Ability of the c-mos product to associate with and phosphorylate tubulin.

The mos proto-oncogene product, pp39mos, is a protein kinase and has been equated with cytostatic factor (CSF), an activity in unfertilized eggs that is thought to be responsible for the arrest of meiosis at metaphase II. The biochemical properties and potential substrates of pp39mos were examined in unfertilized eggs and in transformed cells in order to study how the protein functions both as CSF and in transformation. The pp39mos protein associated with polymers under conditions that favor tubulin oligomerization and was present in an approximately 500-kilodalton "core" complex under conditions that favor depolymerization. beta-Tubulin was preferentially coprecipitated in pp39mos immunoprecipitates and was the major phosphorylated product in a pp39mos-dependent immune complex kinase assay. Immunofluorescence analysis of NIH 3T3 cells transformed with Xenopus c-mos showed that pp39mos colocalizes with tubulin in the spindle during metaphase and in the midbody and asters during telophase. Disruption of microtubules with nocodazole affected tubulin and pp39mos organization in the same way. It therefore appears that pp39mos is a tubulin-associated protein kinase and may thus participate in the modification of microtubules and contribute to the formation of the spindle. This activity expressed during interphase in somatic cells may be responsible for the transforming activity of pp39mos.     

Microtubule gelation-contraction: essential components and relation to slow axonal transport

Preparations of microtubule proteins isolated by assembly and disassembly undergo gelation-contraction after addition of adenosine triphosphate (ATP). A particulate fraction from these preparations that is required, along with purified tubulin, to produce ATP-dependent microtubule gelation-contraction in vitro has been isolated. The particulates exhibited microtubule-stimulated adenosine triphosphatase activity and moved slowly (about 1 micrometer per minute) along microtubule walls in the presence of ATP. The particulates contained tubulin, neurofilament, and spectrin polypeptides. The composition, solubility, and motility of the particulates are consistent with those of slow component a of axonal transport.     

Selective inhibition of tubulin synthesis by amiprophos methyl during flagellar regeneration in Chlamydomonas reinhardi

Amiprophos methyl (APM) is a strong, readily reversible and highly selective inhibitor of tubulin synthesis in Chlamydomonas reinhardi. The extensive induction of tubulin synthesis that accompanies flagellar regeneration in this organism is prevented by 3 to 10 micrometerAPM. When applied after induction has begun, APM causes a rapid cessation of tubulin synthesis. Translation studies in vitro indicate that the lack of tubulin production in APM-treated cells is not due to a direct inhibition of tubulin messenger RNA translation but rather to a selective depletion of tubulin messenger RNA.     

Promotion of tubulin assembly by aluminum ion in vitro

It has been proposed that aluminum ion is a contributing factor in a variety of neurological diseases. In many of these diseases, aberrations in the cytoskeleton have been noted. The effects of aluminum ion on the in vitro assembly of tubulin into microtubules has been examined by determining the association constants for the metal ion-guanosine triphosphate-tubulin ternary complex required for polymerization. The association constant for aluminum ion was approximately 10(7) times that of magnesium ion, the physiological mediator of microtubule assembly. In addition, aluminum ion at 4.0 X 10(-10) mole per liter competed effectively with magnesium ion for support of tubulin polymerization when magnesium ion falls below 1.0 millimole per liter. The microtubules produced by aluminum ion were indistinguishable from those produced by magnesium ion when viewed by electron microscopy, and they showed identical critical tubulin concentrations for assembly and sensitivities to cold-induced depolymerization. However, the rate of guanosine triphosphate hydrolysis and the sensitivity to calcium ion-induced depolymerization, critical regulatory processes of microtubules in vivo, were markedly lower for aluminum ion microtubules than for magnesium ion microtubules.     

An ATP gate controls tubulin binding by the tethered head of kinesin-1

Kinesin-1 is a two-headed molecular motor that walks along microtubules, with each step gated by adenosine triphosphate (ATP) binding. Existing models for the gating mechanism propose a role for the microtubule lattice. We show that unpolymerized tubulin binds to kinesin-1, causing tubulin-activated release of adenosine diphosphate (ADP). With no added nucleotide, each kinesin-1 dimer binds one tubulin heterodimer. In adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable ATP analog, each kinesin-1 dimer binds two tubulin heterodimers. The data reveal an ATP gate that operates independently of the microtubule lattice, by ATP-dependent release of a steric or allosteric block on the tubulin binding site of the tethered kinesin-ADP head.     

Slow transport of tubulin in the neurites of differentiated PC12 cells

In order to study the rate and form of tubulin transport in cultured neuronal cells, the fluorescence recovery after the photobleaching of a fluorescent tubulin analog has been followed within the neuritic processes of differentiated PC12 cells. In these cells, as in peripheral axons, tubulin is transported in coherent, nondiffusing waves at two different slow rates that are within the range of the slow components a and b of axonal transport measured in vivo. Finally, it appears that most, if not all, of the tubulin analog is moving out these processes. Thus, slow neuroplasmic transport in cultured neuron-like cells is a good model of axonal transport, in which experimental manipulations of the system can be performed that would be difficult in the whole animal.     

Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi

Basal bodies isolated from Chlamydomonas reinhardi will serve as initiation centers for the assembly of chick brain microtubule protein subunits (tubulin) into microtubules. The rate of microtubule assembly is tubulin-concentration dependent; this assembly occurs onto both distal and proximal ends of the basal body mnicrotubules, with distal assembly greatly favored. In vitro assembly of brain tubulin also occurs onto the mid-lateral aspects of the basal bodies, presumably onto the fiber connecting the two basal bodies.     

Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues

Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant antibody that specifically recognizes GTP-bound tubulin in microtubules and found that GTP-tubulin was indeed present at the plus end of growing microtubules. Unexpectedly, GTP-tubulin remnants were also present in older parts of microtubules, which suggests that GTP hydrolysis is sometimes incomplete during polymerization. Observations in living cells suggested that these GTP remnants may be responsible for the rescue events in which microtubules recover from catastrophe.     

梭梭ARF1基因的克隆及序列分析

二磷酸腺苷-核糖基化作用因子(ADP-ribosylation factors,ARFs)属小G蛋白超级家族中的Arf亚族,是真核细胞囊泡运输通道的关键组成成分,参与细胞运输和信号传导。本试验采用RT-PCR、RACE等方法从超旱生、耐盐植物梭梭(Haloxylon ammodendron)中扩增出ADP-ribosylation factor(ARF1)基因的cD-NA序列(命名为HaARF1),其开放阅读框为546bp,推测氨基酸序列全长为181个氨基酸残基,具有典型的小GTP结合蛋白结构域。其氨基酸序列与GenBank中已发表同源对比相似度达99%以上,表明ARF1基因在不同物种间高度保守。     

禾谷镰孢菌β2-微管蛋白在大肠杆菌中的可溶性表达及纯化

【目的】在大肠杆菌中表达禾谷镰孢菌（Fusarium graminearum）的β2-微管蛋白,筛选使β2-微管蛋白可溶性表达的诱导因子,并对可溶性蛋白进行纯化。【方法】以构建好的质粒（pET32a+-β2-tubulin）为模板,PCR扩增出β2-微管蛋白基因,将其克隆到pET30a+表达载体上,并转化到表达宿主菌BL21(DE3)及Rossatta（DE3）pLysS,筛选阳性克隆,进行蛋白诱导表达,并筛选蛋白可溶性表达的诱导因子;对纯化后的蛋白进行SDS-PAGE和Western blot验证。【结果】获得了在大肠杆菌中表达效率高的阳性克隆;为了克服常规诱导条件下表达的β2-微管蛋白主要以包涵体形式存在的问题,通过对诱导温度、时间、诱导物浓度、初始诱导时的菌液浓度、培养液及宿主菌等六因子的综合分析,获得了β2-微管蛋白可溶性表达的优化条件;利用HisTrap HP预装柱对β2-微管蛋白进行纯化,可获得纯度很高的可溶性β2-微管蛋白;SDS-PAGE确认表达出的融合蛋白分子量为51.6 kD;Western blot证实表达的融合蛋白能与6×His及β-微管蛋白的单克隆抗体发生特异性反应。【结论】本研究为外源基因在大肠杆菌中的可溶性表达提供了参考;获得的微管蛋白为研究微管蛋白靶标类药物的抗性机制以及生产中开发新的以微管蛋白为靶标的杀菌剂提供了物质基础。     

不同渗透压条件下金钱鱼肾原代细胞的差异蛋白分析

为了研究海水鱼类肾脏的渗透压调节功能,以金钱鱼肾原代细胞作为研究对象,使用低渗、等渗和高渗培养基处理细胞24 h,通过Label-free定量法进行蛋白质组学分析,质谱定性得到蛋白质共计3 787个,低渗组与等渗组比较,得到显著差异蛋白14个;高渗组与等渗组比较,得到显著差异蛋白31个。筛选标准为Ratio+/-2,且P 0. 05。对差异表达蛋白进行Gene Ontology(GO)功能注释,GO富集分析以及蛋白质聚类分析,并挑选了与细胞骨架、能量合成以及蛋白结合相关的蛋白质进行了mRNA水平的定量检测,从基因和蛋白水平了解肾脏细胞在渗透调节中的变化。结果显示:低渗组中,细胞骨架相关蛋白质受到的影响较大,其中包括微管蛋白α-4A链、微管蛋白β链、肌球蛋白-11以及typeⅠcytoskeletal 18-like,而高渗组中差异显著的蛋白质多为细胞外间质蛋白,如纤连蛋白、胶原蛋白等。因此推测低渗刺激后,胞内渗透压高于胞外渗透压,细胞立即膨胀并大量表达细胞骨架蛋白应对低渗刺激,适应细胞形态的改变;而高渗刺激下,细胞处于失水状态,形态骤缩,影响细胞间的粘连,而fn、plod2和arf1升高表明,细胞正在修复受损蛋白。本研究从细胞水平初步分析了金钱鱼肾脏在不同渗透压刺激下的应答机制,为研究细胞水平的渗透压调节机制提供了新的观点。     

Cloning and expression of a rat brain GABA transporter

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.     

Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence

The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein. However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated. A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli. The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides. The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli. A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity. These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process.     

The primary structure and heterogeneity of tau protein from mouse brain

Tau protein is a family of microtubule binding proteins, heterogeneous in molecular weight, that are induced during neurite outgrowth and are found prominently in neurofibrillary tangles in Alzheimer's disease. The predicted amino acid sequences of two forms of tau protein from mouse brain were determined from complementary DNA clones. These forms are identical in their amino-terminal sequences but differ in their carboxyl-terminal domains. Both proteins contain repeated sequences that may be tubulin binding sites. The sequence suggests that tau is an elongated molecule with no extensive alpha-helical or beta-sheet domains. These complementary DNAs should enable the study of various functional domains of tau and the study of tau expression in normal and pathological states.     

Spatial patterns from oscillating microtubules

Microtubules are fibers of the cytoskeleton involved in the generation of cell shape and motility. They can be highly dynamic and are capable of temporal oscillations in their state of assembly. Solutions of tubulin (the subunit protein of microtubules) and guanosine triphosphate (GTP, the cofactor required for microtubule assembly and oscillations) can generate various dissipative structures. They include traveling waves of microtubule assembly and disassembly as well as polygonal networks. The results imply that cytoskeletal proteins can form dynamic spatial structures by themselves, even in the absence of cellular organizing centers. Thus the microtubule system could serve as a simple model for studying pattern formation by biomolecules in vitro.     

Reconstitution of contractile FtsZ rings in liposomes

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation.     

σNS蛋白与宿主TRAM1因子互作对鸭呼肠孤病毒复制的影响

宿主因子TRAM1(translocation-associated membrane protein 1,TRAM1)是一种参与I型膜蛋白质分解的蛋白,通过升高含量来缓解内质网(ER)在应激时的压力。前期研究发现,鸭呼肠孤病毒(duck reovirus,DRV)感染原代鸭成纤维细胞后,TRAM1宿主因子变化异常。为确定宿主细胞TRAM1因子与DRV非结构蛋白σNS是否存在相互作用以及相互作用对DRV复制的影响,本实验首先表达σNS蛋白,通过GST融合蛋白沉降技术(GST pull-down)发现TRAM1蛋白与σNS蛋白在细胞外存在相互作用;通过免疫共沉淀(co-immunoprecipitation, Co-IP)试验证实二者在细胞内存在相互作用。过表达TRAM1能够从转录水平抑制DRV σNS的表达,而抑制TRAM1提高σNS的转录水平。本研究为进一步分析σNS蛋白在DRV复制过程中的功能以及为σNS蛋白与宿主因子TRAM1互作对DRV复制的影响提供试验依据。