脊髓神经元中微管相关蛋白—5的分布及可塑性

用新生Wistar大鼠进行脊髓神经元培养,研究微管相关蛋白-5与其单克隆抗体结合后的分布情况,使用微管解聚药nocodazole处理神经元,应用免疫组织化学染色来观察微管相关蛋白-5的改变,另一组神经元使用nocodazole处理后再用PMA处理,观察微管相关蛋白-5及神经元的改变。结果表明,微管相关蛋白-5在胞浆及突起中均有分布,并随着培养天数的递增而递减。使用nocodazole后神经元中微管相关蛋白-5的分布及数量明显减少。PMA处理神经元后尽管使微管相关蛋白-5的正常结构被破坏,而神经元的伸展却不受影响。     

脊髓培养神经元中微管相关蛋白-5的分布及可塑性

用新生 Wistar大鼠进行脊髓神经元培养 ,研究微管相关蛋白 - 5与其单克隆抗体结合后的分布情况。使用微管解聚药 nocodazole处理神经元 ,应用免疫组织化学染色来观察微管相关蛋白 - 5的改变。另一组神经元使用 nocodazole处理后再用 PMA处理 ,观察微管相关蛋白 - 5及神经元的改变。结果表明 ,微管相关蛋白 - 5在胞浆及突起中均有分布 ,并随着培养天数的递增而递减。使用 nocodazole后神经元中微管相关蛋白 - 5的分布及数量明显减少。PMA处理神经元后尽管使微管相关蛋白 - 5的正常结构被破坏 ,而神经元的伸展却不受影响     

微管解聚酶驱动蛋白家族成员KIF2A研究进展

驱动蛋白家族成员2A(KIF2A)是一种能够与微管相互作用的蛋白,它参与了细胞内物质运输、细胞迁移、细胞形态改变,以及有丝分裂细胞纺锤体动力学等重要的细胞活动。近年来研究发现,KIF2A凭借其独特的微管解聚能力,对神经元中神经突的生长以及细胞有丝分裂中染色体的运动起着重要的调节作用。将主要对KIF2A在脊椎动物神经元发育和细胞有丝分裂中所行使的作用和功能进行综述。     

神经干细胞分化过程中微管蛋白表达变化的光电镜观察

目的对神经干细胞向神经元定向分化过程中微管蛋白的表达变化进行光、电镜观察研究。方法采用细胞培养技术、免疫荧光技术以及免疫电镜技术对神经干细胞向神经元定向分化过程中微管蛋白的表达变化进行观察。结果在神经干细胞向神经元定向分化的不同时期,存在微管蛋白的表达变化,在分化初期以核周附近分布明显,随神经元的成熟散在分布于胞质中及突起内,形成细网状,构成细胞骨架,维持细胞形态。结论在神经干细胞向神经元定向分化过程中伴随有微管蛋白的表达变化,随神经元的成熟而构成细胞骨架,维持细胞形态。     

神经元极化的分子机制

神经元发育过程中轴突和树突的分化和形成是神经元极化建立的标志,也是建立神经信号转导的基础.近年来,神经元极化的分子机制有了重大突破,发现神经元细胞骨架微丝和微管的结构和功能的改变最终调节着极化的建立.其中,细胞内信号转导途径以及一些激酶参与了调节细胞骨架微丝和微管的结构和功能,最终使神经元极化建立.     

激素引起伊贝母愈伤组织微管蛋白变化的免疫细胞化学研究(简报)

微管蛋白聚合形成微管。微管在维持细胞结构、物质运输、分裂及植物细胞壁的建成等过程中起着重要的作用。70年代后期,在微管生物化学研究取得很大进展的基础上,免疫细胞化学技术与微管研究结合起来,使人们能够从整体水平观察以微管蛋白为主要成份的细胞骨架的动态变化。我们采用免疫酶标技术,对生长在含不同激素培养基上的伊贝母愈伤组织的微管及微管蛋白变化进行了观察和分析,结果表明,激素种类和微管的存在形式是相关的。     

综述:Tau蛋白过度磷酸化机制及其在阿尔茨海默病神经元变性中的作用

Tau蛋白是神经元中含量最高的微管相关蛋白,其经典生物学功能是促进微管组装和维持微管的稳定性．在阿尔茨海默病(Alzheimer's disease,AD)患者,异常过度磷酸化的Tau蛋白以配对螺旋丝结构形成神经原纤维缠结并在神经元内聚积．大量研究提示,Tau蛋白异常在AD患者神经变性和学习记忆障碍的发生发展中起重要作用．本课题组对Tau蛋白异常磷酸化的机制及其对细胞的影响进行了系列研究,发现Tau蛋白表达和磷酸化具有调节细胞生存命运的新功能,并由此对AD神经细胞变性的本质提出了新见解．本文主要综述作者实验室有关Tau蛋白的部分研究结果．     

微管解聚相关蛋白质Kinesin-13、Stathmin和Katanin

微管是细胞骨架的主要成份,参与细胞内物质的运输与细胞形态的维持,还与有丝分裂和减数分裂等生命活动密切相关。大多数微管都表现出动力学的不稳定性,处于动态的聚合和解聚及之间的随机转换状态。Kinesin-13、Stathmin和Katanin是三类能够解聚微管的蛋白质,在纺锤体组装、染色体分离和神经元发育过程中起重要作用。本文主要对这三类微管解聚相关蛋白质的结构、功能、解聚机制进行了简要介绍,并对它们的解聚机制进行了比较。     

水稻雄性不育系珍汕97A小孢子发育过程中的微管骨架

水稻（Oryza sativaL.)雄性不育系珍汕97A,保持系珍汕97B和恢复系测64三系小孢子发生过程的研究表明;恢复系测64小孢子母细胞细胞质浓,有明显的微管荧光围绕着细胞核。小孢子母细胞经两次减数分裂形成四分体。四分体和小孢子的微管从细胞核表面向胞质周缘延伸,形成放射性排列格局,花粉发育正常。细胞质中有少量点状微管荧光,保持系珍汕97B小孢子发生过程的细胞形态和微管结构与恢复系测64相似。但细胞质中的点状微管荧光多一些。雄性不育系珍汕97A小孢子发生早期,小孢子母细胞内出现液泡,核中染色质凝集,微管荧光很弱,没有清晰的微管丝结构。细胞质中有许多点状微管荧光等不正常现象。小孢子母细胞经过减数分裂形成的四分体也没有清晰的丝状微管结构。随后,所有的小孢子迅速败育,雄性不育系珍汕97A在小孢子母细胞发生的很早时期,微管结构就明显不正常。     

草丛土毛虫皮层纤毛器的微管胞器研究

本文应用FLUTAX直接荧光标记和抗α-微管蛋白抗体免疫荧光标记．显示了土壤纤毛虫草丛土毛虫（Territricha stramenticola）的皮层纤毛器微管胞器．其中纤毛器基部微管按口围带、波动膜、额腹横棘毛、左右缘棘毛、背触毛等纤毛器图式分布和定位,口围带和波动膜基部含小膜微管托架、小膜附属微管和波动膜微管骨架网;额腹横棘毛基部含前纵微管束、后纵微管束和横微管束：左、右缘棘毛基部含前纵微管束、后纵微管束、横微管束及后微管芽;背触毛基部含前纵微管束、后纵微管柬。横棘毛基部含有较发达的横微管束,缘棘毛基部含后微管芽及其横微管束的定位可能具有本种纤毛虫细胞的特异性。纤毛器微管胞器在细胞表膜下分化形成的基部微管及其微管层使细胞的运动纤毛器与强固的微管骨架结构网相联系．其微管胞器的建构可能是细胞对土壤生存环境的一种适应．是细胞运动胞器的功能活动与环境相互作用的结果。形态发生中,老口围带微管是逐步进行更新的：老棘毛微管胞器对新结构的发生和形成具有定位和物质贡献的作用．并且老结构在新结构分化和成熟期间也经历了行使相应的生理功能及逐渐退化和失去功能的过程．     

Peptide neuroprotection through specific interaction with brain tubulin

This study aimed to identify the neuronal target for the potent neuroprotective peptide NAP. When added to pheochromocytoma cells (neuronal model), NAP was found in the intracellular milieu and was co-localized with microtubules. NAP induced neurite outgrowth and protected primary neurons against microtubule-associated ZnCl2 toxicity. Rapid microtubule reorganization into distinct microtubules ensued after NAP addition to both pheochromocytoma cells and primary cerebral cortical neurons, but not to fibrobalsts. While binding neuronal tubulin and protecting pheochromocytoma cells against oxidative stress, NAP did not bind tubulin extracted from fibroblasts, nor did it protect those cells against oxidative stress. Affinity chromatography identified the brain-specific betaIII-tubulin as a major NAP binding protein. Paclitaxel (a microtubule aggregating agent that interacts with beta-tubulin) reduced NAP tubulin binding. Thus, the underlying mechanism for the neuroprotection offered by NAP is targeting neuronal microtubules that are essential for neuronal survival and function.     

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule‐associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migra tion. Previous in vitro phosphorylation experiments revealed that cyclin‐dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5?/? mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP‐DCX‐WT, or GFP‐DCX‐S332A and analyzed neurite extension and migration. Introduction of GFP‐DCX‐WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP‐DCX‐S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5?/? brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 929–942, 2010     

Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons

Polarized transport by microtubule-based motors is critical for neuronal development and function. Selective translocation of the Kinesin-1 motor domain is the earliest known marker of axonal identity, occurring before morphological differentiation. Thus, Kinesin-1–mediated transport may contribute to axonal specification. We tested whether posttranslational modifications of tubulin influence the ability of Kinesin-1 motors to distinguish microtubule tracks during neuronal development. We detected no difference in microtubule stability between axons and minor neurites in polarized stage 3 hippocampal neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3β decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications.     

β‐Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β‐tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β‐tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β‐tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

α-Synuclein accumulation reduces GABAergic inhibitory transmission in a model of multiple system atrophy

Multiple system atrophy is a neurodegenerative disease caused by abnormal α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. We previously demonstrated that transgenic (Tg) mice that selectively overexpressed human α-syn in oligodendrocytes exhibited neuronal α-syn accumulation. Microtubule β-III tubulin binds to endogenous neuronal α-syn to form an insoluble complex, leading to progressive neuronal degeneration. α-Syn accumulation is increased in the presynaptic terminals of Tg mice neurons and may reduce neurotransmitter release. To clarify the mechanisms underlying its involvement in neuronal dysfunction, in the present study, we investigated the effects of neuronal α-syn accumulation on synaptic function in Tg mice. Using whole-cell patch-clamp recording, we found that the frequency of miniature inhibitory postsynaptic currents was reduced in Tg mice. Furthermore, a microtubule depolymerizing agent restored normal frequencies of miniature inhibitory postsynaptic currents in Tg mice. These findings suggest that α-syn and β-III tubulin protein complex plays roles for regulation of synaptic vesicle release in GABAergic interneurons, and it causes to reduce GABAergic inhibitory transmission.     

Activation of Microtubule Dynamics Increases Neuronal Growth via the Nerve Growth Factor (NGF)- and Gαs-mediated Signaling Pathways

Signals that activate the G protein Gα_s and promote neuronal differentiation evoke Gα_s internalization in rat pheochromocytoma (PC12) cells. These agents also significantly increase Gα_s association with microtubules, resulting in an increase in microtubule dynamics because of the activation of tubulin GTPase by Gα_s. To determine the function of Gα_s/microtubule association in neuronal development, we used real-time trafficking of a GFP-Gα_s fusion protein. GFP-Gα_s concentrates at the distal end of the neurites in differentiated living PC12 cells as well as in cultured hippocampal neurons. Gα_s translocates to specialized membrane compartments at tips of growing neurites. A dominant-negative Gα chimera that interferes with Gα_s binding to tubulin and activation of tubulin GTPase attenuates neurite elongation and neurite number both in PC12 cells and primary hippocampal neurons. This effect is greatest on differentiation induced by activated Gα_s. Together, these data suggest that activated Gα_s translocates from the plasma membrane and, through interaction with tubulin/microtubules in the cytosol, is important for neurite formation, development, and outgrowth. Characterization of neuronal G protein dynamics and their contribution to microtubule dynamics is important for understanding the molecular mechanisms by which G protein-coupled receptor signaling orchestrates neuronal growth and differentiation.     

Molecular interaction of α-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Evidences demonstrate that α-synuclein may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between α-synuclein and tubulin was confirmed by GST pull-down assay and co-immunoprecipitation. The interacting regions within α-synuclein with tubulin were mapped at the residues 60–100 of α-synuclein that is critical for the binding activity with tubulin. Microtubule assembly assays and sedimentation tests demonstrated that α-synuclein influenced the polymerization of tubulin in vitro, revealing an interacting region-dependent feature. Confocal microscopy detected that exposures of α-synuclein proteins inhibited microtubule formation in the cultured cells, with a length-dependent phenomenon. Our data highlight a potential role of α-synuclein in regulating the microtubule dynamics in neurons. The association of α-synuclein with tubulin may further provide insight into the biological and pathophysiological function of synuclein.     

Tau regulates the localization and function of End‐binding proteins 1 and 3 in developing neuronal cells

The axonal microtubule‐associated protein tau is a well‐known regulator of microtubule stability in neurons. However, the putative interplay between tau and End‐binding proteins 1 and 3 (EB1/3), the core microtubule plus‐end tracking proteins, has not been elucidated yet. Here, we show that a cross‐talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E‐115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down‐regulation leads to a reduction of EB1/3 comet length, as observed in shRNA‐stably depleted neuroblastoma cells and TAU?/? neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over‐expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E‐115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non‐neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull‐down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross‐talk between a classical microtubule‐associated protein and a core microtubule plus‐end tracking protein may contribute to the fine‐tuned regulation of microtubule dynamics and stability during neuronal differentiation.

     

The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.