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1.
J Nat Prod ; 79(6): 1576-85, 2016 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-27300079

RESUMEN

M-Phase Phosphoprotein 1 (MPP1), a microtubule plus end directed kinesin, is required for the completion of cytokinesis. Previous studies have shown that MPP1 is upregulated in various types of bladder cancer. This article describes inhibitor screening leading to the identification of a new class of natural product inhibitors of MPP1. Two compounds with structural similarity, norlobaridone (1) and physodic acid (2), were found to inhibit MPP1. Physodic acid is not competitive with ATP, indicating the presence of an allosteric inhibitor-binding pocket. Initial drug-like property screening indicates that physodic acid is more soluble than norlobaridone and has more favorable lipophilicity. However, both suffer from high clearance in human microsomal stability assays mediated by the lability of the lactone ring as well as hydroxylation of the alkyl chains as shown by metabolite identification studies. In cell-based assays physodic acid is a weak inhibitor with EC50 values of about 30 µM in a range of tumor cell lines. The two depsidones identified and characterized here could be used for future improvement of their activity against MPP1 and will be useful chemical probes for studying this unique molecular motor in more depth.


Asunto(s)
Depsidos/aislamiento & purificación , Dibenzoxepinas/aislamiento & purificación , Cinesinas/antagonistas & inhibidores , Lactonas/aislamiento & purificación , Líquenes/química , Algoritmos , Protocolos de Quimioterapia Combinada Antineoplásica , Citocinesis/efectos de los fármacos , Depsidos/química , Depsidos/farmacología , Dibenzoxepinas/química , Dibenzoxepinas/farmacología , Humanos , Cinesinas/efectos de los fármacos , Cinesinas/metabolismo , Lactonas/química , Lactonas/farmacología , Melfalán , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Estructura Molecular , Prednisona , Procarbazina
2.
Nat Prod Rep ; 32(4): 534-42, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25512265

RESUMEN

Various marine natural products that target cytoskeletal proteins have been discovered. A few of these compounds have recently been shown to induce or inhibit protein-protein interactions. Lobophorolide, an actin filament-disrupting macrolide, binds to actin with a unique 2 : 2 stoichiometry in which two lobophorolide molecules cooperate to stabilize an actin dimer. Adociasulfates, merotriterpenoid derivatives, inhibit microtubule-stimulated ATPase activity of a motor protein kinesin by blocking both the binding of microtubules and the processive motion of kinesin along microtubules. The antitumor macrolide aplyronine A synergistically binds to tubulin in association with actin, and prevents spindle formation and mitosis. In this highlight, we address recent chemical biology studies on these mechanistically-attractive marine natural products. These findings may be useful for the design and development of new pharmacological tools and therapeutic agents.


Asunto(s)
Productos Biológicos/farmacología , Microtúbulos/efectos de los fármacos , Actinas/metabolismo , Productos Biológicos/química , Cinesinas/efectos de los fármacos , Macrólidos/farmacología , Biología Marina , Estructura Molecular , Piranos/farmacología , Tubulina (Proteína)/metabolismo
3.
Assay Drug Dev Technol ; 12(8): 470-80, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25383721

RESUMEN

The kinesin superfamily of motor proteins are involved in the active transport of a large number of cargos such as organelles, proteins, and RNAs from the neuronal cell body to distal neuronal processes. Previously, we have shown that kinesin-mediated axonal transport of proteins and RNAs are important for long-term memory storage. Identification of small molecules that can activate or inhibit kinesins is of specific interest due to the significance of kinesin-mediated functions in neuronal health and plasticity. Here, we describe a high-throughput screening assay designed to specifically identify compounds that inhibit or activate adenosine triphosphatase activity of the kinesin 5B of humans. The luminescence-based assay that we developed is highly reproducible and robust. Using this approach, we screened a pharmacologically characterized compound collection and have identified small molecules with either activator or inhibitor-like activity. To further characterize screening hits, we also developed an orthogonal assay based on absorbance and a counter screen assay based on luminescence. Development of such assays is important to help identify small molecules that can be used in potential drug development efforts targeted at modulating the function of kinesin.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Cinesinas/efectos de los fármacos , Adenosina Trifosfatasas/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Indicadores y Reactivos , Luminiscencia , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas
4.
J Med Chem ; 56(16): 6314-6, 2013 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-23899248

RESUMEN

Talapatra et al. elucidated the molecular basis of resistance by characterizing the binding interactions between Eg5 and the allosteric inhibitor SB743921. The investigation, employing biochemical, biophysical, and structural analyses, made path-breaking revelations in Eg5 studies and discussed a novel phenomenon "resistance by allostery", which could have far-reaching consequences from a rational drug design perspective.


Asunto(s)
Diseño de Fármacos , Cinesinas/efectos de los fármacos , Regulación Alostérica , Benzamidas/química , Benzamidas/farmacología , Cromonas/química , Cromonas/farmacología , Humanos , Cinesinas/química , Modelos Moleculares
5.
J Med Chem ; 56(16): 6317-29, 2013 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-23875972

RESUMEN

Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 (1), a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of 1 by several 1000-fold. Surprisingly, the structures of wild-type and mutant Eg5 in complex with 1 display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to 1 is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called "resistance by allostery".


Asunto(s)
Benzamidas/farmacología , Cromonas/farmacología , Cinesinas/fisiología , Mitosis , Regulación Alostérica , Humanos , Cinesinas/química , Cinesinas/efectos de los fármacos , Cinética , Modelos Moleculares
6.
Endocrinology ; 153(4): 1734-42, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22334720

RESUMEN

Intracellular cargo transport relies on microtubules and motor proteins such as kinesins and dyneins. Currently we have ample knowledge of the mechanisms by which motor proteins propel themselves along the microtubules, but little is known about intracellular factors that regulate motor speed. Here we show that proadrenomedullin N-terminal 20 peptide (PAMP) increases kinesin velocity and ATP consumption in a dose-dependent manner, using a variety of human kinesins. Structure-activity studies found that the terminal amide of PAMP is required for modulating kinesin activity and that the smallest peptide fragment retaining this role is PAMP12₋20. On the other hand, peptide fragments as small as PAMP18₋20 maintained the ability of delaying tubulin polymerization, another function previously described for PAMP, indicating that these two activities depend on different regions of the molecule. To demonstrate that these observations are also relevant in vivo, hippocampal neurons were isolated from mice lacking the gene coding for PAMP and from wild type littermates. Intravital stains followed by time-lapse microscopy analysis revealed that mitochondrial speed inside neurons lacking PAMP was significantly slower than in cells expressing the peptide. External addition of synthetic PAMP reversed this phenotype in PAMP-null neurons. Besides the obvious implications for better understanding cell biology, these results may be also relevant for the rapidly evolving discipline of nanotechnology because PAMP may be used as an accelerator of nanodevices based on microtubules and motor proteins.


Asunto(s)
Adrenomedulina/farmacología , Adrenomedulina/fisiología , Cinesinas/efectos de los fármacos , Cinesinas/fisiología , Adenosina Trifosfatasas/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Adrenomedulina/deficiencia , Animales , Relación Dosis-Respuesta a Droga , Hipocampo/citología , Humanos , Técnicas In Vitro , Ratones , Ratones Noqueados , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Modelos Animales , Neuronas/efectos de los fármacos , Neuronas/fisiología , Polimerizacion
7.
Diabetes ; 60(1): 320-30, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20870970

RESUMEN

OBJECTIVE: Suppression of Kinesin-1 by antisense oligonucleotides, or overexpression of dominant-negative acting kinesin heavy chain, has been reported to affect the sustained phase of glucose-stimulated insulin secretion in ß-cells in vitro. In this study, we examined the in vivo physiological role of Kinesin-1 in ß-cell development and function. RESEARCH DESIGN AND METHODS: A Cre-LoxP strategy was used to generate conditional knockout mice in which the Kif5b gene is specifically inactivated in pancreatic ß-cells. Physiological and histological analyses were carried out in Kif5b knockout mice as well as littermate controls. RESULTS: Mice with ß-cell specific deletion of Kif5b (Kif5b(fl/)⁻:RIP2-Cre) displayed significantly retarded growth as well as slight hyperglycemia in both nonfasting and 16-h fasting conditions compared with control littermates. In addition, Kif5b(fl/)⁻:RIP2-Cre mice displayed significant glucose intolerance, which was not due to insulin resistance but was related to an insulin secretory defect in response to glucose challenge. These defects of ß-cell function in mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or ß-cell size. However, compared with controls, pancreas of Kif5b(fl/)⁻:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in ß-cells. CONCLUSIONS: In addition to being essential for maintaining glucose homeostasis and regulating ß-cell function, Kif5b may be involved in ß-cell development by regulating ß-cell proliferation and insulin vesicle synthesis.


Asunto(s)
Células Secretoras de Insulina/fisiología , Insulina/metabolismo , Cinesinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Genotipo , Humanos , Insulina/deficiencia , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinesinas/química , Cinesinas/deficiencia , Cinesinas/efectos de los fármacos , Cinesinas/genética , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Fragmentos de Péptidos/química
8.
J Am Chem Soc ; 130(24): 7584-91, 2008 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-18491908

RESUMEN

Human kinesin spindle protein (KSP)/hsEg5, a member of the kinesin-5 family, is essential for mitotic spindle assembly in dividing human cells and is required for cell cycle progression through mitosis. Inhibition of the ATPase activity of KSP leads to cell cycle arrest during mitosis and subsequent cell death. Ispinesib (SB-715992), a potent and selective inhibitor of KSP, is currently in phase II clinical trials for the treatment of multiple tumor types. Mutations that attenuate Ispinesib binding to KSP in vitro have been identified, highlighting the need for inhibitors that target different binding sites and inhibit KSP activity by novel mechanisms. We report here a small-molecule modulator, KSPA-1, that activates KSP-catalyzed ATP hydrolysis in the absence of microtubules yet inhibits microtubule-stimulated ATP hydrolysis by KSP. KSPA-1 inhibits cell proliferation and induces monopolar-spindle formation in tumor cells. Results from kinetic analyses, microtubule (MT) binding competition assays, and hydrogen/deuterium-exchange studies show that KSPA-1 does not compete directly for microtubule binding. Rather, this compound acts by driving a conformational change in the KSP motor domain and disrupts productive ATP turnover stimulated by MT. These findings provide a novel mechanism for targeting KSP and perhaps other mitotic kinesins.


Asunto(s)
Adenosina Trifosfato/metabolismo , Hidrocarburos Fluorados/farmacología , Cinesinas/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Pirroles/farmacología , Adenosina Difosfato/metabolismo , Unión Competitiva , Línea Celular , Proliferación Celular/efectos de los fármacos , Deuterio/metabolismo , Humanos , Hidrógeno/metabolismo , Hidrólisis/efectos de los fármacos , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Ligandos , Maleatos/farmacología , Microtúbulos/metabolismo , Huso Acromático/efectos de los fármacos
9.
Anticancer Drugs ; 18(7): 773-9, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17581299

RESUMEN

HsEg5 (Eg5) is a kinesin required for proper execution of mitosis. Several compounds that specifically block Eg5 are in clinical development and have the potential to be used in the treatment of breast cancer. In this study, we investigated the interaction between Eg5 and estrogen receptor signaling. We observed decreased Eg5 expression after treatment of estrogen receptor-positive human breast cancer MCF-7 cells with the estrogen receptor downregulator fulvestrant. Downregulation of Eg5 expression in response to fulvestrant was also observed in another estrogen receptor-positive cell line ZR-75, but not in the estrogen receptor-negative breast cancer cell line MDA-231. Moreover, in MCF-7 cells previously arrested in the G0/G1 phase of the cell cycle by fulvestrant, addition of estrogen increased Eg5 expression. This upregulation correlated with progression through S-phase. Nevertheless, the effect of fulvestrant in Eg5 expression could not be explained solely by cell cycle arrest, because treatments that blocked cell cycle progression did not consistently decrease Eg5 expression. Pharmacological inhibition of Eg5 function, with either S-trityl-L-cysteine or monastrol, prevented growth of estrogen-treated MCF-7 cells with an IC50 of 0.46 and 29.71 micromol/l, respectively. Simultaneous inhibition of estrogen receptor function with fulvestrant increased the IC50 for S-trityl-L-cysteine to 2.30 micromol/l and for monastrol to 112.69 micromol/l. Our results suggest that pharmacological inhibition of Eg5 may be an effective treatment for estrogen receptor-positive breast cancer, even without concomitant hormonal therapy.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Cinesinas/metabolismo , Receptores de Estrógenos/antagonistas & inhibidores , Western Blotting , Neoplasias de la Mama/fisiopatología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cisteína/administración & dosificación , Cisteína/análogos & derivados , Cisteína/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Estradiol/farmacología , Estrógenos , Femenino , Citometría de Flujo , Fulvestrant , Humanos , Concentración 50 Inhibidora , Cinesinas/antagonistas & inhibidores , Cinesinas/efectos de los fármacos , Mitosis/efectos de los fármacos , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Tionas/administración & dosificación , Tionas/farmacología , Regulación hacia Arriba
10.
Arch Toxicol ; 78(10): 575-83, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15205888

RESUMEN

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 microM, and inhibition is complete at about 10 microM. In this range, the tubulin assembly is fully (up to 6 microM) or partially (~6-10 microM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 microM Hg2+, the IC50 5.8 microM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 microM and a complete inhibition is reached at 1 microM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 microM HgCl2. Between 15 and 20 microM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 microM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 microM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.


Asunto(s)
Cloruro de Mercurio/toxicidad , Micronúcleos con Defecto Cromosómico/inducido químicamente , Proteínas de Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Mutagénesis/efectos de los fármacos , Mutágenos/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/patología , Cromosomas/efectos de los fármacos , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Cinesinas/efectos de los fármacos , Cinesinas/metabolismo , Pruebas de Micronúcleos , Microscopía por Video , Proteínas de Microtúbulos/ultraestructura , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/efectos de los fármacos , Proteínas Motoras Moleculares/metabolismo , Mutagénesis/genética , Nivel sin Efectos Adversos Observados , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismo
11.
Mol Biol Cell ; 15(1): 219-33, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14528012

RESUMEN

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.


Asunto(s)
Cromátides/metabolismo , Posicionamiento de Cromosoma/fisiología , Drosophila melanogaster/metabolismo , Cinesinas/metabolismo , Huso Acromático/metabolismo , Anafase/fisiología , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Clonación Molecular , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Cinesinas/efectos de los fármacos , Cinesinas/genética , Metafase/fisiología , Microinyecciones , Microscopía Fluorescente , Microtúbulos/metabolismo , Modelos Moleculares , Mutación , ARN Interferente Pequeño/farmacología , Telofase/fisiología
12.
Arch Toxicol ; 78(1): 49-57, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14513206

RESUMEN

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 micro M was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 micro M, and no-effect-concentrations were between 0.001 and 0.005 micro M. Clearly enhanced MN rates were found at 0.1 micro M and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37 degrees C was seen above the no-effect-concentration of 2 mM, with an IC(50) of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 micro M and reaching complete inhibition of motility at 30 micro M, whereas benzonitrile up to 200 micro M did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 micro M. This points to the relevance of interactions with the cellular spindle apparatus.


Asunto(s)
Pruebas de Micronúcleos , Mutágenos/toxicidad , Nitrilos/toxicidad , Nitrobencenos/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Citoesqueleto/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Cinesinas/efectos de los fármacos , Mutágenos/clasificación , Rojo Neutro/metabolismo , Tubulina (Proteína)/efectos de los fármacos
13.
J Cell Biol ; 162(6): 1045-55, 2003 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-12975348

RESUMEN

Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1-YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.


Asunto(s)
Transporte Axonal/fisiología , Polaridad Celular/fisiología , Cinesinas/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Axones/metabolismo , Axones/ultraestructura , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Células Cultivadas , Dendritas/metabolismo , Dendritas/ultraestructura , Feto , Cinesinas/efectos de los fármacos , Ratones , Proteínas Asociadas a Microtúbulos , Microtúbulos/ultraestructura , Proteínas Motoras Moleculares/fisiología , Neuronas/citología , Paclitaxel/farmacología , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión
14.
Mol Biol Cell ; 14(11): 4458-69, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12937278

RESUMEN

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.


Asunto(s)
Caenorhabditis elegans/metabolismo , Proteínas de Insectos/metabolismo , Cinesinas/metabolismo , Meiosis/fisiología , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Animales , Caenorhabditis elegans/fisiología , Centrosoma/metabolismo , Centrosoma/fisiología , Cromosomas/metabolismo , Cromosomas/fisiología , Femenino , Proteínas de Insectos/efectos de los fármacos , Proteínas de Insectos/fisiología , Cinesinas/efectos de los fármacos , Cinesinas/fisiología , Microtúbulos/fisiología , ARN Interferente Pequeño/farmacología , Huso Acromático/fisiología
15.
J Cell Sci ; 116(Pt 7): 1209-17, 2003 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-12615964

RESUMEN

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for neurite extension and is known to occur mainly at the growth cone. This report examines whether this is a regulated event controlled by growth factors. The Golgi complex and nascent PPVs of hippocampal neurons in culture were pulse-labeled with fluorescent ceramide. We studied the dynamics of labeled PPVs upon arrival at the axonal growth cone. In controls and cultures stimulated with brain-derived neurotrophic factor (BDNF), PPV clusters persisted in growth cones with a half-life (t(1/2)) of >14 minutes. Upon challenge with IGF-1, however, fluorescent elements cleared from the growth cones with a t(1/2) of only 6 minutes. Plasmalemmal expansion was measured directly as externalization of membrane glycoconjugates in resealed growth cone particles (GCPs) isolated from fetal forebrain. These assays demonstrated that membrane expansion could be stimulated by IGF-1 in a dose-dependent manner but not by BDNF, even though intact, functional BDNF receptor was present on GCPs. Because both BDNF and IGF-1 are known to enhance neurite growth, but BDNF did not stimulate membrane expansion at the growth cone, we studied the effect of BDNF on the IGF-1 receptor. BDNF was found to cause the translocation of the growth-cone-specific IGF-1 receptor subunit beta(gc) to the distal axon, in a KIF2-dependent manner. We conclude that IGF-1 stimulates axonal assembly at the growth cone, and that this occurs via regulated exocytosis of PPVs. This mechanism is affected by BDNF only indirectly, by regulation of the beta(gc) level at the growth cone.


Asunto(s)
Membrana Celular/metabolismo , Exocitosis/fisiología , Conos de Crecimiento/metabolismo , Sustancias de Crecimiento/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Células Cultivadas , Ceramidas , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Feto , Colorantes Fluorescentes , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/ultraestructura , Sustancias de Crecimiento/farmacología , Hipocampo/citología , Hipocampo/embriología , Hipocampo/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Cinesinas/efectos de los fármacos , Cinesinas/metabolismo , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/metabolismo , Receptor trkB/agonistas , Receptor trkB/metabolismo , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/ultraestructura
16.
Nat Cell Biol ; 4(10): 790-7, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12360289

RESUMEN

The molecular motor kinesin travels processively along a microtubule in a stepwise manner. Here we have studied the chemomechanical coupling of the hydrolysis of ATP to the mechanical work of kinesin by analysing the individual stepwise movements according to the directionality of the movements. Kinesin molecules move primarily in the forward direction and only occasionally in the backward direction. The hydrolysis of a single ATP molecule is coupled to either the forward or the backward movement. This bidirectional movement is well described by a model of Brownian motion assuming an asymmetric potential of activation energy. Thus, the stepwise movement along the microtubule is most probably due to Brownian motion that is biased towards the forward direction by chemical energy stored in ATP molecules.


Asunto(s)
Adenosina Trifosfato/metabolismo , Transporte Biológico/fisiología , Células Eucariotas/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Adenosina Trifosfato/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Bovinos , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Cinesinas/efectos de los fármacos , Cinética , Modelos Biológicos , Proteínas Motoras Moleculares/efectos de los fármacos , Factores de Tiempo
17.
Mutat Res ; 499(1): 73-84, 2002 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-11804606

RESUMEN

The role of microtubule-based motors in the induction of abnormal centrosome integrity by dimethylarsinic acid (DMAA) was investigated with the use of monastrol, a specific inhibitor of mitotic kinesin, and vanadate, an inhibitor of dynein ATPase. Cytoplasmic dynein co-localized with multiple foci of gamma-tubulin in mitotic cells arrested by DMAA. Disruption of microtubules caused dispersion of dynein while multiple foci of gamma-tubulin were coalesced to a single dot. Vanadate also caused dispersion of dynein, which had been co-localized with multiple foci of gamma-tubulin by DMAA, without affecting spindle organization. However, the dispersion of dynein did not prohibit the induction of abnormal centrosome integrity by DMAA. Inhibition of mitotic kinesin by monastrol resulted in monoastral cells with non-migrated centrosomes in the cell center. Monastrol, when applied to mitotic cells with abnormal centrosome integrity, rapidly reduced the incidence of cells with the centrosome abnormality. Moreover, monastrol completely inhibited reorganization of abnormal centrosomes that had been coalesced to a single dot by microtubule disruption. These results suggest that abnormal centrosome integrity caused by DMAA is not simply due to dispersion of fragments of microtubule-organizing centers, but is dependent on the action of kinesin. In addition, the results suggest that kinesin plays a role not only in the induction of mitotic centrosome abnormality, but also in maintenance.


Asunto(s)
Ácido Cacodílico/farmacología , Centrosoma/efectos de los fármacos , Dineínas/metabolismo , Cinesinas/metabolismo , Huso Acromático/fisiología , Animales , Células Cultivadas , Cricetinae , Cricetulus , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dineínas/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Cinesinas/antagonistas & inhibidores , Cinesinas/efectos de los fármacos , Masculino , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Pirimidinas/farmacología , Huso Acromático/efectos de los fármacos , Tionas/farmacología , Tubulina (Proteína)/metabolismo , Vanadatos/farmacología
18.
J Cell Biol ; 154(6): 1125-33, 2001 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-11564753

RESUMEN

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.


Asunto(s)
Cinesinas/fisiología , Microtúbulos/fisiología , Huso Acromático/química , Proteínas de Xenopus , Adenosina Trifosfatasas/farmacología , Adenilil Imidodifosfato/farmacología , Animales , Citoplasma/química , Complejo Dinactina , Humanos , Cinesinas/efectos de los fármacos , Cinesinas/metabolismo , Cinética , Microscopía Fluorescente/métodos , Proteínas Asociadas a Microtúbulos/farmacología , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/antagonistas & inhibidores , Proteínas Motoras Moleculares/metabolismo , Proteínas Motoras Moleculares/fisiología , Movimiento , Pirimidinas/farmacología , Huso Acromático/metabolismo , Huso Acromático/fisiología , Tionas/farmacología , Xenopus
19.
Science ; 286(5441): 971-4, 1999 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-10542155

RESUMEN

Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.


Asunto(s)
Cinesinas/efectos de los fármacos , Mitosis/efectos de los fármacos , Pirimidinas/farmacología , Huso Acromático/efectos de los fármacos , Tionas/farmacología , Proteínas de Xenopus , Actinas/efectos de los fármacos , Animales , Bovinos , Línea Celular , Citoesqueleto/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Proteínas Motoras Moleculares/efectos de los fármacos , Fenotipo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Células Tumorales Cultivadas , Xenopus , Nucleolina
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