小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca^2＋]cyt的关系

以小麦叶肉细胞原生质体为材料,通过免疫荧光标记和Ca^2＋荧光染料的装载并结合药物学试验,借助激光共聚焦扫描显微镜观察,探讨微管骨架和Ca^2＋之间的内在联系。试验结果表明,[Ca^2＋]cyt的升高能够诱发微管骨架的解聚;而微管骨架的解聚也会促使胞外Ca^2＋内流,进而造成[Ca^2＋]cyt的升高。     

Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonas mutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15 axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.     

小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca2+]cyt的关系

以小麦叶肉细胞原生质体为材料,通过免疫荧光标记和Ca~(2 )荧光染料的装载并结合药物学试验,借助激光共聚焦扫描显微镜观察,探讨微管骨架和Ca~(2 )之间的内在联系。试验结果表明,[Ca~(2 )]_(cyt)的升高能够诱发微管骨架的解聚;而微管骨架的解聚也会促使胞外Ca~(2 )内流,进而造成[Ca~(2 )]_(cyt)的升高。     

Neuritogenesis in mouse NB2a/d1 neuroblastoma cells: triggering by calcium influx and involvement of actin and tubulin dynamics

Ionophore (A23187)-mediated calcium influx induced rapid neurite outgrowth in NB2a/d1 cells. This outgrowth was prevented by colchicine but not by cycloheximide, demonstrating a requirement for microtubule assembly but not de novo synthesis. Cytochalasin B induced rapid, colchicine-sensitive outgrowth, indicating that depolymerization of the submembrane actin network may be sufficient to allow neurite outgrowth under conditions which permitted microtubule assembly. Neurites induced by serum-deprivation or calcium influx were rapidly retracted by colchicine unless cytochalasin B was first added, indicating that the actin network may provide the retractile force which mediates neurite retraction following microtubule depolymerization. We conclude that neurite outgrowth can be initiated in NB2a/d1 cells by calcium influx, and may involve alterations in actin and microtubule dynamics.     

Calcium signaling is involved in dynein-dependent microtubule organization

The microtubule cytoskeleton supports cellular morphogenesis and polar growth, but the underlying mechanisms are not understood. In a screen for morphology mutants defective in microtubule organization in the fungus Ustilago maydis, we identified eca1 that encodes a sarcoplasmic/endoplasmic calcium ATPase. Eca1 resides in the endoplasmic reticulum and restores growth of a yeast mutant defective in calcium homeostasis. Deletion of eca1 resulted in elevated cytosolic calcium levels and a severe growth and morphology defect. While F-actin and myosin V distribution is unaffected, Deltaeca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca(2+)/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in Deltaeca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

The C terminus of tubulin, a versatile partner for cationic molecules: binding of Tau, polyamines, and calcium

The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.     

Overexpression of Arabidopsis translationally controlled tumor protein gene AtTCTP enhances drought tolerance with rapid ABA-induced stomatal closure

Translationally controlled tumor protein (TCTP), also termed P23 in human, belongs to a family of calcium- and tubulin-binding proteins, and it is generally regarded as a growth-regulating protein. Recently, Arabidopsis TCTP (AtTCTP) has been reported to function as an important growth regulator in plants. On the other hand, plant TCTP has been suggested to be involved in abiotic stress signaling such as aluminum, salt, and water deficit by a number of microarray or proteomic analyses. In this study, the biological functions of AtTCTP were investigated by using transgenic Arabidopsis plants overexpressing AtTCTP. Interestingly, AtTCTP overexpression enhanced drought tolerance in plants. The expression analysis showed that AtTCTP was expressed in guard cells as well as in actively growing tissues. Physiological studies of the overexpression lines showed increased ABA- and calcium-induced stomatal closure ratios and faster stomatal closing responses to ABA. Furthermore, in vitro protein-protein interaction analysis confirmed the interaction between AtTCTP and microtubules, and microtubule cosedimentation assays revealed that the microtubule binding of AtTCTP increased after calcium treatment. These results demonstrate that the overexpression of AtTCTP confers drought tolerance to plants by rapid ABA-mediated stomatal closure via the interaction with microtubules in which calcium binding enhances the interaction. Collectively, the present results suggest that the plant TCTP has molecular properties similar to animal TCTPs, such as tubulin- and calcium-binding, and that it functions in ABA-mediated stomatal movement, in addition to regulating the growth of plants.     

Alteration in calcium channel properties is responsible for the neurotoxic action of a familial frontotemporal dementia tau mutation

Tau, a microtubule binding protein, is not only a major component of neurofibrillary tangles in Alzheimer's disease, but also a causative gene for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show here that an FTDP-17 tau mutation (V337M) in SH-SY5Y cells reduces microtubule polymerization, increases voltage-dependent calcium current (ICa) density, and decreases ICa rundown. The reduced rundown of ICa by V337M was significantly inhibited by nifedipine (L-type Ca channel blocker), whereas omega-conotoxin GVIA (N-type Ca channel blocker) showed smaller effects, indicating that tau mutations affect L-type calcium channel activity. The depolarization-induced increase in intracellular calcium was also significantly augmented by the V337M tau mutation. Treatment with a microtubule polymerizing agent (taxol), an adenylyl cyclase inhibitor, or a protein kinase A (PKA) inhibitor, counteracted the effects of mutant tau on ICa. Taxol also attenuated the Ca2+ response to depolarization in cells expressing mutant tau. Apoptosis in SH-SY5Y cells induced by serum deprivation was exacerbated by the V337M mutation, and nifedipine, taxol, and a PKA inhibitor significantly protected cells against apoptosis. Our results indicate that a tau mutation which decreases its microtubule-binding ability augments calcium influx by depolymerizing microtubules and activating adenylyl cyclase and PKA.     

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional γ-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular “cocoon”. Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Effect of modification of cell calcium status on lectin activity

Effects of oryzalin (10 microM), an inhibitor of microtubule polymerization, on the activity of soluble and cell wall lectins were studied in 7 day-old seedlings of unhardened (23 degrees C) and cold acclimated (7 days at 2-3 degrees C) winter wheat (Triticum aestivum L.). Seedlings were grown in the presence of 25 microM and 1 mM Ca2+, 500 microM verapamil, 250 microM chlorpromazine or without modifiers of calcium status in the medium. Inhibitor of the microtubule polymerization inhibitor, likely as inhibitors of Ca(2+)-signal, decreased the activity of soluble lectins and increased that of cell wall lectins. Apparently, injury of microtubule phosphorylation results in a more considerable microtubule disorganization, than that observed after oryzalin effect. A low Ca2+ concentration (25 microM) depressed, while a high concentration (1 mM) prompted microtubule sensibility to oryzalin. Such an effect of high Ca2+ concentration may be related to destabilizative action of Ca(2+)-calmodulin in these conditions, because chlorpromazine decreased oryzalin-induced increase in the activity of cell wall lectins with 1 mM Ca2+. It is concluded that the activity of cell wall lectins depends on the microtubule status that is regulated by calcium signal.     

AKAP220 protein organizes signaling elements that impact cell migration

Cell movement requires the coordinated reception, integration, and processing of intracellular signals. We have discovered that the protein kinase A anchoring protein AKAP220 interacts with the cytoskeletal scaffolding protein IQGAP1 to influence cell motility. AKAP220/IQGAP1 networks receive and integrate calcium and cAMP second messenger signals and position signaling enzymes near their intended substrates at leading edges of migrating cells. IQGAP1 supports calcium/calmodulin-dependent association of factors that modulate microtubule dynamics. AKAP220 suppresses GSK-3β and positions this kinase to allow recruitment of the plus-end microtubule tracking protein CLASP2. Gene silencing of AKAP220 alters the rate of microtubule polymerization and the lateral tracking of growing microtubules and retards cell migration in metastatic human cancer cells. This reveals an unappreciated role for this anchored kinase/microtubule effector protein network in the propagation of cell motility.     

A novel tubulin-dependent protein kinase forming a paired helical filament epitope on tau

From rat brain microtubule proteins, we purified a protein kinase that phosphorylated tau, one of microtubule-associated proteins. The electrophoretic mobility of the phosphorylated tau on SDS-polyacrylamide gel decreased. The enzyme was not activated by cyclic nucleotides, calmodulin, or phospholipids, and was inhibited by the calcium ions. The kinase bound to tau. The phosphorylation of tau was stimulated by tubulin under the condition of microtubule formation. From these results we propose an idea that the phosphorylation could occur concomitantly with microtubule formation in the brain. Human tau phosphorylated by the kinase carried an epitope of the paired helical filaments that accumulate in the brain in Alzheimer's disease.     

Some Factors Involved in the Control of Microtubule Assembly in Sea Urchins

Some recent evidence is discussed which suggests that the controlof microtubule assembly during the formation of the mitoticapparatus in sea urchins involves three factors. These include:nucleation sites such as the kinetochores, a mechanism for regulatingintracellular calcium, and a heat stable substance which preventsspontaneous, non-nucleated microtubule polymerization.     

Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

The association of the cytoskeleton with the cadherin--catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the E-cadherin-catenin-actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non- or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.     

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.     

Microtubules and beta cell function: effect of colchicine on microtubules and insulin secretion in vitro by mouse beta cells

A monolayer culture system was developed to study the role of microtubules in insulin secretion. Cultured cells were obtained to study the role of microtubules in insulin secretion. Cultured cells were obtained by enzymatic digestion of pancreases from C57BL-KsJ mice 6-12 wk of age. On day 4 of culture, the medium was changed, control or treatment medium added, and frequent samples were removed for insulin assay. Microtubules and beta cells were identified by indirect immunofluorescence with monospecific antibodies to tubulin and insulin. An extensive microtubule network radiates from the perinuclear region of the beta cell to the plasma membrane. Although alterations in the calcium concentration of the medium did not affect the microtubule pattern, the absence of calcium or glucose in the medium inhibited insulin secretion (P less than 0.001). Optimum insulin release occurred at a calcium concentration of 2.5 mM. Colchicine, in concentrations of 10(-10) M, did not affect the microtubule immunofluorescent pattern, whereas concentrations of 1 and 5 x 10(-7) M decreased the number of microtubules, and microtubules could not be identified in cultures treated with 10(-6) M colchicine for 2 h. After a 2-h preincubation, the prolonged release of insulin at either 2.0 or 4.5 mg/ml of glucose was decreased by 10(-6) M colchicine (P less than 0.02). The immediate release of insulin was similar to that in control plates and occurred in cultures with no identifiable microtubules. Microtubules and insulin secretion were not altered by 10(-6) M lumicolchicine and prolonged insulin secretion recovered 24 h after removal of colchicine. These studies show that the microtubules facilitate sustained secretion of insulin but are not required for the immediate release of the hormone. Alterations in the extracellular calcium concentration which play an essential role in insulin secretion do not alter the microtubule pattern in the beta cell.     

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.     

激发子诱发小麦叶肉细胞原生质体微管骨架排列格局与[Ca2+]cyt的变化

以小麦叶肉细胞原生质体-激发子互作为研究体系,通过免疫荧光标记和Ca~(2 )荧光染料的装载并结合药理学试验,借助激光共聚焦扫描显微镜观察,探讨小麦抵抗叶锈菌侵染过程中微管骨架和Ca~(2 )之间的内在联系。试验结果表明,激发子处理可引起抗性品种原生质体[Ca~(2 )]_(cyt)的升高并诱发微管骨架的解聚,预解聚微管骨架,再用激发子处理,可使抗性品种原生质体[Ca~(2 )]_(cyt)的升高幅度增加。