RGS2 promotes formation of neurites by stimulating microtubule polymerization

Regulator of G-protein signaling (RGS) proteins interact with subunits of heterotrimeric G-proteins via the RGS domain and attenuate their activity by accelerating GTPase activity. RGS2, a member of the RGS family, regulates synaptic development via hereto unknown mechanism. In this study, we found that RGS2 directly interacted with tubulin via a short region at the N-terminus: amino acids 41–60. RGS2 enhanced microtubule polymerization in vitro, and the tubulin binding region was necessary and sufficient for this activity. In Vero cells, polymerization of microtubule was stimulated when peptides containing the tubulin binding region were microinjected. Immunocytochemical analysis showed that endogenous RGS2 was localized at the termini of neurites in differentiated PC12 cells. Over-expression of RGS2 enhanced the nerve growth factor-induced neurite outgrowth in PC12 cells, while specific knock-down of endogenous RGS2 suppressed the neurite outgrowth. These findings demonstrate that RGS2 contributes to the neuronal cell differentiation via regulation of microtubule dynamics.     

Tyrosination State of α-Tubulin in Regenerating Peripheral Nerve

Abstract: Certain modifications of the neuronal cytoskeleton that are associated with development also occur during regeneration of adult mammalian peripheral nerve. The aim of the present study was to examine one such modification, the tyrosination of a-tubulin. Adult rats were anaesthetized and the left or right sciatic nerve randomly selected and crushed to induce regeneration. In certain instances nerves were crushed then ligatured about the crush, to prevent regeneration. Five days later the rats were killed and the regenerating (or ligatured) and the contralateral (control) nerves were removed. Quantitative immunoblotting of nerve homogenates with antibodies that recognize tyrosinated a-tubulin and total a-tubulin revealed a significant increase (p < 0.01) in the proportion of a-tubulin that was tyrosinated in nerve pieces distal (peripheral) to a nerve crush compared with nerve pieces proximal (central) to a nerve crush and to uncrushed nerve. No such difference occurred in ligatured (crushed but nonregenerating) nerve, implying that the increase was related to the presence of regenerating fibres; nor was there any gradient in tyrosination of α-tubulin in control nerves. This effect was confirmed by cytofluorimetric scanning and fluorescence confocal laser scanning microscopy of fixed sections of control and regenerating nerve, stained with antibodies directed against tyrosinated a-tubulin. When nerves were separated into fractions containing assembled and nonassembled tubulin, a significant (p < 0.01) increase was found in the proportion of tyrosinated α-tubulin in the nonassembled tubulin fraction in nerve pieces containing regenerating fibres. This occurred in the absence of a change in the proportion of assembled and nonassembled tubulin. Measurements of tubulin:tyrosine ligase activity, by incorporation of [³H] tyrosine into endogenous nerve tubulin in vitro, indicated a decrease in tyrosine incorporation into tubulin from nerve pieces distal, compared with those proximal to a nerve crush. There was no such difference in ligatured nerves. It is proposed that the increased amount of tyrosinated a-tubulin is related to an alteration in assembly rate of microtubules required for neurite outgrowth and that the apparent decrease in the tubulin:tyrosine ligase activity in vitro reflects the increased tyrosination in vivo.     

Pharmacology and in vivo efficacy of pyridine-pyrimidine amides that inhibit microtubule polymerization

Microtubule-targeting agents are important tools in cancer treatment. Generating novel microtubule targeting agents with novel pharmacology could dramatically expand the utility of this class of drugs. Here we characterize the pharmacology of recently described small molecule microtubule polymerization inhibitors. Pharmacokinetic experiments show oral bioavailability through gastric absorption. In vitro assays designed to predict absorption, distribution, metabolism, and excretion (ADME) and safety reveal a scaffold that is metabolically stable, evades P-glycoprotein, does not inhibit CYP enzymes, occurs as a significant free fraction in serum, and has exceptionally high cellular permeability. Together with in vivo efficacy models, pharmacology supports further development as a treatment for solid tumors.     

Uptake pathways of clinical and healthy animal isolates of Campylobacter jejuni into INT-407 cells

Campylobacter jejuni isolates obtained from human and animal sources showed different invasion levels into human embryonic intestinal (INT-407) cells. There was no significant relation between the degree of invasion and cytotoxins production. The depolymerization of both microfilaments by cytochalasin-D and microtubules by colchicine, demecolcine and nocodazole or stabilization of microtubules by paclitaxel reduced the invasiveness of C. jejuni, although microfilament depolymerization showed greater inhibition than microtubule depolymerization. Interference with receptor-mediated endocytosis by G-strophanthin and monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular C. jejuni cells. Furthermore inhibition of only host protein kinases by staurosporine, but not phosphoinositide 3-kinase by wortmannin or protein kinase-C by calphostin-C, significantly reduced invasion of epithelial cells by C. jejuni. These data suggest that the internalization mechanism triggered by C. jejuni is strikingly different from the microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria such as enteroinvasive strains of Escherichia coli, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica and Yersinia pseudotuberculosis.     

Impairment of microtubule system increases alpha-synuclein aggregation and toxicity

The accumulation of fibrillar form of α-synuclein (α-syn) has been implicated in Parkinson’s disease. Here we show that tubulin can stimulate α-syn fibrillization in vitro in different ways depending on its oligomeric status. The physiological significance of tubulin-seeded α-syn fibrillization is demonstrated by using Saccharomyces cerevisiae as a model system. Perturbation of microtubule system either by treating benomyl that inhibits microtubule assembly or by deleting genes involved in microtubule biogenesis, stimulates α-syn aggregation and toxicity. These results suggest that impairment of the microtubule system may act as a risk factor deteriorating the α-syn-mediated neurodegeneration by increasing the chance of tubulin-seeded α-syn aggregation.     

Organisation and structure of microtubules and microtubule-motor protein complexes

We present a short overview of the current status of work on the organisation and structure of microtubules and of microtubule-motor protein complexes. At present there is great interest in obtaining structural information that can help us to understand the movement of the kinesin family of microtubule associated molecular motors. Using electron cryomicroscopy and image reconstruction methods three dimensional maps of microtubule-motor complexes have been obtained in the presence of different nucleotides. We address a number of principles involved in different aspects of this work. Received: 21 January 1998 / Revised version: 2 March 1998 / Accepted: 7 March 1998     

Tubulin,actin and heterotrimeric G proteins: Coordination of signaling and structure

G proteins mediate signals from membrane G protein coupled receptors to the cell interior, evoking significant regulation of cell physiology. The cytoskeleton contributes to cell morphology, motility, division, and transport functions. This review will discuss the interplay between heterotrimeric G protein signaling and elements of the cytoskeleton. Also described and discussed will be the interplay between tubulin and G proteins that results in atypical modulation of signaling pathways and cytoskeletal dynamics. This will be extended to describe how tubulin and G proteins act in concert to influence various aspects of cellular behavior. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters.This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization

Previously, it has been shown that chronic melatonin exposure in MT1-CHO cells results in receptor desensitization while at the same time producing drastic morphological changes. The addition of a depolymerizing agent during the melatonin pretreatment period prevents MT1 receptor desensitization and the changes in cellular morphology. The lack of morphological change in the presence of a depolymerizing agent is easily explained by the inability of the microtubules to polymerize, however, the prevention of receptor desensitization is a little more complex and may involve G-protein activation. The goal of this study was to determine whether melatonin-induced MT1 receptor desensitization is regulated by proteins known to regulate G-protein activation states, beta-tubulin and RGS4,using anti sense knockdown approaches. The expression of RGS4 mRNA in CHO cells was confirmed using RT PCR and successful knockdown of each was confirmed by western blot analysis or quantitative PCR. Pretreatment of MT1-CHO cells, transfected with the nonsense probes and exposed to melatonin, resulted in a desensitization of the receptor, an increase in forskolin-induced cAMP accumulation, an increase in 2-[125I]-iodomelatonin binding and no change in the affinity of melatonin for the MT1 receptor. However, knockdown of either beta-tubulin or RGS4 in MT1-CHO cells followed by pretreatment with melatonin attenuated the desensitization of melatonin receptors, decreased total 2-[125I]-iodomelatonin binding, and did not affect neither the forskolin response nor the affinity of melatonin for the MT1 receptor. Perhaps RGS4 and beta-tubulin modulate Galpha-GDP and Galpha-GTP states thus modulating MT1 melatonin receptor function.     

萱草花粉微管蛋白的体外聚合及电镜观察

微管(microtubule)作为细胞骨架的主要成分,在植物体内,微管除决定细胞的形状外,还参与很多重要的细胞功能。但有关微管蛋白生物化学的研究绝大多数来自动物脑组织材料,对植物微管蛋白的研究除培养细胞外所知甚少,我们纯化了毫克数量的萱草(Hemer-ocallis fulvaL.)花粉微管蛋白,利用紫杉醇作为促进剂,在Mg2 、GTP等存在下体外聚合成功,并观察了其电镜下的形态。     

Identification of Endogenous Calmodulin-Dependent Kinase and Calmodulin-Binding Proteins in Cold-Stable Microtubule Preparations from Rat Brain

Calmodulin-dependent kinase activity was investigated in cold-stable microtubule fractions. Calmodulin-dependent kinase activity was enriched approximately 20-fold over cytosol in cold-stable microtubule preparations. Calmodulin-dependent kinase activity in cold-stable microtubule preparations phosphorylated microtubule-associated protein-2, alpha- and beta-tubulin, an 80,000-dalton doublet, and several minor phosphoproteins. The endogenous calmodulin-dependent kinase in cold-stable microtubule fractions was identical to a previously purified calmodulin-dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin-binding properties, (4) subunit autophosphorylation, (5) calmodulin-binding subunit composition on high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on beta-tubulin, and (9) phosphopeptide mapping. Endogenous cold-stable calmodulin-dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin-binding, autophosphorylating rho and sigma subunits of the previously purified kinase. The rho and sigma subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and non-denaturing procedures. These results indicate that calmodulin-dependent kinase is a major calmodulin-binding enzyme system in cold-stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.     

One-pot synthesis and biological evaluation of N-(aminosulfonyl)-4-podophyllotoxin carbamates as potential anticancer agents

A series of N-(aminosulfonyl)-4-podophyllotoxin carbamates were synthesized via the Burgess-type intermediate, and their antiproliferative activities were evaluated. Most of them possessed more potent cytotoxic effects against four human tumor cell lines (HeLa, A-549, HCT-8 and HepG2) and less toxic to normal human fetal lung fibroblast WI-38 cells than etoposide. In particular, N-(morpholinosulfonyl)-4-podophyllotoxin carbamate (9) exhibited the most potent activity towards these four tumor cells with IC₅₀ values in the range of 0.5–16.5 μM. Furthermore, immunofluorescence analysis revealed that 9 induced cell apoptosis by up-regulating the expression of p53 and ROS. Meanwhile, 9 effectively inhibited tubulin polymerization and microtubule assembly at cellular levels in HeLa cells. In addition, 9 could induce cell cycle arrest in the G2/M phase in HeLa cells by up-regulating levels of cyclinB1 and cdc2 and decreasing the expression of p-cdc2. These results indicated that 9 had potential for further development as anticancer agents.     

Subcellular Localization of Functionally Differentiated Microtubules in Squid Neurons: Regional Distribution of Microtubule-Associated Proteins and β-Tubulin Isotypes

The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immunologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high-molecular-weight microtubule-associated proteins [MAP A of approximately 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of beta-tubulin isotypes (beta 1 and beta 2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two beta-tubulin isotypes (beta 1 and beta 2); (3) axolinin and the beta 1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the beta 1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the beta 1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonal microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.     

Regulation of 109Cd2+ Uptake into Isolated Neurohypophysial Peptidergic Nerve Terminals

Cadmium (109Cd2+) uptake was studied in a preparation of isolated neurohypophysial nerve terminals. By use of a filter separation method, together with a permeabilizing agent (Triton X-100), two cellular Cd2+ pools have been distinguished. The uptake into the intraterminal pool was governed mainly by a process that displayed saturable kinetics, with a Vmax of 0.15 nmol of Cd2+/mg of protein/min and a Km of 0.18 mM, consistent with a transport system. The superficially bound Cd2+ pool (Triton insensitive), which represented 30-50% of the total Cd2+ bound to the cellular system, was very sensitive to the ionic composition of the incubation medium. Reducing the extracellular Ca2+ or Na+ concentration caused a significant increase in the size of the Triton-insensitive Cd2+ pool. Whereas Na+ did not affect Cd2+ uptake, Ca2+ induced a small, but significant, increase of Cd2+ uptake into the terminals. It is concluded that there is a significant intraterminal uptake of Cd2+, which could explain several physiological effects of this ion.     

The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive effect on microtubule assembly in vitro

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Some evidences demonstrate that PrP may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between PrP and tubulin was confirmed using pull-down assays, immunoprecipitation and ELISA. The interacting regions within PrP with tubulin were mapped in the N-terminus of PrP spanning residues 23-50 and 51-91. PrP octapeptide repeats are critical for the binding activity with tubulin, that the binding activity of PrP with tubulin became stronger along with the number of the octapeptide repeats increased. Microtubule assembly assays, sedimental tests and transmission electron microscopy demonstrated that the full-length PrP (aa 23-231) obviously inhibited the microtubule polymerization processes in vitro, whereas the N- (aa 23-91) and C- (aa 91-231) terminal peptides of PrP did not affect microtubule polymerization. Moreover, the familial Cruetzfeldt Jacob disease (fCJD) related PrP mutants with inserted or deleted octapeptide repeats showed much stronger inhibitive capacities on the microtubule dynamics in vitro than wild-type PrP. Our data highlight a potential role of PrP in regulating the microtubule dynamics in neurons.     

Expression of Nerve Growth Factor and Nerve Growth Factor Receptor Genes in Human Tissues and in Prostatic Adenocarcinoma Cell Lines

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.     

Regulation by Interleukin-1 of Nerve Growth Factor Secretion and Nerve Growth Factor mRNA Expression in Rat Primary Astroglial Cultures

Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38-h incubation). Incubation of astrocytes with interleukin-1 (IL-1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL-1. The stimulatory action of IL-1 on astrocytes prepared from cortex was dose- and time-dependent. Concentrations of IL-1 causing half-maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively). Maximal NGF secretion induced by IL-1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2(+)-free conditions; however, the percent stimulation of NGF secretion by IL-1 was the same in the absence or presence of Ca2+. The stimulatory action of IL-1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL-1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL-1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.     

The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl

Incorporation of leucine-C¹⁴ into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C¹⁴ in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions.     

Demonstration of microtubule-like structures formed with (−)-rhazinilam from purified tubulin outside of cells and a simple tubulin-based assay for evaluation of analog activity

(−)-Rhazinilam was spontaneously generated from a natural product during isolation. In cultured cells, it causes microtubule bundle formation, like those caused by paclitaxel. With tubulin, (−)-rhazinilam causes formation of an aberrant spiral polymer. Using glutamate and GTP, we developed an assay for spiral formation and applied it to 17 new (±)-rhazinilam analogs with either a modified side chain or a different size D ring. There was reasonable correlation between spiral formation and inhibition of human MCF-7 breast carcinoma cell growth. Only one side chain analog was as active as (±)-rhazinilam. During these studies, we observed that omitting GTP from the reaction mixture caused a major change in the morphology of the (−)-rhazinilam-induced polymer, with half the observed polymer being microtubule-like and half being spirals. This mixed polymer slowly disassembled at 0 °C, but there was no apparent difference in the lability of the microtubules versus the spirals.