松胞素对百合花粉原生质体内肌动蛋白微丝的影响

用B_5培养基酶解分离出百合花粉原生质体。原生质体经松胞素(5μg/ml)分别处理5、10、15、30、60 min,再用荧光标记的鬼笔碱染色,共焦激光扫描镜观察,跟踪了原生质体内的肌动蛋白微丝从一个组织复杂严密的网络转变为无数颗粒体的过程。松胞素处理过的原生质体移回至不含松胞素的培养基中后继续培养15 min,肌动蛋白颗粒快速地再延伸出微丝,重组成新的网络。存在于花粉原生质体中生殖细胞的微丝网络,在经松胞素处理后同样都形成为颗粒体。之后,如果原生质体再放入不含松胞素的培养基内继续培养,生殖细胞内的颗粒体同样会再延伸出微丝,重新组成网络。从原生质体胞质以及生殖细胞内所见到的微丝和颗粒相互转化的情况,可以断定,颗粒体不但具有凝聚微丝的功能,同时也具有重组微丝的功能。     

Diazepam inhibits cell respiration and induces fragmentation of mitochondrial reticulum

Diazepam (70-150 micrograms/ml) significantly inhibits oxygen consumption by pig kidney embryo cells and causes the cellular ATP level to fall. The maximum inhibitory effect develops after 1.5-2.5 h of diazepam treatment. In isolated mitochondria diazepam inhibits respiration in state 2 and 3u with glutamate and in state 3u with succinate. Ethylrhodamine staining and electron microscopic study reveal fragmentation of mitochondria in living cells.     

Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets. Evidence of a role for a GTP-binding protein in platelet activation

Human platelets containing granule-bound [14C]serotonin were permeabilized, equilibrated at 0 degrees C with ATP and with various Ca2+ buffers and guanine nucleotides, and then incubated at 25 degrees C with or without a stimulatory agonist. Ca2+ alone induced the ATP-dependent secretion of [14C]serotonin (50% at a pCa of 5.1) but the sensitivity of secretion to Ca2+ was greatly enhanced by guanine nucleotides [6-fold by 100 microM GTP, 100-fold by 100 microM guanyl-5'-yl imidodiphosphate and greater than 500-fold by 100 microM guanosine 5'-O-(3-thiotriphosphate)] or by stimulatory agonists (10-fold by 2 units thrombin/ml and 4-fold by 1 microM 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine). When both GTP and a stimulatory agonist were added, they had synergistic effects on secretion. Cyclic GMP and GMP acted similarly to GTP. The effects of all these guanine nucleotides were inhibited by guanosine 5'-O-(2-thiodiphosphate), whereas those of stimulatory agonists were not. Our results demonstrate the presence in platelets of guanine nucleotide-dependent and independent mechanisms regulating the sensitivity of secretion to Ca2+.     

Isolation and characterization of plant myosin from pollen tubes of lily

Summary A plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 m/s with an average of 7.7 m/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazin-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate     

玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的比较研究

本文对玉米花粉肌动蛋白和兔骨骼肌肌动蛋白进行了比较研究。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白具有相同的分子量(42KD)。玉米花粉肌动蛋白可与兔抗鸡胃肌动蛋白抗血清产生免疫沉淀反应。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的氨基酸组成以及胰蛋白酶水解所得到的肽谱都相似。它们的羧基未端氨基酸顺序完全一致,其顺序都是Lys.Cys.Phe(COOH)。它们的圆二色谱基本相同,由圆二色谱计算得到的二级结构数据也相近。以上的结果表明了玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的相似性。     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.     

Identification of sucrose synthase as an actin-binding protein

Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.     

Cortical actin filaments fragment and aggregate to form chloroplast-associated and free F-actin rings in mechanically isolatedZinnia mesophyll cells

Summary Changes in F-actin organization following mechanical isolation ofZinnia mesophyll cells were documented by rhodamine-phalloidin staining. Immediately after isolation, most cells contained irregular cortical actin fragments of varying lengths, and less than 5% of cells contained intact cortical filaments. During the first 8 h of culture, filament fragments were replaced by actin rings, stellate actin aggregates, and bundled filament fragments. Some of these aggregates had no association with organelles (free actin aggregates). Other aggregates were associated with chloroplasts, which changed in shape and location at the same time actin aggregates appeared. F-actin was concentrated within or around the nucleus in a small percentage of cells. After 12 h in culture, the percentage of cells with free actin rings and chloroplast-associated actin aggregates began to decline and the percentage of cells having intact cortical actin filaments increased greatly. Intermediate images were recorded that strongly indicate that free actin rings, chloroplast-associated actin rings, and other actin aggregates self-assemble by successive bundling of actin filament fragments. The fragmentation and bundling of F-actin observed in mechanically isolatedZinnia cells resembles changes in F-actin distribution reported after diverse forms of cell disturbance and appears to be an example of a generalized response of the actin cytoskeleton to cell stress.Abbreviations FITC fluorescein isothiocyanate - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - RhPh tetramethylrhodamine isothiocyanate-phalloidin     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

A high-throughput assay shows that DNase-I binds actin monomers and polymers with similar affinity

Previous conflicting reports suggest that DNase-I binds F-actin with either equal or drastically different K(D) values compared to G-actin. We developed a high-throughput DNase-I inhibition assay to determine the K(D) of DNase-I for F-actin. We confirmed that phalloidin-stabilized F-actin is protected from depolymerization by DNase-I and that the critical concentration at the pointed end of phalloidin-F-actin is 45.5+/-13.9 nM. We found that DNase-I inhibition by actin follows ultrasensitive mechanics. Using varying lengths of gelsolin-capped phalloidin-F-actin, we concluded that the affinities of DNase-I for G- and the pointed end subunits of F-actin are almost indistinguishable, such that DNase-I may not distinguish between G- and F-actin conformations.     

Pollen tube extract supports the movement of actin filaments in vitro

Summary Muscle actin filaments labeled with rhodamine-phalloidin were observed to move on the surface coated with a crude extract of pollen tubes ofLilium longiflorum with an average velocity of 1.99±0.55 m/sec. The movement required both Mg²⁺ and ATP. These results indicate that the extract of pollen tubes contains a myosin-like translocatorAbbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride     

Staggered movement of an actin filament sliding on myosin molecules in the presence of ATP

An actin filament sliding on myosin molecules in the presence of an extremely low concentration of ATP exhibited a staggered movement. Longitudinally sliding movement of the filament was frequently interrupted by its non-sliding, fluctuating movements both in the longitudinal and transversal directions. Intermittent sliding movements of an actin filament indicate establishment of a coordination of ATP-mediated active sites distributed along the filament.     

花粉管微丝骨架的研究

用PHEM缓冲液对萌发花粉进行吸胀处理,经0.15%Tritonx-100提取,0.2%考马斯亮兰R250染色后,在光学显微镜下观察到具有蛋白质性质的骨架结构。对从花粉管吸胀出的凝胶状物质进行临界点干燥,电子显微镜扫描??结果表明,花粉管原生质中镶嵌着大量相互交错的丝状物质。兔肌重酶解肌球蛋白标记结果证明,这些丝状物质主要为F-肌动蛋白,其直径为5—7nm,半螺距为370A。本实验结果表明,花粉管原生质中存在着大量微丝骨架结构,这些微丝骨架是由F-肌动蛋白组成的。     

小麦胚乳细胞中肌动蛋白纤丝分布格局的荧光显微观察

以异硫氰四甲基罗丹明－鬼笔环肽为探针,结合电视显微镜术对呈现原生质胞间运动的小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）胚乳细胞胞质中肌动蛋白纤丝（ＡＦｓ）的存在与分布格局进行了观察与分析。结果表明,ＡＦｓ以多种形式在胚乳细胞内呈区域性分布：胞核为ＡＦｓ网络所包围,ＡＦｓ成束由核周向四处辐射直抵周质层,大量纤细ＡＦｓ在胞质皮层内呈无序的密集分布以及腹沟区细胞内ＡＦｓ梭状体的大量汇集。联系胞质组分在胞间运动中的动态与胞质纤索对ＣＢ处理的反应,讨论了ＡＦｓ的不同存在状态与胞质纤索的伸缩穿越和细胞基质的集流迁移这两种胞间运动方式的关系     

Crenactin from Pyrobaculum calidifontis is closely related to actin in structure and forms steep helical filaments

Polymerising proteins of the actin family are nearly ubiquitous. Crenactins, restricted to Crenarchaea, are more closely related to actin than bacterial MreB. Crenactins occur in gene clusters hinting at an unknown, but conserved function. We solved the crystal structure of crenactin at 3.2 Å resolution. The protein crystallises as a continuous right-handed helix with 8 subunits per complete turn, spanning 419 Å. The structure of crenactin shows several loops that are longer than in actin, but overall, crenactin is closely related to eukaryotic actin, with an RMSD of 1.6 Å. Crenactin filaments imaged by electron microscopy showed polymers with very similar helical parameters.