirmed that Wdr78 is the vertebrate orthologue of IC3 Wdr78 associated with Dynein-f subunits such as Dnah2 (a HC) and Wdr63 (IC140 orthologue). It was expressed as a motile cilium-specific protein in mammalian cells. Depletion of Wdr78 or Dnah2 by RNAi paralyzed mouse ependymal cilia. Zebrafish Wdr78 morphants displayed ciliopathy-related phenotypes, such as curved bodies, hydrocephalus, abnormal otolith, randomized left-right asymmetry, and pronephric cysts, accompanied with paralyzed pronephric cilia. Furthermore, all the HCs and ICs of Dynein-f failed to localize in the Wdr78-depleted mouse ependymal cilia. Therefore, both the functions and subunit dependency of Dynein-fare altered in evolution, probably to comply with ciliary roles in higher organisms.展开更多
The dynein motor protein family is involved in a wide variety of functions in eukaryotic cells. The axonemal dynein class and cytoplasmic dynein-1 subclass have been well characterized. However, the cytoplasmic dynein...The dynein motor protein family is involved in a wide variety of functions in eukaryotic cells. The axonemal dynein class and cytoplasmic dynein-1 subclass have been well characterized. However, the cytoplasmic dynein-2 subclass of the family has only recently begun to be understood. We describe the entire dynein family but focus on cytoplasmic dynein-2. Dynein-2 consists of a heavy, an intermediate, a light intermediate, and a light chain. The complex appears to function primarily as the retrograde motor for intraflagellar transport. This process is important for the formation and maintenance of cilia and flagella. Additionally, dynein-2 has roles in the control of ciliary length and in non-ciliary functions. Mutations in the human dynein-2 heavy chain lead to cilia-related diseases.展开更多
The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins,which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors(DNAAFs)before being transported to and assembled o...The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins,which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors(DNAAFs)before being transported to and assembled on the ciliary axoneme.Here,we characterize the function of WDR92 in Chlamydomonas.Loss of WDR92,a cytoplasmic protein,in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella,disappearance of axonemal dynein arms,and diminishment of dynein arm heavy chains in the cytoplasm,suggesting that WDR92 is a DNAAF.Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBLl,RPAP3,MOT48,ODA7,and DYX1C.The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBLl/2 and RPAP3,while PF13,another PIH1 domain-containing protein with function in dynein preassembly,did not.Interestingly,the third PIH1 domain-containing protein TWI1 was not related to flagellar motility.WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs.Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.展开更多
Chordotonal neurons are responsible for sound sensation in Drosophila.However,little is known about how they respond to sound with high sensitivity.Using genetic labeling,we found one of the Drosophila axonemal dynein...Chordotonal neurons are responsible for sound sensation in Drosophila.However,little is known about how they respond to sound with high sensitivity.Using genetic labeling,we found one of the Drosophila axonemal dynein heavy chains,CG9492(DNAH5),was specifically expressed in larval chordotonal neurons and showed a distribution restricted to proximal cilia.While DNAH5 mutation did not affect the cilium morphology or the trafficking of Inactive,a candidate auditory transduction channel,larvae with DNAH5 mutation had reduced startle responses to sound at low and medium intensities.Calcium imaging confirmed that DNAH5 functioned autonomously in chordotonal neurons for larval sound sensation.Furthermore,disrupting DNAH5 resulted in a decrease of spike firing responses to low-level sound in chordotonal neurons.Intriguingly,DNAH5 mutant larvae displayed an altered frequency tuning curve of the auditory organs.All together,our findings support a critical role of DNAH5 in tuning the frequency selectivity and the sound sensitivity of larval auditory neurons.展开更多
Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects.Most cases are due to mutations in genes encoding the components of sperm flagella,which have an ultrastructure similar to that of m...Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects.Most cases are due to mutations in genes encoding the components of sperm flagella,which have an ultrastructure similar to that of motile cilia.Coiled-coil domain containing 103(CCDC103)is an outer dynein arm assembly factor,and pathogenic variants of CCDC103 cause primary ciliary dyskinesia(PCD).However,whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined.Whole-exome sequencing(WES)was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family.A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified(ENST00000035776.2,c.461A>C,p.His154Pro).CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD,though the reproductive phenotype of these PCD individuals is unknown.Transmission electron microscopy(TEM)of affected individuals’spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms,similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation.Thus,our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.展开更多
Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules.As a molecular motor protein,dynein performs a variety of basic cellular ...Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules.As a molecular motor protein,dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly.In the nervous system,dynein has been demonstrated to be responsible for axonal retrograde transport.Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis,Charcot-Marie-Tooth disease,Alzheimer’s disease,Parkinson’s disease and Huntington’s disease.Among them,a number of mutant proteins involved in various neurodegenerative diseases interact with dynein.Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases.Dynein heavy chain mutant mice also show features of neurodegenerative diseases.Moreover,defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases.Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases.In this review,we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.展开更多
基金This work was supported by a grant from the National Natural Science Foundation of China (No. 30170481).
文摘Objective To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level. Methods Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level. Results In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 mmol/L SOV and control groups (P<0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 mmol/L. In incon- tinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 mmol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P<0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed. Conclusion Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or MⅠto anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.
文摘We propose a dynamic mechanism for the processive motility of dynein on microtubules (MTs). The force generated for the motion of dynein is purely mechanical in origin. When a dynein monomer binds to a MT, the AAA ring of dynein might fit into one of the trenches on the outer surface of the MT, with the linker domain leaning on the ratchet-shaped protofilament. At room temperature, the dynein molecule exhibits random thermal motion on the outer surface of the MT. The collision between the asymmetric ratchet teeth and the linker exerts a reactive impulsive force on the dynein molecule. The probability of producing an impulse with a longitudinal component pointing to either end of the MT depends on the instantaneous motion of dynein, the shape of the linker, and the mass distribution of the dynein with/without a load. In the dynamic mechanism, dynein monomers can move independently and processively toward either end of the MT. Many observations of the motility of dynein can be reproduced in a simulation system.
基金Acknowledgments The authors thank Qiongping Huang, Lirong Liu, and Wei Bian for technical assistance. We are grateful to Drs G Chan （Cross Cancer Institute, University of Alberta, Edmonton Alberta, Canada） for antibodies to human ZW 10 and Rod, KH Choo （Murdoch Children＇s Research Institute, Royal Children＇s Hospital, Melbourne, Australia） for anti-CREST serum, and E Fuchs （Rockefeller University, USA） for mRFP cDNA. This work was supported by the National Science Foundation of China （30330330, 30421005, and 30623003）, Ministry of Science and Technology of China （2005CB522703 and 2007CB914501）, and the Shanghai Municipal Council for Science and Technology （S048014317, 06DZ22032, and 058014578）.
基金the National Natural Science Foundation of China (NSFC31330045)+2 种基金the National Key R&D Program of China (2017YFA0503500)Chinese Academy of Sciences (XDBl9020000)NSFC (31471323) to X.Y.
文摘Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that drive ciliary beat have been extensively studied in unicellular Chlamydomonas, to what extent such knowledge can be applied to vertebrate is poorly known. In Chlamydomonas, Dynein-f controls flagellar waveforms but is dispensable for beating. The flagellar assembly of its heavy chains (HCs) requires its intermediate chain (IC) IC140 but not IC138. Here we show that, unlike its Chlamydomonas counte『part, vertebrate Dynein-f is essential for ciliary beat. We confirmed that Wdr78 is the vertebrate orthologue of IC3 Wdr78 associated with Dynein-f subunits such as Dnah2 (a HC) and Wdr63 (IC140 orthologue). It was expressed as a motile cilium-specific protein in mammalian cells. Depletion of Wdr78 or Dnah2 by RNAi paralyzed mouse ependymal cilia. Zebrafish Wdr78 morphants displayed ciliopathy-related phenotypes, such as curved bodies, hydrocephalus, abnormal otolith, randomized left-right asymmetry, and pronephric cysts, accompanied with paralyzed pronephric cilia. Furthermore, all the HCs and ICs of Dynein-f failed to localize in the Wdr78-depleted mouse ependymal cilia. Therefore, both the functions and subunit dependency of Dynein-fare altered in evolution, probably to comply with ciliary roles in higher organisms.
基金We thank all the past and present members of the Asai and Wilkes laboratories who have contributed to our knowledge of cytoplasmic dynein-2 in Tetrahymena, Paramecium, and rat. Our laboratory is supported by a grant from the National Science Foundation.
文摘The dynein motor protein family is involved in a wide variety of functions in eukaryotic cells. The axonemal dynein class and cytoplasmic dynein-1 subclass have been well characterized. However, the cytoplasmic dynein-2 subclass of the family has only recently begun to be understood. We describe the entire dynein family but focus on cytoplasmic dynein-2. Dynein-2 consists of a heavy, an intermediate, a light intermediate, and a light chain. The complex appears to function primarily as the retrograde motor for intraflagellar transport. This process is important for the formation and maintenance of cilia and flagella. Additionally, dynein-2 has roles in the control of ciliary length and in non-ciliary functions. Mutations in the human dynein-2 heavy chain lead to cilia-related diseases.
基金the National Key R&D Program of China(2017YFA0503500)the National Natural Science Foundation of China(31330044 and 31671387)to J.P.
文摘The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins,which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors(DNAAFs)before being transported to and assembled on the ciliary axoneme.Here,we characterize the function of WDR92 in Chlamydomonas.Loss of WDR92,a cytoplasmic protein,in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella,disappearance of axonemal dynein arms,and diminishment of dynein arm heavy chains in the cytoplasm,suggesting that WDR92 is a DNAAF.Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBLl,RPAP3,MOT48,ODA7,and DYX1C.The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBLl/2 and RPAP3,while PF13,another PIH1 domain-containing protein with function in dynein preassembly,did not.Interestingly,the third PIH1 domain-containing protein TWI1 was not related to flagellar motility.WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs.Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.
基金the National Key R&D Program of China Project(2017YFA0103900 and 2016YFA0502800)the National Natural Science Foundation of China(31571083 and 31970931)+2 种基金the Program for Professor of Special Appointment(Eastern Scholar of Shanghai,TP2014008)the Shanghai Municipal Science and Technology Major Project(2017SHZDZX01 and 2018SHZDZX01)ZJLab,and the Shanghai Rising-Star Program(14QA1400800)。
文摘Chordotonal neurons are responsible for sound sensation in Drosophila.However,little is known about how they respond to sound with high sensitivity.Using genetic labeling,we found one of the Drosophila axonemal dynein heavy chains,CG9492(DNAH5),was specifically expressed in larval chordotonal neurons and showed a distribution restricted to proximal cilia.While DNAH5 mutation did not affect the cilium morphology or the trafficking of Inactive,a candidate auditory transduction channel,larvae with DNAH5 mutation had reduced startle responses to sound at low and medium intensities.Calcium imaging confirmed that DNAH5 functioned autonomously in chordotonal neurons for larval sound sensation.Furthermore,disrupting DNAH5 resulted in a decrease of spike firing responses to low-level sound in chordotonal neurons.Intriguingly,DNAH5 mutant larvae displayed an altered frequency tuning curve of the auditory organs.All together,our findings support a critical role of DNAH5 in tuning the frequency selectivity and the sound sensitivity of larval auditory neurons.
基金supported by the National Natural Science Foundation of China(No.81971446).
文摘Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects.Most cases are due to mutations in genes encoding the components of sperm flagella,which have an ultrastructure similar to that of motile cilia.Coiled-coil domain containing 103(CCDC103)is an outer dynein arm assembly factor,and pathogenic variants of CCDC103 cause primary ciliary dyskinesia(PCD).However,whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined.Whole-exome sequencing(WES)was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family.A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified(ENST00000035776.2,c.461A>C,p.His154Pro).CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD,though the reproductive phenotype of these PCD individuals is unknown.Transmission electron microscopy(TEM)of affected individuals’spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms,similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation.Thus,our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.
基金the National Natural Science Foundation of China(81330026,31271259,30990261,30871425 to Liu Yaobo30870873,81171187 to Chen Xiang-Jun)+2 种基金the National Basic Research Program,Ministry of Sci-ence and Technology of China(2013CB945604)the Chinese Academy of Sciences(KSCX2-EW-Q-11)for funding supportthe Queensland Government’s National and International Research Alliances Program and the National Health and Medical Research Council of Aus-tralia for financial support
文摘Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules.As a molecular motor protein,dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly.In the nervous system,dynein has been demonstrated to be responsible for axonal retrograde transport.Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis,Charcot-Marie-Tooth disease,Alzheimer’s disease,Parkinson’s disease and Huntington’s disease.Among them,a number of mutant proteins involved in various neurodegenerative diseases interact with dynein.Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases.Dynein heavy chain mutant mice also show features of neurodegenerative diseases.Moreover,defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases.Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases.In this review,we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.
