Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.     

Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons.

Neurons develop a highly polarized morphology consisting of dendrites and a long axon. Both axons and dendrites contain microtubules and microtubule-associated proteins (MAPs) with characteristic structures. Among MAPs, MAP2 is specifically expressed in dendrites whereas MAP2C and tau are abundant in the axon. But the influence of MAP2, MAP2C and tau on the organization of microtubule domains in dendrites versus axons is unknown. Both MAP2 and tau induce microtubule bundle formation in fibroblasts after transfection of complementary DNAs, and a long process resembling an axon is extended in Sf9 cells infected with recombinant baculovirus expressing tau. We have now expressed MAP2 and MAP2C in Sf9 cells in order to compare their morphology and the arrangement of their microtubules to that found in Sf9 cells expressing tau. We report here that the spacing between microtubules depends on the MAP expressed: in cells expressing MAP2, the distance is similar to that found in dendrites, whereas the spacing between microtubules in cells expressing MAP2C or tau is similar to that found in axons.     

Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule-associated proteins MAP2 and tau

Here we report that the microtubule-associated proteins MAP2 and tau share two separable functional domains. One is the microtubule-binding site which serves to nucleate microtubule assembly; the second is a short C-terminal alpha-helical sequence which can crosslink microtubules by means of a hydrophobic zipper interaction into dense stable parallel arrays characteristic of axons or dendrites. Thus, interactions between molecules of a single type are capable of drastically reorganizing microtubules and completely suppressing their dynamic properties.     

MAP2c confers drug stability to microtubulesin vivo

Microtubules in the axons of nerve cells have unusual stability. To investigate the role of MAP2c in the stabilization of microtubules in neurites, a baculovirus vector has been used to express high levels of MAP2c in insect ovarian Sf9 cells. The cells respond by extending neu-rite-like processes that contain dense bundles of microtubules. The infected cells have been treated with cold, colchicine and nocodazole, respectively. Results showed that all of the polymers were depolymerized within the first 30 min in cold, while no MT depolymerization was detected after 12 h in colchicine or 6 h in nocodazole. The findings strongly suggest that MAP2c is a factor in conferring drug stability to microtubules in neurons, but factors other than MAP2c or in addition to MAP2c are required for cold stability.     

Retrograde transport by the microtubule-associated protein MAP 1C

Microtubules are involved in several forms of intracellular motility, including mitosis and organelle movement. Fast axonal transport is a highly ordered form of organelle motility that operates in both the anterograde (outwards from the cell body) and retrograde (from the periphery towards the cell body) direction. Similar microtubule-associated movement is observed in non-neuronal cells, and might be involved in secretion, endocytosis and the positioning of organelles within the cell. Kinesin is a mechanochemical protein that produces force along microtubules in an anterograde direction. We recently found that the brain microtubule-associated protein MAP 1C (ref. 7) is a microtubule-activated ATPase and, like kinesin, can translocate microtubules in an in vitro assay for microtubule-associated motility. MAP 1C seemed to be related to the ciliary and flagellar ATPase, dynein, which is thought to produce force in a direction opposite to that observed for kinesin. Here we report that MAP 1C, in fact, acts in a direction opposite to kinesin, and has the properties of a retrograde translocator.     

Interaction of brain cytoplasmic dynein and MAP2 with a common sequence at the C terminus of tubulin

Two main types of microtubule-associated proteins (MAPs) have been identified in neuronal cells. The fibrous MAPs, including MAP2 and tau, serve to organize and regulate the assembly of microtubules. A second distinct class of force-producing MAPs, including kinesin, dynein and dynamin, are involved in microtubule-based movement. These proteins are mechanochemical ATPases which seem to be responsible for the bidirectional transport of organelles and perhaps also the movement of chromosomes. Here we report that MAP2 inhibits microtubule gliding on dynein-coated coverslips, as well as the microtubule-activated ATPase of dynein, indicating that MAP2 and other fibrous MAPs could be important modulators of microtubule-based motility in vivo. By proteolytic modification of tubulin, we found that dynein interacts with microtubules at the C termini of alpha- and beta-tubulin, the regions previously reported to be the sites for the interaction of MAP2. The use of site-directed antibodies implicates a small region of alpha- and beta-tubulin, containing the sequence Glu-Gly-Glu-Glu, as the site of the interaction of dynein and MAP2 with the microtubule.     

Gigaxonin-controlled degradation of MAP1B light chain is critical to neuronal survival

Giant axonal neuropathy (GAN) is a devastating sensory and motor neuropathy caused by mutations in the GAN gene, which encodes the ubiquitously expressed protein gigaxonin. Cytopathological features of GAN include axonal degeneration, with accumulation and aggregation of cytoskeletal components. Little is currently known about the molecular mechanisms underlying this recessive disorder. Here we show that gigaxonin controls protein degradation, and is essential for neuronal function and survival. We present evidence that gigaxonin binds to the ubiquitin-activating enzyme E1 through its amino-terminal BTB domain, while the carboxy-terminal kelch repeat domain interacts directly with the light chain (LC) of microtubule-associated protein 1B (MAP1B). Overexpression of gigaxonin leads to enhanced degradation of MAP1B-LC, which can be antagonized by proteasome inhibitors. Ablation of gigaxonin causes a substantial accumulation of MAP1B-LC in GAN-null neurons. Moreover, we show that overexpression of MAP1B in wild-type cortical neurons leads to cell death characteristic of GAN-null neurons, whereas reducing MAP1B levels significantly improves the survival rate of null neurons. Our results identify gigaxonin as a ubiquitin scaffolding protein that controls MAP1B-LC degradation, and provide insight into the molecular mechanisms underlying human neurodegenerative disorders.     

高碳钢小方坯的一次枝晶臂间距的影响因素

高碳钢小方坯显微凝固组织的研究表明:一次枝晶臂在凝固过程中不断粗化变短;碳含量高,容易形成长宽比小的一次枝晶,并且一次枝晶臂间距增宽,容易形成粗大的柱状晶组织;过热度高,易形成粗大的一次枝晶,一次枝晶臂间距增宽;拉速慢,容易形成细长的一次枝晶,一次枝晶臂间距减少;二次冷却水流量比增加,易形成细长的一次枝晶,一次枝晶臂间距降低.结晶器电磁搅拌可显著降低一次枝晶臂长宽比,并减小一次枝晶臂间距;搅拌电流增加,则一次枝晶臂间距减小效果更明显.     

小麦幼苗微管结合蛋白MAP65s体外对微管聚合影响的研究

微管结合蛋白与微管特异地结合在一起,参与调节微管的结构与功能,目前已成为植物细胞骨架研究的前沿领域。以"临优2018"小麦幼苗为材料,通过免疫印迹实验鉴定了小麦幼苗中微管结合蛋白MAP65s的存在并利用紫外吸收法和SDS-聚丙烯酰胺凝胶电泳法初步探究了小麦幼苗MAP65s体外对微管聚合的影响,为进一步研究MAP65s蛋白的生理功能提供了重要的依据。     

MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function

The basic unit of skeletal muscle in all metazoans is the multinucleate myofibre, within which individual nuclei are regularly positioned. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle, including centronuclear myopathies, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein ensconsin (Ens)/microtubule-associated protein 7 (MAP7) and kinesin heavy chain (Khc)/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins interact physically and that expression of the Kif5b motor domain fused to the MAP7 microtubule-binding domain rescues nuclear positioning defects in MAP7-depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally, we show that myonuclear positioning is physiologically important. Drosophila ens mutant larvae have decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell-autonomous fashion.     

Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2

Synaptic transmission from excitatory nerve cells in the mammalian brain is largely mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors located at the surface of dendritic spines. The abundance of postsynaptic AMPA receptors correlates with the size of the synapse and the dimensions of the dendritic spine head. Moreover, long-term potentiation is associated with the formation of dendritic spines as well as synaptic delivery of AMPA receptors. The molecular mechanisms that coordinate AMPA receptor delivery and spine morphogenesis are unknown. Here we show that overexpression of the glutamate receptor 2 (GluR2) subunit of AMPA receptors increases spine size and density in hippocampal neurons, and more remarkably, induces spine formation in GABA-releasing interneurons that normally lack spines. The extracellular N-terminal domain (NTD) of GluR2 is responsible for this effect, and heterologous fusion proteins of the NTD of GluR2 inhibit spine morphogenesis. We propose that the NTD of GluR2 functions at the cell surface as part of a receptor-ligand interaction that is important for spine growth and/or stability.     

Structural basis for the regulation of tubulin by vinblastine

Vinblastine is one of several tubulin-targeting Vinca alkaloids that have been responsible for many chemotherapeutic successes since their introduction in the clinic as antitumour drugs. In contrast with the two other classes of small tubulin-binding molecules (Taxol and colchicine), the binding site of vinblastine is largely unknown and the molecular mechanism of this drug has remained elusive. Here we report the X-ray structure of vinblastine bound to tubulin in a complex with the RB3 protein stathmin-like domain (RB3-SLD). Vinblastine introduces a wedge at the interface of two tubulin molecules and thus interferes with tubulin assembly. Together with electron microscopical and biochemical data, the structure explains vinblastine-induced tubulin self-association into spiral aggregates at the expense of microtubule growth. It also shows that vinblastine and the amino-terminal part of RB3-SLD binding sites share a hydrophobic groove on the alpha-tubulin surface that is located at an intermolecular contact in microtubules. This is an attractive target for drugs designed to perturb microtubule dynamics by interfacial interference, for which tubulin seems ideally suited because of its propensity to self-associate.     

The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons.

The dendrites of many types of neurons contain voltage-dependent Na+ and Ca2+ conductances that generate action potentials (see ref. 1 for review). The function of these spikes is not well understood, but the Ca2+ entry stimulated by spikes probably affects Ca(2+)-dependent processes in dendrites. These include synaptic plasticity, cytotoxicity and exocytosis. Several lines of evidence suggest that dendritic spikes occur within subregions of the dendrites. To study the mechanism that govern the spread of spikes in the dendrites of hippocampal pyramidal cells, we imaged Ca2+ entry with Fura-2 (ref. 9) and Na+ entry with a newly developed Na(+)-sensitive dye. Our results indicate that Ca2+ entry into dendrites is triggered by Na+ spikes that actively invade the dendrites. The restricted spatial distribution of Ca2+ entry seems to depend on the spread of Na+ spikes in the dendrites, rather than on a limited distribution of Ca2+ channels. In addition, we have observed an activity-dependent process that modulates the invasion of spikes into the dendrites and progressively restricts Ca2+ entry to more proximal dendritic regions.     

Raf-1 activates MAP kinase-kinase.

The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.     

层次移动IPv6域内切换与实现

针对移动主机在外地网络中频繁的域内切换的性能问题,提出了移动IPv6的层次型管理的切换模型并给予实现.层次移动IPv6采用移动锚点,可以减少移动主机同本地域以外节点的信令交互,并对层次移动IPv6域内切换的原理进行了研究;组建了层次移动IPv6实验床和物理网络拓扑结构与之相似的移动IPv6实验床,对比移动IPv6的切换机制对层次移动IPv6的域内切换机制进行测试、分析,结果表明层次移动IPv6能够显著提高域内切换的性能.     

A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor

Atrial natriuretic peptide (ANP) is a polypeptide hormone whose effects include the induction of diuresis, natriuresis and vasorelaxation. One of the earliest events following binding of ANP to receptors on target cells is an increase in cyclic GMP concentration, indicating that this nucleotide might act as a mediator of the physiological effects of the hormone. Guanylate cyclase exists in at least two different molecular forms: a soluble haem-containing enzyme consisting of two subunits and a non-haem-containing transmembrane protein having a single subunit. It is the membrane form of guanylate cyclase that is activated following binding of ANP to target cells. We report here the isolation, sequence and expression of a complementary DNA clone encoding the membrane form of guanylate cyclase from rat brain. Transfection of this cDNA into cultured mammalian cells results in expression of guanylate cyclase activity and ANP-binding activity. The ANP receptor/guanylate cyclase represents a new class of mammalian cell-surface receptors which contain an extracellular ligand-binding domain and an intracellular guanylate cyclase catalytic domain.     

In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte.

The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.     

Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein

Dynein, an ATPase, is the force-generating protein in cilia and flagella. It has long been speculated that cytoplasmic microtubules contain a related enzyme involved in cell division or in intracellular organelle transport. A 'cytoplasmic dynein' has been described in sea urchin eggs, but because the egg stockpiles precursors for both cytoplasmic and ciliary microtubules, the role of this enzyme in the cell has remained unresolved. We recently found that the microtubule-associated protein (MAP) 1C (ref. 6) from brain is a microtubule-activated ATPase that produces force in the direction corresponding to retrograde organelle transport in the cell. MAP 1C has several similar properties to ciliary and flagellar dynein. Here we show directly, using scanning transmission electron microscopy, that MAP 1C is structurally equivalent to the ciliary and flagellar enzyme and is the long-sought cytoplasmic analogue of this enzyme.     

Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo

Many lines of evidence suggest that memory in the mammalian brain is stored with distinct spatiotemporal patterns. Despite recent progresses in identifying neuronal populations involved in memory coding, the synapse-level mechanism is still poorly understood. Computational models and electrophysiological data have shown that functional clustering of synapses along dendritic branches leads to nonlinear summation of synaptic inputs and greatly expands the computing power of a neural network. However, whether neighbouring synapses are involved in encoding similar memory and how task-specific cortical networks develop during learning remain elusive. Using transcranial two-photon microscopy, we followed apical dendrites of layer 5 pyramidal neurons in the motor cortex while mice practised novel forelimb skills. Here we show that a third of new dendritic spines (postsynaptic structures of most excitatory synapses) formed during the acquisition phase of learning emerge in clusters, and that most such clusters are neighbouring spine pairs. These clustered new spines are more likely to persist throughout prolonged learning sessions, and even long after training stops, than non-clustered counterparts. Moreover, formation of new spine clusters requires repetition of the same motor task, and the emergence of succedent new spine(s) accompanies the strengthening of the first new spine in the cluster. We also show that under control conditions new spines appear to avoid existing stable spines, rather than being uniformly added along dendrites. However, succedent new spines in clusters overcome such a spatial constraint and form in close vicinity to neighbouring stable spines. Our findings suggest that clustering of new synapses along dendrites is induced by repetitive activation of the cortical circuitry during learning, providing a structural basis for spatial coding of motor memory in the mammalian brain.