Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.     

Synapsin I bundles F-actin in a phosphorylation-dependent manner

Synapsin I is a neuron-specific phosphoprotein localized to the cytoplasmic surface of synaptic vesicles. This phosphoprotein is a major substrate for cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases. Its state of phosphorylation can be altered both in vivo and in vitro by a variety of physiological and pharmacological manipulations known to affect synaptic function. Recent direct evidence suggests that it may be involved in the regulation of neurotransmitter release from the nerve terminal. In the nerve terminal, synaptic vesicles are embedded in a cytoskeletal network, consisting in part of actin. We report here the ability of the dephospho-form of synapsin I to bundle F-actin. This bundling activity is reduced when synapsin I is phosphorylated by cAMP-dependent protein kinase and virtually abolished when it is phosphorylated by calcium/calmodulin-dependent protein kinase II or by both kinases. These results, demonstrating an interaction of synapsin I with actin in vitro, support the possibility that synapsin I is involved in clustering of synaptic vesicles at the presynaptic terminal and that the phosphorylation of synapsin I may be involved in regulating the translocation of synaptic vesicles to their sites of release.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Phosphorylation of tubulin enhances its interaction with membranes

Tubulin, the main component of intracellular microtubules, is also a major protein in subcellular membrane preparations and can interact with biological and artificial membranes in vitro. Of particular interest is the association of tubulin with postsynaptic junctional lattices enriched in a polypeptide of relative molecular mass (Mr) 50,000 (50K), recently identified as the major subunit of the calmodulin-dependent protein kinase. Phosphorylation of tubulin with a calmodulin-dependent protein kinase similar to that found in postsynaptic densities inhibits its ability to self-assemble into microtubules in a reversible fashion. This involves the phosphorylation of residues in its 4K carboxy-terminal region, a domain that seems to regulate its self-assembly. The results presented here suggest that the phosphorylation of tubulin with this kinase enhances its ability to interact with membranes. This effect is reversible.     

RIM1alpha is required for presynaptic long-term potentiation.

Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.     

The Ndc80 kinetochore complex forms oligomeric arrays along microtubules

The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by Aurora B kinase. The complex's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.     

TCP1 complex is a molecular chaperone in tubulin biogenesis.

A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.     

Assembly of microtubules from nucleotide-depleted tubulin

In vitro assembly of microtubules from tubulin is considered to have an absolute requirement for added GTP (or a non-hydrolysable GTP-analogue) involving binding at the E(exchangeable)-site located on the beta-subunit of the tubulin dimer. By contrast, GDP inhibits assembly. Nucleotide hydrolysis has been implicated in the dynamic properties of microtubules, treadmilling and mechanical coupling. Here we demonstrate that assembly is not necessarily dependent on the presence of GTP at the E-site; microtubules can be formed efficiently in the absence of GTP in the presence of pyrophosphate. These microtubules, which have normal morphology and lability at cold temperatures, contain N(non-exchangeable)-site GTP and a significant proportion of E-site GDP. This demonstrates the possibility of direct incorporation of GDP-containing tubulin dimer during assembly which probably derives from microtubule-associated protein (MAP)-containing oligomers. This finding has important implications for the mechanism of microtubule elongation. The effects of pyrophosphate suggest that charge neutralization by the bidentate ligand is an essential step in promoting microtubule assembly, and that this interaction involves only a minimal conformational change in the protein.     

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.     

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.     

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.     

In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule "seeds" is usually necessary for plant tubulin assembly in vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule "seed" system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerized in vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical "parabola". The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in the presence of taxol.     

In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule “seeds” is usually necessary for plant tubulin assemblyin vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule “seed” system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerizedin vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical “parabola”. The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in ihe presence of taxol.     

New features of microtubule behaviour observed in vivo

The microtubule cytoskeleton is thought to be intimately involved in generating and maintaining cell polarity and can generate many different morphological structures from a few structural elements. The mechanism by which these structures are generated has been partially elucidated from studies of microtubule polymerization both in vitro and in vivo. Microtubules in vitro exist in growing (polymerizing) and shrinking (depolymerizing) populations that interconvert infrequently. This behaviour, termed dynamic instability, permits microtubules in the cell rapidly to explore different arrangements and allows selective stabilization of specific morphologies. To investigate the regulation of these processes, we have implemented techniques for direct observation of fluorescently labelled microtubules and developed them to observe the dynamic behaviour of individual microtubules in single living cells. Sammak and Borisy recently used this technique to show that the dynamics of microtubules in fibroblasts is explained by dynamic instability. Although we also conclude here that dynamic instability explains much of microtubule behaviour in vivo, we find significant deviations from the properties of tubulin in vitro. These results suggest that local cytoplasmic factors strongly influence microtubule dynamics; such control has important implications for cellular morphogenesis.     

聚合硫酸铁铝硅混凝剂的制备及其性能

研究以硫铁矿烧渣、铝酸钙粉、水玻璃、硫酸为原料，通过酸溶、中和、聚合过程，制备新型复合混凝剂——聚合硫酸铁铝硅(PFASS)的方法．考察了制备工艺对产品稳定性的影响，并对其混凝性能进行了研究．试验结果表明，当Fe^3 Al^3 的摩尔浓度为2．4mol／L、Fe^3 Al^3 ／SiO2摩尔比为10时，产品会有较好的贮存稳定性(即产品保持良好的流动性，无胶凝现象)．聚合硫酸铁铝硅与聚合硫酸铁(PFS)的混凝对比结果表明，PFASS具有更优的混凝除浊性能，具有较宽的pH适用范围．     

Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo

Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. Here, we test directly the role of Syt I during endocytosis in vivo. We use quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (synapto-pHluorin) to measure the kinetics of endocytosis in sytI-null Drosophila. We then combine live imaging of the synapto-pHluorins with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, we demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein.     

Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans

Microtubules, which are essential for mitosis and many other cytoskeletal functions, are composed primarily of alpha- and beta-tubulin. The properties of microtubules are due, in part, to proteins other than tubulins that are part of, or interact with, microtubules and the identification and characterization of such proteins is important to understanding how microtubules function. Analyses of mutations at the mipA (microtubule interacting protein) locus of Aspergillus nidulans have suggested that the product of mipA interacts specifically, probably physically, with beta-tubulin in vivo and is involved in microtubule function. We have cloned and sequenced the wild-type mipA gene as well as complementary DNA copies of its messenger RNA. Comparisons of the predicted product of mipA with tubulins from diverse organisms reveal that mipA is a previously undiscovered member of the tubulin superfamily of genes; the only member yet discovered that does not encode alpha- or beta-tubulin. We propose that the product of mipA be called gamma-tubulin.     

Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin

Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. Here we report the X-ray crystal structure of the Drosophila spastin AAA domain and provide a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, our data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin-tubulin interactions within the microtubule lattice. Our work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients.     

Actin-dependent organelle movement in squid axoplasm.

Studies of organelle movement in axoplasm extruded from the squid giant axon have led to the basic discoveries of microtubule-dependent organelle motility and the characterization of the microtubule-based motor proteins kinesin and cytoplasmic dynein. Rapid organelle movement in higher animal cells, especially in neurons, is considered to be microtubule-based. The role of actin filaments, which are also abundant in axonal cytoplasm, has remained unclear. The inhibition of organelle movement in axoplasm by actin-binding proteins such as DNase I, gelsolin and synapsin I has been attributed to their ability to disorganize the microtubule domains where most of the actin-filaments are located. Here we provide evidence of a new type of organelle movement in squid axoplasm which is independent of both microtubules and microtubule-based motors. This movement is ATP-dependent, unidirectional, actin-dependent, and probably generated by a myosin-like motor. These results demonstrate that an actomyosin-like mechanism can be directly involved in the generation of rapid organelle transport in nerve cells.