植物驱动蛋白: 从微管阵列到生理活动调控

驱动蛋白(kinesin)是以微管为轨道的分子马达, 其催化ATP水解为ADP, 将贮藏在ATP分子中的化学能高效地转化为机械能, 在细胞形态建成、细胞分裂、细胞运动、胞内物质运输和信号转导等多种生命活动中发挥重要作用。对植物驱动蛋白的研究落后于动物和真菌, 其原因不仅由于植物进化出独有的驱动蛋白家族, 而且其家族成员数量远多于动物驱动蛋白。该文主要总结了驱动蛋白在微管阵列动态组织, 包括周质微管和有丝分裂早前期微管带、纺锤体及成膜体中的角色和功能, 以及其对植物生理活动的调控作用。同时对重要经济作物大豆(Glycine max)中的驱动蛋白进行了系统分析、分类及功能预测, 发现大豆驱动蛋白数量庞大。结合公共数据库中大豆转录组数据, 对部分大豆驱动蛋白进行功能预测, 以期对大豆及其它作物驱动蛋白功能研究提供线索和启示。     

Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.     

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.     

Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus

The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIF1, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus-directed microtubule movement up to 2 microm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.     

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

Fractionation and characterization of kinesin II species in vertebrate brain

Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide-dependent, microtubule-binding proteins from 13-day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

Expression of a novel HsMCAK mRNA splice variant,tsMCAK gene,in human testis

Identification of specifically expressed genes in the adult or fetal testis is very important for the study of genes related to the development and function of the testis. In this study, a human adult testis cDNA microarray was constructed and hybridized with 33P-labeled human adult and embryo testis cDNA probes, respectively. After differential display analyzing, a number of new genes related to the development of testis and spermatogenesis had been identified. One of these new genes is tsMCAK. tsMCAK was expressed 2.62 folds more in human adult testis than fetal testis. The full length of tsMCAK is 2401 bp and contains a 2013 bp open reading frame, encoding a 671-amino-acid protein. Sequence analysis showed that it has a central kinesin motor domain and is homologous to HsMCAK gene of the somatic cells. Blasting human genome database localized tsMCAK to human chromosome 1P34 and further investigation showed that it is a splice variant of HsMCAK. The tissue distribution of tsMCAK was determined by RT-PCR and it is expressed highly and specifically in the testis. Southern blot studies of its expression in patients with infertility indicated its specific expression in spermatogenic cells and its correlation with male infertility. The above results suggested that tsMCAK is a candidate gene for the testis-specific KRPs and its specific expression in the testis was correlated with spermatogenesis and may be correlated with male infertility.