This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in...This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable an- tisense cenpb transfected cell line, named HACPB, was ob- tained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scat- tered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2/M phase into next G1 phase were observed in HACPB cells. Furthermore, the ex- pression of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors (CKIs) were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centro- mere/kinetochore complex, and plays important roles in cell cycle regulation.展开更多
In eukaryotic cells, the course of the cell cycle depends on correct cytoskeleton arrangement. The cell cycle consists of several phases, and in each of them the cytoskeleton has a unique structure and set of characte...In eukaryotic cells, the course of the cell cycle depends on correct cytoskeleton arrangement. The cell cycle consists of several phases, and in each of them the cytoskeleton has a unique structure and set of characteristics. The dynamics of the cytoskeleton together with its binding proteins greatly contribute to progression of the cell cycle. Here, we mainly review recent research on the dynamic distribution of the actin cytoskeleton and actin-binding proteins, and the mechanisms by which they affect the progression of the plant cell cycle.展开更多
The phragmoplast is a special apparatus that functions in establishing a cell plate in dividing plant cells. It is known that microfilaments (MFs) are involved in constituting phragmoplast structure, but the dynamic d...The phragmoplast is a special apparatus that functions in establishing a cell plate in dividing plant cells. It is known that microfilaments (MFs) are involved in constituting phragmoplast structure, but the dynamic distribution and role of phragmoplast MFs are far from being understood. In this study, the precise structure and dynamics of MFs during the initiation and the late lateral expansion of the phragmoplast were observed by using a tobacco BY-2 cell line stably expressing the microfilament reporter construct GFP-fABD2. Three-dimensional imaging showed that the phragmoplast MFs were initiated by two populations of MFs emerging between the reconstituting daughter nuclei at anaphase, which migrated to the mid-zone and gave rise to two layers of microfilament arrays. FM4-64 stained vesicles accumulated and fused with the cell plate between the two populations of MFs. The two layers of microfilament arrays of phragmoplast with ends overlapped always surrounded the centrifugally expanding cell plate. Partial disruption of MFs at metaphase by low concentration of latrunculin B resulted in the inhibition of the cell plate consolidation and the blockage of cell plate lateral expansion, whereas high concentration of latrunculin B restrained the progression of the cell cycle. Treating the cell after the initiation of phragmoplast led to the cease of the expansion of the cell plate. Our observations provide new insights into the precise structure and dynamics of phragmoplast MFs during cytokinesis and suggest that dynamic phragmoplast MFs are important in cell plate formation.展开更多
Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies....Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1 FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FHIFH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro, in addition, AtFH16 FHIFH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FHIFH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.展开更多
The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results sho...The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0±1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 1013 ions/cm2. Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.展开更多
Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs (Arabidopsis thaliana formin homologs) and their effects on actin organizati...Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs (Arabidopsis thaliana formin homologs) and their effects on actin organization is poorly understood, in this study, we characterized the bundling activity of AtFH8 in vitro and in vivo. Biochemical analysis showed that AtFH8(FH1FH2) could form dimers and bundle preformed actin filaments or induce stellar structures during actin polymerization. Expression of truncated forms of AtFH8 and immunolocalization analysis showed that AtFH8 localized primarily to nuclear envelope in interphase and to the new cell wall after cytokinesis, depending primarily on its N-terminal transmembrane domain. GUS histochemical staining showed AtFH8 was predominantly expressed in Arabidopsis root meristem, vasculature, and outgrowth points of lateral roots. The primary root growth and lateral root initiation of atfh8 could be decreased by latrunculin B (LatB). Analysis of the number of dividing cells in Arabidopsis root tips showed that much fewer dividing cells in Lat B-treated atfh8 plants than wild-type plants, which indicates that AtFH8 was involved in cell division. Actin cytoskeleton in root meristem of atfh8-1 was more sensitive to LatB treatment than that of wild-type. Altogether, our results indicate that AtFH8 is an actin filament nucleator and bundler that functions in cell division and root development.展开更多
Immunomodulatory signaling imposes tight regulations on metabolic programs within immune cells and consequentially determines immune response outcomes.Although the glucocorticoid receptor(GR)has been recently implicat...Immunomodulatory signaling imposes tight regulations on metabolic programs within immune cells and consequentially determines immune response outcomes.Although the glucocorticoid receptor(GR)has been recently implicated in regulating the function of myeloid-derived suppressor cells(MDSCs),whether the dysregulation of GR in MDSCs is involved in immune-mediated hepatic diseases and how GR regulates the function of MDSCs in such a context remains unknown.Here,we revealed the dysregulation of GR expression in MDSCs during innate immunological hepatic injury(IMH)and found that GR regulates the function of MDSCs through modulating HIF1α-dependent glycolysis.Pharmacological modulation of GR by its agonist(dexamethasone,Dex)protects IMH mice against inflammatory injury.Mechanistically,GR signaling suppresses HIF1αand HIF1α-dependent glycolysis in MDSCs and thus promotes the immune suppressive activity of MDSCs.Our studies reveal a role of GR-HIF1αin regulating the metabolism and function of MDSCs and further implicate MDSC GR signaling as a potential therapeutic target in hepatic diseases that are driven by innate immune cell-mediated systemic inflammation.展开更多
Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require furthe...Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 (RAN1) regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type (WT) and transgenic lines misexpressing RAN1 (msRAN1) gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 (MINI3)-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.展开更多
Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio ...Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg2+ and K+, the fluorescent actin analogue polymerized into filaments in vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.展开更多
Dear Editor, As an essential element for pollen tube tip growth, actJn cytoskeleton has been a focus for decades. Recently, both rapid-freeze and room-temperature fixation have revealed the presence of a subapical co...Dear Editor, As an essential element for pollen tube tip growth, actJn cytoskeleton has been a focus for decades. Recently, both rapid-freeze and room-temperature fixation have revealed the presence of a subapical cortical F-actin (actin fringe) as a consistent feature of lily pollen tubes (Lovy-Wheeler et al., 2005). The actin fringe consists of a palisade of short longitudinally oriented actin cables that start about 1-5~m behind the extreme apex and extend basally for an additional 5-10pm (Lovy-Wheeler et al., 2005). MeanwhJJe, observations in live cells using Lifeact-mEGFP, an actin probe consisting of the first 17 amino acids from the budding yeast ABP-140 fused to GFP, which does not affect the dynamics of actin, compellingly support the presence of a cortical actin fringe in lilv pollen tubes ~Vidali et al.. 2009~.展开更多
Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin d...Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.展开更多
The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attract...The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, func-tions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.展开更多
The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule 'seeds' is usually necessary for plant tubulin assembly in vit...The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule 'seeds' is usually necessary for plant tubulin assembly in vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule 'seed' system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerized in vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical 'parabola'. The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/展开更多
Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization.Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process...Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization.Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process of its germination and polarized growth.The native structures and dynamics of actin are subtly modulated by many factors among which numerous actin binding proteins(ABPs)are the most direct and significant regulators.Upstream signals such as Ca^(2+),PIP_(2)(phosphatidylinositol-4,5-bis-phosphate)and GTPases can also indirectly act on actin organization through several ABPs.Under such elaborate regulation,actin structures show dynamically continuous modulation to adapt to the in vivo biologic functions to mediate secretory vesicle transportation and fusion,which lead to normal growth of the pollen tube.Many encouraging progress has been made in the connection between actin regulation and pollen tube growth in recent years.In this review,we summarize different factors that affect actin organization in pollen tube growth and highlight relative research progress.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
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
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
Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In ...Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In this study, a ser- ine/argine-rich (SR) protein cDNA with 28kDa protein (LISR28) was isolated from a lily (Lilium Iongiflorum) expression library. Protein domain analysis showed that LISR28 had similar structures to Arabidopsis SR45 (AtSR45), and LISR28 could complement the phenotype of loss of AtSR45 function. Therefore, overexpression of LISR28 and AtSR45 mutant (atsr45-1) were used in the following experiments. Overexpression of LISR28 in Arabidopsis completely inhibited pollen germina- tion. In contrast, the pollen germination of atsr45-1 was earlier than that of wild-type. In addition, pollen of atsr45-1 contained less F-actin at the corresponding hydration stage during pollen germination compared to that of wild-type. Alternative splicing analysis showed that Arabidopsis villinl (AtVLN1) transcript encoding the full-length protein was increased, and that encoding the truncated protein was decreased in atst45-1. Moreover, the mRNA expression level of other actin-binding proteins (ABPs) abundant in Arabidopsis pollen was also changed in atsr45-1. In conclusion, we hypothesize that LISR28 alters F-actin dynamics probably through its alternative splicing activities to affect directly or indirectly the alternative splicing of AtVLN1 and the expression of different ABPs, which then affects the pollen germination.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.39430080).
文摘This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable an- tisense cenpb transfected cell line, named HACPB, was ob- tained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scat- tered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2/M phase into next G1 phase were observed in HACPB cells. Furthermore, the ex- pression of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors (CKIs) were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centro- mere/kinetochore complex, and plays important roles in cell cycle regulation.
基金supported by the National Natural Science Foundation of China (30870211, 30970174)the National Basic Research Program of China (2007CB108700)the Chinese Transgenic Project (2009ZX08009-059B) to H. Ren
文摘In eukaryotic cells, the course of the cell cycle depends on correct cytoskeleton arrangement. The cell cycle consists of several phases, and in each of them the cytoskeleton has a unique structure and set of characteristics. The dynamics of the cytoskeleton together with its binding proteins greatly contribute to progression of the cell cycle. Here, we mainly review recent research on the dynamic distribution of the actin cytoskeleton and actin-binding proteins, and the mechanisms by which they affect the progression of the plant cell cycle.
基金Supported by National Natural Science Foundation of China(Grant Nos.30630005,30470176)National Key Basic Research and Development Program of China(Grant Nos.2006CB100100,2007CB108700)
文摘The phragmoplast is a special apparatus that functions in establishing a cell plate in dividing plant cells. It is known that microfilaments (MFs) are involved in constituting phragmoplast structure, but the dynamic distribution and role of phragmoplast MFs are far from being understood. In this study, the precise structure and dynamics of MFs during the initiation and the late lateral expansion of the phragmoplast were observed by using a tobacco BY-2 cell line stably expressing the microfilament reporter construct GFP-fABD2. Three-dimensional imaging showed that the phragmoplast MFs were initiated by two populations of MFs emerging between the reconstituting daughter nuclei at anaphase, which migrated to the mid-zone and gave rise to two layers of microfilament arrays. FM4-64 stained vesicles accumulated and fused with the cell plate between the two populations of MFs. The two layers of microfilament arrays of phragmoplast with ends overlapped always surrounded the centrifugally expanding cell plate. Partial disruption of MFs at metaphase by low concentration of latrunculin B resulted in the inhibition of the cell plate consolidation and the blockage of cell plate lateral expansion, whereas high concentration of latrunculin B restrained the progression of the cell cycle. Treating the cell after the initiation of phragmoplast led to the cease of the expansion of the cell plate. Our observations provide new insights into the precise structure and dynamics of phragmoplast MFs during cytokinesis and suggest that dynamic phragmoplast MFs are important in cell plate formation.
基金supported by the National Natural Science Foundation of China (31130005)the National Basic Research Program of China (2013CB126902) to H. R.
文摘Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1 FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FHIFH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro, in addition, AtFH16 FHIFH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FHIFH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.
文摘The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0±1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 1013 ions/cm2. Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.
文摘Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs (Arabidopsis thaliana formin homologs) and their effects on actin organization is poorly understood, in this study, we characterized the bundling activity of AtFH8 in vitro and in vivo. Biochemical analysis showed that AtFH8(FH1FH2) could form dimers and bundle preformed actin filaments or induce stellar structures during actin polymerization. Expression of truncated forms of AtFH8 and immunolocalization analysis showed that AtFH8 localized primarily to nuclear envelope in interphase and to the new cell wall after cytokinesis, depending primarily on its N-terminal transmembrane domain. GUS histochemical staining showed AtFH8 was predominantly expressed in Arabidopsis root meristem, vasculature, and outgrowth points of lateral roots. The primary root growth and lateral root initiation of atfh8 could be decreased by latrunculin B (LatB). Analysis of the number of dividing cells in Arabidopsis root tips showed that much fewer dividing cells in Lat B-treated atfh8 plants than wild-type plants, which indicates that AtFH8 was involved in cell division. Actin cytoskeleton in root meristem of atfh8-1 was more sensitive to LatB treatment than that of wild-type. Altogether, our results indicate that AtFH8 is an actin filament nucleator and bundler that functions in cell division and root development.
基金This research is supported by grants from the National Natural Science Foundation for General Programs of China(31671524,31171407 and 81273201,GL)the Key Basic Research Project of the Science and Technology Commission of Shanghai Municipality(12JC1400900,GL)+2 种基金the Innovation Program of Shanghai Municipal Education Commission(14ZZ009,GL)the Excellent Youth Foundation of Chinese Academy of Sciences(KSCX2-EW-Q-7,GL)R21AI117547,1R01AI114581,V2014-001 from the V-foundation,and 128436-RSG-15-180-01-LIB from the American Cancer Society(RW).
文摘Immunomodulatory signaling imposes tight regulations on metabolic programs within immune cells and consequentially determines immune response outcomes.Although the glucocorticoid receptor(GR)has been recently implicated in regulating the function of myeloid-derived suppressor cells(MDSCs),whether the dysregulation of GR in MDSCs is involved in immune-mediated hepatic diseases and how GR regulates the function of MDSCs in such a context remains unknown.Here,we revealed the dysregulation of GR expression in MDSCs during innate immunological hepatic injury(IMH)and found that GR regulates the function of MDSCs through modulating HIF1α-dependent glycolysis.Pharmacological modulation of GR by its agonist(dexamethasone,Dex)protects IMH mice against inflammatory injury.Mechanistically,GR signaling suppresses HIF1αand HIF1α-dependent glycolysis in MDSCs and thus promotes the immune suppressive activity of MDSCs.Our studies reveal a role of GR-HIF1αin regulating the metabolism and function of MDSCs and further implicate MDSC GR signaling as a potential therapeutic target in hepatic diseases that are driven by innate immune cell-mediated systemic inflammation.
文摘Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 (RAN1) regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type (WT) and transgenic lines misexpressing RAN1 (msRAN1) gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 (MINI3)-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.
文摘Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg2+ and K+, the fluorescent actin analogue polymerized into filaments in vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.
文摘Dear Editor, As an essential element for pollen tube tip growth, actJn cytoskeleton has been a focus for decades. Recently, both rapid-freeze and room-temperature fixation have revealed the presence of a subapical cortical F-actin (actin fringe) as a consistent feature of lily pollen tubes (Lovy-Wheeler et al., 2005). The actin fringe consists of a palisade of short longitudinally oriented actin cables that start about 1-5~m behind the extreme apex and extend basally for an additional 5-10pm (Lovy-Wheeler et al., 2005). MeanwhJJe, observations in live cells using Lifeact-mEGFP, an actin probe consisting of the first 17 amino acids from the budding yeast ABP-140 fused to GFP, which does not affect the dynamics of actin, compellingly support the presence of a cortical actin fringe in lilv pollen tubes ~Vidali et al.. 2009~.
文摘Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.
基金This work was supported by the National Natural Science Foundation for Distinguished Young Scholars (Grant No. 30325005)the National Natural Science Foundation of China (Grant No. 30470176)the National Basic Research Program of China (Grant No. 2006CB 100100).
文摘The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, func-tions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.
文摘The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule 'seeds' is usually necessary for plant tubulin assembly in vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule 'seed' system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerized in vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical 'parabola'. The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/
基金the National Natural Science Foundation of China(Grant Nos.30970174,30325005,30870211)the National Basic Research Program of China(No.2007CB108700)to HR.
文摘Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization.Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process of its germination and polarized growth.The native structures and dynamics of actin are subtly modulated by many factors among which numerous actin binding proteins(ABPs)are the most direct and significant regulators.Upstream signals such as Ca^(2+),PIP_(2)(phosphatidylinositol-4,5-bis-phosphate)and GTPases can also indirectly act on actin organization through several ABPs.Under such elaborate regulation,actin structures show dynamically continuous modulation to adapt to the in vivo biologic functions to mediate secretory vesicle transportation and fusion,which lead to normal growth of the pollen tube.Many encouraging progress has been made in the connection between actin regulation and pollen tube growth in recent years.In this review,we summarize different factors that affect actin organization in pollen tube growth and highlight relative research progress.
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supported by the National Natural Science Foundation of China(32270774,31671400,81971439,32070694,31571436,31872822,and 31301153)the National Key Research and Development Program of China(2017YFA0503502,2016YFA0500903,2021YFC2700200,2017YFA0504600,and 2019YFA0508700)funded by the National Institutes of Health Office of Research Infrastructure Programs(P40 OD010440)。
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.
基金the National Natural Science Foundation of China,by the National Basic Research Program of China (2013CB126902)to H.R
文摘Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In this study, a ser- ine/argine-rich (SR) protein cDNA with 28kDa protein (LISR28) was isolated from a lily (Lilium Iongiflorum) expression library. Protein domain analysis showed that LISR28 had similar structures to Arabidopsis SR45 (AtSR45), and LISR28 could complement the phenotype of loss of AtSR45 function. Therefore, overexpression of LISR28 and AtSR45 mutant (atsr45-1) were used in the following experiments. Overexpression of LISR28 in Arabidopsis completely inhibited pollen germina- tion. In contrast, the pollen germination of atsr45-1 was earlier than that of wild-type. In addition, pollen of atsr45-1 contained less F-actin at the corresponding hydration stage during pollen germination compared to that of wild-type. Alternative splicing analysis showed that Arabidopsis villinl (AtVLN1) transcript encoding the full-length protein was increased, and that encoding the truncated protein was decreased in atst45-1. Moreover, the mRNA expression level of other actin-binding proteins (ABPs) abundant in Arabidopsis pollen was also changed in atsr45-1. In conclusion, we hypothesize that LISR28 alters F-actin dynamics probably through its alternative splicing activities to affect directly or indirectly the alternative splicing of AtVLN1 and the expression of different ABPs, which then affects the pollen germination.