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1.
J Cell Biol ; 88(1): 234-40, 1981 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-7193677

RESUMEN

Microtubule proteins and tubulin have been purified from brain and labeled with dichlorotriazinyl fluorescein (DTAF). This procedure compromises neither the polymerizability of the proteins nor their affinities for unlabeled proteins. Within 15 min after microinjection of either DTAF-microtubule proteins or DTAF-tubulin into cultured gerbil fibroma cells, there was an evolution of a fluorescent fibrillar pattern with a distribution similar to that of the microtubular network seen after staining with fluorescent antitubulin. These filaments were colchicine sensitive and could be seen to elongate with time. DTAF-labeled microtubule accessory proteins from brain were not incorporated into filaments and appeared to label autophagic vacuoles.


Asunto(s)
Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Línea Celular , Fibroblastos , Colorantes Fluorescentes , Gerbillinae , Microinyecciones , Microscopía Fluorescente , Microtúbulos/ultraestructura
2.
J Cell Biol ; 97(6): 1762-76, 1983 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-6315742

RESUMEN

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.


Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Endocitosis , Glicoproteínas/metabolismo , Neoplasias Hepáticas/fisiopatología , Asialoglicoproteínas , Línea Celular , Endocitosis/efectos de los fármacos , Fluoresceína-5-Isotiocianato , Fluoresceínas , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Cinética , Monensina/farmacología , Tiocianatos
3.
J Cell Biol ; 99(3): 1167-72, 1984 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-6432803

RESUMEN

We have developed an accurate and practical method for measuring intracellular Ca2+ concentration [( Ca2+]i) in single cells in monolayer culture using the fluorescent Ca2+-binding dye quin2. Quin2 was loaded into cells as a membrane-permeant ester which is hydrolyzed in the cytoplasm to the impermeant free acid, which is the indicator form (Tsien, R.Y., T. Pozzan, and T.J. Rink, 1982, J. Cell Biol., 94:325-334). The method involves the measurement of fluorescence at 340-nm excitation (I340), where dye fluorescence is dependent on Ca2+, and at 360-nm excitation (I360), where dye fluorescence is independent of Ca2+. The ratio of these two values (I340/I360) is thus related to the concentration of Ca2+ but independent of dye concentration and can be used as a measure of [Ca2+]. To test the ratio method in the microscope, we measured [Ca2+]i in GH3 cells in monolayer culture. We found a resting [Ca2+]i of 44 +/- 28 nM (mean +/- SD, n = 34), as compared with a suspension value (Gershengorn, M., and C. Thaw, 1983, Endocrinology, 113:1522-1524) of 118 +/- 18 nM. We also measured [Ca2+]i during stimulation of the cells with thyrotropin-releasing hormone (TRH) and found a 2.4-fold increase above resting levels within 20 s, a trough at 73% of resting at 90-100 s, and a peak slightly above resting at 3 min. Depolarization of the plasma membrane with KCl produced a sustained increase in [Ca2+]i. All of these data are in good agreement with the results of Gershengorn and Thaw on suspension cultures. When measuring both resting [Ca2+]i and the effects of TRH and KCl on small groups of cells, we found some variation among experiments. Using an image intensifier-video camera, we videotaped cells during TRH stimulation. Digital image analysis of these pictures demonstrated that there was a large variation in responsiveness from cell to cell. The microscope ratio method offers the possibility of resolving regions of differing [Ca2+] within the cytoplasm.


Asunto(s)
Calcio/metabolismo , Hormona Liberadora de Tirotropina/farmacología , Aminoquinolinas , Animales , Línea Celular , Colorantes Fluorescentes , Cinética , Magnesio/farmacología , Neoplasias Hipofisarias , Cloruro de Potasio/farmacología , Conejos , Ratas , Sodio/farmacología , Espectrometría de Fluorescencia/métodos
4.
Science ; 235(4786): 337-9, 1987 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-2432662

RESUMEN

In order to study the rate and form of tubulin transport in cultured neuronal cells, the fluorescence recovery after the photobleaching of a fluorescent tubulin analog has been followed within the neuritic processes of differentiated PC12 cells. In these cells, as in peripheral axons, tubulin is transported in coherent, nondiffusing waves at two different slow rates that are within the range of the slow components a and b of axonal transport measured in vivo. Finally, it appears that most, if not all, of the tubulin analog is moving out these processes. Thus, slow neuroplasmic transport in cultured neuron-like cells is a good model of axonal transport, in which experimental manipulations of the system can be performed that would be difficult in the whole animal.


Asunto(s)
Neuronas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Transporte Axonal , Transporte Biológico , Feocromocitoma , Ratas , Espectrometría de Fluorescencia , Grabación en Video
5.
Int Rev Cytol ; 205: 77-147, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11336394

RESUMEN

The sculpting and maintenance of axonal and dendritic arbors is largely under the control of molecules external to the cell. These factors include both substratum-associated and soluble factors that can enhance or inhibit the outgrowth of axons and dendrites. A large number of factors that modulate axonal outgrowth have been identified, and the first stages of the intracellular signaling pathways by which they modify process outgrowth have been characterized. Relatively fewer factors and pathways that affect dendritic outgrowth have been described. The factors that affect axonal arbors form an incompletely overlapping set with those that affect dendritic arbors, allowing selective control of the development and maintenance of these critical aspects of neuronal morphology.


Asunto(s)
Axones/fisiología , Dendritas/fisiología , Animales , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Matriz Extracelular/química , Inmunohistoquímica , Factores de Crecimiento Nervioso/metabolismo , Neurotransmisores/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología
6.
Genetics ; 157(3): 979-90, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11238388

RESUMEN

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.


Asunto(s)
Cromosomas/genética , Cósmidos/genética , Biblioteca de Genes , Genoma Fúngico , Neurospora crassa/genética , Bacteriófago lambda/genética , Mapeo Cromosómico , ADN Complementario/genética , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada , Ligamiento Genético , Vectores Genéticos , Cariotipificación , Modelos Genéticos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Mapeo Físico de Cromosoma , Análisis de Secuencia de ADN
7.
Eur J Cell Biol ; 47(1): 94-100, 1988 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-3229421

RESUMEN

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.


Asunto(s)
Calmodulina/análogos & derivados , Mitosis/efectos de los fármacos , Fenotiazinas/farmacología , Análisis de Varianza , Animales , Calcio/análisis , Calmodulina/antagonistas & inhibidores , Calmodulina/farmacología , Línea Celular , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Imidazoles/farmacología , Sulfonamidas/farmacología
8.
Biotechniques ; 27(4): 722-6, 728, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10524313

RESUMEN

We present a unique design for a flow cell with a small working volume that allows rapid displacement of media viewed under high power and short working distance objectives. The flow cell has a small internal depth (ca. 0.033 cm) and volume (ca. 0.05 mL) and is easy to handle. Made of Delrin, the flow cell is biologically inert. We have used the flow cell for fluorescence imaging of PC12 cells loaded with tetramethylrhodamine dextran (TMRD) and other dyes.


Asunto(s)
Técnicas de Cultivo de Célula , Fenómenos Fisiológicos Celulares , Técnicas Citológicas , Animales , AMP Cíclico/metabolismo , Técnicas Citológicas/instrumentación , Fluoresceína , Colorantes Fluorescentes , Humanos , Ratones , Microscopía Fluorescente , Células PC12 , Perfusión , Ratas , Rodaminas , Células Tumorales Cultivadas
9.
Biotechniques ; 25(5): 858-60, 862-4, 866, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9821588

RESUMEN

When thin optically transparent specimens are grown on reflective substrates, contrast in reflection confocal microscopy is markedly enhanced. This enhanced contrast allows for the visualization of the thin filopodia and organelles contained within the neuritic processes of PC12 cells in culture. The characteristics of this contrast enhancement suggest that it arises because of interference between light scattered from the specimen and coherently backscattered illumination reflected off the substrate. This technique provides a method for visualizing living cells or other similarly transparent objects on opaque substrates in a nondestructive manner.


Asunto(s)
Microscopía Confocal/métodos , Animales , Medios de Cultivo/química , Medios de Cultivo/farmacología , Oro/farmacología , Técnicas Histológicas , Luz , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Células PC12 , Ratas
10.
J Neurosci Methods ; 39(2): 141-52, 1991 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-1798344

RESUMEN

The local control of intracellular microtubule polymerization equilibria has been hypothesized to be an important factor in the determination of neurite extension and other examples of cellular asymmetry. Provided that the quantum yield of the fluorophore remains constant, the combination of fluorescent analogue cytochemistry, differential extraction protocols, and quantitative video microscopy makes it possible to measure local fractions of cytoskeletal protein in polymer, even when it is impossible to resolve individual fibrils of the polymer. We have developed appropriate quantitative video microscopic techniques for measuring the fluorescence of a fluorescent analogue-injected neurite before and after extraction under microtubule-stabilizing conditions. We have used these methods to demonstrate that tetramethylrhodamine-n-hydroxysuccinimide tubulin is an appropriate fluorescent analogue, allowing us to measure fractions of tubulin in polymer locally within PC12 neurites. As would be expected, the fraction of tubulin fluorescent analogue in polymer approaches 1.0 in neurites exposed to the microtubule-stabilizing drug taxol and is close to 0 in neurites injected and extracted in the cold, or extracted under microtubule-destabilizing conditions. We have, therefore, developed a tool that allows us to measure microtubule polymerization equilibria out the neurites of cells in culture, which will allow us to test hypotheses that factors which affect neurite outgrowth do so by means of effects on microtubule polymerization equilibria.


Asunto(s)
Microscopía Fluorescente/métodos , Microtúbulos/metabolismo , Neuronas/metabolismo , Tubulina (Proteína)/metabolismo , Neoplasias de las Glándulas Suprarrenales/patología , Animales , Axones/ultraestructura , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Neuronas/ultraestructura , Feocromocitoma/patología , Polímeros , Ratas , Rodaminas , Succinimidas , Televisión , Células Tumorales Cultivadas
12.
Cell Motil Cytoskeleton ; 7(1): 1-9, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-3545503

RESUMEN

Calcium and calmodulin are believed to play a significant role in the regulation of mitosis, because they are both localized in the mitotic spindle and because they can potentiate microtubule depolymerization in the test tube and in the living cell. It has been hypothesized, specifically, that calcium-saturated calmodulin drives the shortening of the kinetochore microtubules that must occur during prometaphase, when the chromosomes congress to the metaphase plate, and during anaphase A, when the half-spindles shorten. We have examined the role of calmodulin in mitosis by observing the consequences of calmodulin microinjection on the progress of mitosis and morphology of the mitotic spindle in PtK2 cells. We have found that the injection of excess calcium-saturated calmodulin during early prometaphase significantly prolongs the time required for the cell to go into anaphase, and that neither calcium-depleted calmodulin nor buffer alone produce a similar perturbation. Calcium ion alone produces a similar but much smaller retardation of mitosis. Immunofluorescence and fluorescent analogue cytochemical studies of spindle morphology reveal that the immediate (less than 5-min) effect of calcium-saturated calmodulin on prometaphase spindles is a significant shortening of the kinetochore fibers and "interpolar" microtubules but not the astral microtubules. After this perturbation, however, the spindle quickly recovers its normal form. An equivalent transient shortening of the spindle fibers is seen following the injection of calcium chloride solutions but not after the injection of calcium-depleted calmodulin or buffer alone. Taken together, these observations suggest that calcium-saturated calmodulin plays a significant role in the regulation of mitosis, and that this regulatory pathway involves more than spindle fiber shortening.


Asunto(s)
Calcio/farmacología , Calmodulina/farmacología , Mitosis/efectos de los fármacos , Animales , Línea Celular , Dipodomys , Técnica del Anticuerpo Fluorescente , Metafase/efectos de los fármacos , Microinyecciones , Profase/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructura
13.
Cell Motil Cytoskeleton ; 17(2): 95-105, 1990.
Artículo en Inglés | MEDLINE | ID: mdl-2257634

RESUMEN

We have injected process-bearing PC12 cells with colchicine-tubulin mixed with either fluorescein-dextran or a rhodamine-labelled tubulin analogue to determine the role of microtubule polymerization in neurite elongation. Colchicine-tubulin is a specific, substoichiometric poison of microtubule assembly. We have shown that colchicine-tubulin does not cause existing PC12 microtubules to disassemble, and yet can inhibit the assembly of rhodamine-tubulin injected along with it. In population studies of neurite outgrowth in injected and uninjected cells, we find that colchicine-tubulin substantially inhibits neurite extension from injected cells over a wide variety of concentrations. In acute time-course studies of injected cells, we find that colchicine-tubulin does not block neurite outgrowth until the injectate reaches the neurite tip. Thereafter, however, it blocks process elongation completely. Thus we can conclude that microtubule polymerization in the region of the growth cone is an important element in neurite elongation. While polymerization at the cell body may be important in supplying subunits to the distal neurite, it does not play a direct role in process extension.


Asunto(s)
Axones/fisiología , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Animales , Axones/efectos de los fármacos , Línea Celular , Colchicina/farmacología , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Microscopía Fluorescente , Microtúbulos/metabolismo , Rodaminas , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacología
14.
J Neurochem ; 54(4): 1258-68, 1990 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-2179472

RESUMEN

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.


Asunto(s)
Feocromocitoma/metabolismo , Polímeros/metabolismo , Tubulina (Proteína)/metabolismo , Axones/ultraestructura , Tampones (Química) , Técnica del Anticuerpo Fluorescente , Técnicas Histológicas , Microscopía Electrónica , Microtúbulos/ultraestructura , Tubulina (Proteína)/análogos & derivados , Células Tumorales Cultivadas
15.
J Neurosci Res ; 52(5): 599-611, 1998 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-9632316

RESUMEN

Glutamate can both facilitate and inhibit dendrite outgrowth in vitro. The major effects of low levels of glutamate occur only on the dendrites (not the axon) of pyramidal neurons and may be important for modulating dendrite outgrowth during neuronal development in vivo. Cytoskeletal changes resulting from glutamate exposure must underlie these changes in dendrite outgrowth. In the present study, hippocampal neuron cultures were used to measure the outgrowth of both axons and immature dendrites in the presence or absence of 50 microM glutamate. Subsequently, neurons were extracted and fixed for immunofluorescent labeling of microtubules and rhodamine phalloidin labeling of microfilaments. Additionally, neurons were prepared for electron microscopy to examine dendritic microtubules at the ultrastructural level. Glutamate led to increased dendrite outgrowth in the short term (4 hr) and dendrite retraction in the long term (8 hr). After short-term glutamate exposures, no obvious morphological changes occur in either the microtubules or microfilaments. However, longer glutamate exposure causes a decrease in the number of microtubules in the distal region of retracting dendrites, and causes an increase in microtubule number in the dendritic shaft of both retracting and growing dendrites. Thus, the microtubule cytoskeleton may be involved in producing the changes in dendrite outgrowth caused by glutamate exposure.


Asunto(s)
Dendritas/fisiología , Dendritas/ultraestructura , Ácido Glutámico/farmacología , Microtúbulos/ultraestructura , Citoesqueleto de Actina/ultraestructura , Animales , Axones/fisiología , Axones/ultraestructura , Células Cultivadas , Dendritas/efectos de los fármacos , Hipocampo/citología , Microscopía Electrónica , Células Piramidales/ultraestructura , Ratas , Ratas Sprague-Dawley
16.
Cell Motil Cytoskeleton ; 25(4): 345-57, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8402955

RESUMEN

We have examined the effects of various means of photobleaching on the recovery of fluorescence, movement, and morphology of the microtubules in the neurites of rhodamine-tubulin-injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low-power beam for an extended period, fluorescence in polymer recovers very little after 1 hour. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high-power laser, for 0.5-5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate-stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high-intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20-30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extend through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in PC12 neurites, and both are likely to be involved in neurite maintenance and growth.


Asunto(s)
Luz , Microtúbulos/fisiología , Neuritas/fisiología , Animales , Citoplasma/ultraestructura , Fluorescencia , Rayos Láser , Métodos , Microscopía Electrónica , Microtúbulos/efectos de la radiación , Microtúbulos/ultraestructura , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Nocodazol/farmacología , Células PC12 , Tubulina (Proteína)/análisis
17.
J Protozool ; 35(1): 123-9, 1988 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-3284998

RESUMEN

A mouse monoclonal anti-alpha-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines--J774 macrophages, BSC-1, and Vero cells--infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Citoesqueleto/ultraestructura , Microtúbulos/ultraestructura , Trypanosoma cruzi/ultraestructura , Tubulina (Proteína)/inmunología , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Macrófagos , Microscopía Electrónica , Trypanosoma cruzi/inmunología , Células Vero
18.
J Neurosci Res ; 47(5): 555-60, 1997 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-9067865

RESUMEN

The temporal dynamics of the intracellular second messenger cyclic AMP (cAMP) were monitored in living PC12 cells by digital fluorescence ratio imaging using FlCRh, a single-excitation dual-emission cAMP indicator. When the cells were depolarized by exposure to high K+, the free cAMP concentration was elevated, and then slowly decreased back to resting levels when the depolarizing stimulus was removed. Furthermore, the cAMP elevation due to depolarization decreased with successive depolarizations.


Asunto(s)
Potenciales de Acción/fisiología , AMP Cíclico/fisiología , Células PC12/fisiología , Potasio/fisiología , Animales , Fluorescencia , Ratas
19.
Proc Natl Acad Sci U S A ; 82(3): 800-4, 1985 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-3856233

RESUMEN

Using a fluorescence ratio method, we have studied the intracellular free calcium levels in individual quin-2-loaded mitotic cells under the microscope. We have found that intracellular free calcium concentrations in Pt K2 epithelial cells drop by approximately 50% as they pass through mitosis. Calcium levels in interphase cells were 53 +/- 7 nM. During prophase, free cytoplasmic calcium begins to decrease, reaching 28 +/- 3 nM in prometaphase. Calcium levels remain low until the nuclear envelope is re-formed in late telophase, when they increase again to interphase levels. This decrease in overall free calcium in mitosis suggests that the mitotic cell has mechanisms for the general sequestration, and perhaps local release, of calcium ions.


Asunto(s)
Calcio/análisis , Riñón/citología , Mitosis , Aminoquinolinas/farmacología , Animales , Línea Celular , Dipodomys , Epitelio/análisis , Microscopía Fluorescente , Mitosis/efectos de los fármacos
20.
J Protozool ; 39(4): 463-70, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1403981

RESUMEN

The disruption of vimentin and actin filaments of host BSC-1 fibroblast cells by Trypanosoma cruzi was investigated using a mouse monoclonal anti-vimentin antibody and rhodamine phalloidin, respectively. Indirect immunofluorescence microscopy demonstrated that infection of BSC-1 cells by T. cruzi caused disruption of both cytoskeletal components. The disruption was greater as infection progressed. Mechanisms other than mechanical ones may play a role in the disruption since disrupted cytoskeletal elements were well removed from the parasites. In the determination of intracellular calcium concentrations using Fura-2 AM, infected and uninfected cells both showed an initial increase in intracellular calcium levels. At later times of infection (3 to 5 days), intracellular calcium levels of infected cells were significantly lower than those of control cells. There was no specific localization of intracellular calcium in the infected host cells as determined by image analysis.


Asunto(s)
Calcio/metabolismo , Citoesqueleto/patología , Trypanosoma cruzi/patogenicidad , Actinas/metabolismo , Animales , Anticuerpos Monoclonales , Línea Celular , Chlorocebus aethiops , Fibroblastos/parasitología , Ratones , Microscopía Fluorescente , Faloidina , Fotomicrografía , Rodaminas , Vimentina/metabolismo
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