Effects of nocodazole on structures of calf brain tubulin

Lee, James C.; Field, Deborah J.; Lee, Lucy L. Y.

doi:10.1021/bi00567a041

Cited by 101 publications

(55 citation statements)

References 31 publications

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“…Aliquots were kept at -20°C and diluted to final concentrations in PEM buffer [0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9,2 mM EGTA, 1 mM MgSO4I for in vitro studies or in cell culture medium for in vivo studies. These working dilutions were kept on ice and used within 4 h to minimize the likelihood of the nocodazole precipitating out of solution (Lee et al, 1980). Porcine brain tubulin and axoneme fragments from Strongylocentrotus purpuratus sperm were prepared as described previously (Vasquez, et al 1994).…”

Section: Materials and Methods Stock Solutions And Protein Purificationmentioning

confidence: 99%

“…It is still widely used to study MT-dependent processes because of its ability to rapidly depolymerize MTs in vivo when administered at micromolar concentrations. Early studies demonstrated that, at concentrations equal to or greater than those of tubulin, nocodazole inhibits tubulin polymerization in vitro (Hoebeke et al, 1976;Friedman and Platzer, 1978;Ireland, 1979;Lee et al, 1980) and in vivo . Cultured cells treated with high concentrations of nocodazole quickly depolymerize their MTs and are prevented from proceeding through mitosis .…”

Section: Introductionmentioning

confidence: 99%

“…Cultured cells treated with high concentrations of nocodazole quickly depolymerize their MTs and are prevented from proceeding through mitosis . Other studies have demonstrated that nocodazole competes with colchicine for tubulin binding but that nocodazole exhibits more rapid binding to, and dissociation from, tubulin than does colchicine (Hoebeke, 1976;Lee et al, 1980). Recent studies of several MT drugs indicate that, at low concentrations relative to tubulin, these drugs can disrupt MT-dependent processes by altering MT dynamics, without resulting in significant MT depolymerization (Jordan and Wilson, 1990;Jordan et al, 1992;Toso et al, 1993;Tanaka et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

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Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Vasquez

Howell

Yvon

et al. 1997

MBoC

408

328

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Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 ,uM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.

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Section: Materials and Methods Stock Solutions And Protein Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Vasquez

Howell

Yvon

et al. 1997

MBoC

408

328

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“…The excitation and emission wavelengths were 488 nm and 505 nm for BODIPY FL-C5, and 543 nm and 580 nm for TAMRA-succinimidyl ester, respectively. Nocodazole (methyl-(5-[thienylcarbonyl]-1 H-benzimidazol-2-YL) carbamate; Sigma Aldrich, St. Louis, Missouri), an agent that interferes with structure and function of microtubules (Lee et al, 1980), was added to the media (5 g/ml) 30 minutes before the addition of apoptotic bodies to modulate phagocytosis.…”

Section: Phagocytosismentioning

confidence: 99%

Apoptotic Body Engulfment by a Human Stellate Cell Line Is Profibrogenic

Canbay

Taimr

Torok

et al. 2003

Laboratory Investigation

375

274

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SUMMARY:Hepatocyte apoptosis and stellate cell activation are both features of chronic liver diseases, but a relationship between these events has not been explored. In macrophages, engulfment of apoptotic bodies induces expression of transforming growth factor-␤ (TGF-␤), a profibrogenic cytokine. We examined whether a similar response occurs in stellate cells. Fluorescently labeled hepatocyte apoptotic bodies were added to cultures of primary and immortalized human stellate cells. Stellate cells, but not hepatocytes, readily engulfed apoptotic bodies in a time-dependent manner as assessed by confocal microscopy. The activation of primary and immortalized human stellate cells after incubation with apoptotic bodies, as well as their fibrogenic activity, was indicated by an increase in ␣-smooth muscle actin (primary cells), TGF-␤1, and collagen ␣1(I) mRNA (primary and immortalized cells). The profibrogenic response was dependent upon apoptotic body engulfment, because nocodazole, a microtubule-inhibiting agent, blocked both the engulfment and the increase of TGF-␤1 and collagen ␣1(I) mRNA. As described in primary rodent stellate cells, up-regulation of collagen ␣1(I) mRNA was inhibited by a PI-3K inhibitor (LY294002) and a p38 mitogen-activated protein kinase inhibitor (SB203580) in LX-1 cells. In conclusion, these data support a model in which engulfment of hepatocyte apoptotic bodies by stellate cells leads to a fibrogenic response by eliciting a kinase-signaling pathway. (Lab Invest 2003, 83:655-663).L iver fibrosis is a cardinal feature of chronic liver injury. Indeed, despite the wide array of metabolic, genetic, viral, and cholestatic pathologies causing human liver injury, fibrosis is a common, if not generic, response of the liver to these injury processes. Extensive liver fibrosis results from the deposition and accumulation of type I collagen within the liver parenchyma and is recognized clinically as cirrhosis. The clinical complications of cirrhosis include portal hypertension and chronic liver failure. For many patients with advanced liver cirrhosis, the only viable treatment option is liver transplantation, highlighting the clinical severity of this fibrotic process. Insights into the mechanisms initiating and promoting liver fibrosis are, therefore, of broad scientific and clinical importance.Activated hepatic stellate cells have been established as the source of type I collagen in the liver (de Leeuw et al, 1984;Friedman et al, 1985;McGee and Patrick, 1972). Under basal conditions, hepatic stellate cells are in a quiescent state and function to store retinoids within the liver (Blomhoff et al, 1990;Geerts, 2001). However, upon activation, these cells undergo transformation, assuming a myofibroblast morphology and expressing smooth muscle actin (Ramadori et al, 1990;Schmitt-Graff et al, 1991). These activated stellate cells secrete collagen types I and III, the principle matrix protein responsible for the development of liver fibrosis and cirrhosis (Friedman, 2000;Kent et al, 1976). Over time, activ...

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“…Microtubule polymerization was modulated by either a depolymerization protocol using nocodazole or colchicine (Sigma Chemical Co.), or by promoting microtubule assembly with taxol, a plant-derived drug that has been reported to enhance microtubule assembly and stabilize microtubules against depolymerizing conditions (Garland, 1978;Schiff et al, 1979;Lee et al, 1980 (Loeb et al, 1985;Pollock et al, 1991;Ishii et al, 1991;Nunokawa et al, 1993 collected and the acid-extracted cultures were solubilized in 1.0 N NaOH. The HCl extract was directly analysed for cyclic GMP by radioimmunoassay using the automated Gammaflow system and the protein concentration of the NaOH-solubilized samples was determined, as described by Marczin et al (1992Marczin et al ( , 1993.…”

Section: Modulation Of Microtubule and Microfilament Assemblymentioning

confidence: 99%

Cytoskeleton‐dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells

Marczin¹,

Jilling²,

Papapetropoulos³

et al. 1996

British J Pharmacology

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1 Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin-and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2 Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)-and interleukin-lfl-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3 Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4 Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5 In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell-and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6 Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.

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Effects of nocodazole on structures of calf brain tubulin

Cited by 101 publications

References 31 publications

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Apoptotic Body Engulfment by a Human Stellate Cell Line Is Profibrogenic

Cytoskeleton‐dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells

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