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V E Centonze

Publications and source records attributed to V E Centonze.

At least 19 recordsLinked to original sources
PubMed2003

Quantitative imaging of protein-protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera.

Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. Recent developments in multiphoton FLIM methods are reviewed and a novel multiphoton FLIM system using a streak camera is described. An example of a typical application of the system is provided in which the fluorescence resonance energy transfer between a donor-acceptor pair of fluorescent proteins within a cellular specimen is measured.

Animals↗
PubMed2001

Short-term culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium.

OBJECTIVE: To evaluate the initial adhesion of endometrium to the peritoneum. DESIGN: Descriptive study using light and confocal laser-scanning microscopy, immunohistochemistry, and transmission electron microscopy. SETTING: University-based laboratory. PATIENT(S): Women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Explants of peritoneum (n = 20), prepared from four patients, were cultured for 1 hour with mechanically dispersed proliferative or secretory endometrium. Peritoneum was cultured with endometrium from the same patient. Specimens were fixed and serially sectioned for hematoxylin and eosin stain, immunohistochemistry using an anti-cytokeratin monoclonal antibody, and transmission electron microscopy. RESULT(S): In 17 of 20 explants, endometrium was adherent to intact mesothelium. There was no evidence of transmesothelial invasion at any sites of attachment. Although in most cases endometrium was adherent to mesothelium via endometrial stroma, there were many sites of endometrial epithelium-mesothelium attachment. Confocal laser scanning microscopy demonstrated an intact monolayer of cytokeratin-positive cells below the sites of endometrial implantation. Transmission electron microscopy demonstrated intact, viable, mesothelial cells below sites of attachment. CONCLUSION(S): This study demonstrates that endometrium rapidly adheres to intact peritoneal mesothelium. In addition, this study demonstrates that endometrial epithelial cells, as well as stroma, can attach to mesothelium. Further studies are needed that characterize the mechanism of endometrial-mesothelial cell adhesion.

Cell Adhesion↗
PubMed2000

Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line.

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 microM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 microM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 microM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 microM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 microM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

Animals↗
PubMed2000

Reovirus nonstructural protein muNS binds to core particles but does not inhibit their transcription and capping activities.

Previous studies provided evidence that nonstructural protein muNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455-466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of muNS. Given the possible role of muNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant muNS can bind to cores in vitro. The muNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of muNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of muNS were bound. Thus, the muNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the muNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to muNS. The muNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5' ends of the transcripts. In vitro competition assays showed that mixing muNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins mu1 and sigma3. These findings are consistent with the presence of muNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, muNS may block or delay outer-capsid assembly and allow continued transcription by these particles.

Animals↗
PubMed1999

Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells.

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.

3T3 Cells↗
PubMed1999

Reovirus virion-like particles obtained by recoating infectious subvirion particles with baculovirus-expressed sigma3 protein: an approach for analyzing sigma3 functions during virus entry.

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein sigma3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed sigma3 protein to infectious subvirion particles (ISVPs) that lack sigma3. A level of sigma3 binding approaching 100% of that in native virions was routinely achieved. The sigma3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study sigma3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52 degrees C. Since rcISVPs contain virion-like levels of sigma3 but contain outer-capsid protein mu1/mu1C mostly cleaved at the delta-phi junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that sigma3, and not the delta-phi cleavage of mu1/mu1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric sigma3 proteins to localize the primary determinants of a strain-dependent difference in sigma3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.

Animals↗
PubMed1998

Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.

Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.

Animals↗
PubMed1995

Imaging of cytoskeletal elements by low-temperature high-resolution scanning electron microscopy.

Actin filaments and microtubules, both in situ and in vitro, were imaged using high-resolution scanning electron microscopy (HRSEM) at low temperature. For visualization of cytoskeletal elements in situ, fibroblasts were first extracted and fixed; for cytoskeletal elements in vitro, purified proteins were polymerized and fixed. Both types of specimen were then subjected to plunge freezing, controlled freeze-drying, cryo-sputter coating with a thin chromium layer, cryo-transferring and cryo-observation in an FESEM. The three-dimensional architecture of the cytoskeleton was well preserved, permitting examination of the structural relationships among cytoskeletal elements. Actin filaments and microtubules were identified by their characteristic helical features. Two periodicities of actin filaments, the short pitch of the left-handed helix measured at 5.5 nm and the 37-nm-long pitch helix, were revealed. Individual protofilaments were seen in microtubules as well as the characteristic 4-nm repeat of tubulin subunits along the protofilament. Clathrin cages were also observed. This technique provides a powerful approach for direct imaging of macromolecular structures with high contrast and high signal-to-noise ratio at a resolution of 2-3 nm.

3T3 Cells↗
PubMed1994

Calcium dynamics during starfish oocyte maturation and fertilization.

Intracellular free calcium levels in starfish oocytes have been monitored during meiotic maturation and fertilization using calcium-sensitive fluorescent dyes combined with confocal laser scanning microscopy or fura ratioing techniques. In time-lapse analyses of prophase-arrested and maturing oocytes, calcium transients were elicited by inositol 1,4,5-trisphosphate (IP3), ryanodine, or caffeine, indicating that both the IP3-sensitive and IP3-insensitive receptors of the oocyte's calcium release channels could be stimulated to mobilize calcium ions. Fertilization also triggered a global calcium wave that appeared to travel faster around the cortex than through the center of the oocyte, and maturing oocytes developed normally after their fertilization-induced calcium waves had been imaged. Prophase-arrested specimens, on the other hand, did not undergo germinal vesicle breakdown or cleavage after displaying a fertilization-induced calcium transient throughout their cytoplasm and nucleus, confirming previous observations that calcium spikes are not sufficient to induce development in immature oocytes. In addition, although the calcium spikes triggered by sperm or caffeine reached similar normalized peak heights, fertilization-induced calcium waves in maturing oocytes tended to be more prolonged than the fertilization waves observed in prophase-arrested oocytes or the caffeine-triggered spikes elicited at any stage of maturation. Collectively, such findings suggest that the total amount of releasable calcium does not vary appreciably during maturation, but the patterns of the calcium transients can differ depending on the stage of maturation and/or the type of calcium-releasing agent. Possible artifacts affecting these findings are assessed, and the results are discussed relative to the functioning of calcium release pathways during starfish oocyte maturation and fertilization.

Animals↗
PubMed1994

Inhibition of microtubule nucleation at the neuronal centrosome compromises axon growth.

We tested the dependence of axon growth on microtubule (MT) nucleation from the neuronal centrosome. Nocodazole diminished MTs in freshly plated neurons by > 99%. Within 5 min of drug removal, MTs reassembled at the centrosome. This response was inhibited in cells microinjected with gamma-tubulin antibody. Within 2 hr of drug removal, uninjected neurons grew > 500 microns of axon. In roughly half of the antibody-injected cells, axon growth was abolished and MT levels were reduced by approximately 87% compared with uninjected cells. In the other antibody-injected cells, axon growth was compromised but not abolished, and MT levels were reduced by approximately 38%. Thus inhibition of MT nucleation at the centrosome hindered MT reassembly, and depending on the severity of this response, axon growth was either compromised or abolished.

Animals↗
PubMed1993

Microtubule nucleation and release from the neuronal centrosome.

We have proposed that microtubules (MTs) destined for axons and dendrites are nucleated at the centrosome within the cell body of the neuron, and are then released for translocation into these neurites (Baas, P. W., and H. C. Joshi. 1992. J. Cell Biol. 119:171-178). In the present study, we have tested the capacity of the neuronal centrosome to act as a generator of MTs for relocation into other regions of the neuron. In cultured sympathetic neurons undergoing active axonal outgrowth, MTs are present throughout the cell body including the region around the centrosome, but very few (< 10) are directly attached to the centrosome. These results indicate either that the neuronal centrosome is relatively inactive with regard to MT nucleation, or that most of the MTs nucleated at the centrosome are rapidly released. Treatment for 6 h with 10 micrograms/ml nocodazole results in the depolymerization of greater than 97% of the MT polymer in the cell body. Within 5 min after removal of the drug, hundreds of MTs have assembled in the region of the centrosome, and most of these MTs are clearly attached to the centrosome. A portion of the MTs are not attached to the centrosome, but are aligned side-by-side with the attached MTs, suggesting that the unattached MTs were released from the centrosome after nucleation. In addition, unattached MTs are present in the cell body at decreasing levels with increasing distance from the centrosome. By 30 min, the MT array of the cell body is indistinguishable from that of controls. The number of MTs attached to the centrosome is once again diminished to fewer than 10, suggesting that the hundreds of MTs nucleated from the centrosome after 5 min were subsequently released and translocated away from the centrosome. These results indicate that the neuronal centrosome is a highly potent MT-nucleating structure, and provide strong indirect evidence that MTs nucleated from the centrosome are released for translocation into other regions of the neuron.

Animals↗
PubMed1992

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.

Aniline Compounds↗
PubMed1991

Pole-to-chromosome movements induced at metaphase: sites of microtubule disassembly.

Metaphase spindles can be induced to shrink by treating cells with microtubule-depolymerizing agents. During treatment, the paired sister chromatids remain at the metaphase plate and the poles move toward them. The question we asked is whether this pole-to-chromosome movement was accompanied by a loss of subunits from the kinetochore ends of the microtubules, the polar ends, or both ends. LLC-PK cells were injected at late prometaphase with Xrhodamine tubulin and at metaphase the fluorescent spindles were marked by photobleaching a bar between one pole and the chromosomes. Nocodazole at low concentrations was briefly applied to the cells to induce the shortening of the spindle and movement of the poles inward toward the chromosomes. In the induced shortening, the distance between the photobleached bar and the chromosomes decreased substantially while the distance between the bar and the pole showed a smaller change. Upon reversal from nocodazole, new polymer was added to the spindle as determined by recovery of fluorescence, and the cells progressed through mitosis and cytokinesis. We conclude that the movement of the poles to the chromosomes induced by nocodazole treatment during metaphase is similar to the chromosome-to-pole movement occurring during anaphase in that under both conditions the primary site for kinetochore microtubule disassembly is at the kinetochore.

Anaphase↗
PubMed1991

Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis.

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 x 10(3) Mr, 350 x 10(3) Mr, a 230-240 X 10(3) Mr doublet, 210 x 10(3) Mr and 120 x 10(3) Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined. The phosphorylated 210 x 10(3) Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 x 10(3) Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies.

Animals↗
PubMed1990

Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope.

At the onset of mitosis a class of proteins appears that possess a phosphorylated epitope recognized by the monoclonal antibody MPM-2. Immunofluorescence staining shows that a subset of these proteins is associated with the centrosomes of the mitotic apparatus. The appearance of these proteins coincides with the increased microtubule nucleating capacity of the centrosomes. We have tested whether growth of microtubules from mitotic centrosomes in a lysed cell model is dependent on the availability of the phosphorylated epitope by blocking the epitope with a specific antibody or by modifying it by removal of the phosphate. Centrosomes incubated with purified tubulin nucleate microtubule asters. However, preincubating the centrosomes with MPM-2 blocks all microtubule nucleation. Pretreating mitotic centrosomes with alkaline phosphatase also inhibits nucleation. These data suggest that the phosphorylated epitope recognized by MPM-2 is important for microtubule nucleation.

Animals↗
PubMed1986

Structure and composition of the cytoskeleton of nucleated erythrocytes: III. Organization of the cytoskeleton of Bufo marinus erythrocytes as revealed by freeze-dried platinum-carbon replicas and immunofluorescence microscopy.

Platinum-carbon (Pt-C) replicas of freeze-dried erythrocyte cytoskeletons of the toad, Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule-containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross-bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface-associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt-C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the cytoskeleton.

Animals↗
PubMed1986

A protein factor from Bufo marinus erythrocytes cross-bridges microtubules in vitro.

A microtubule cross-bridging factor was isolated from erythrocytes of the toad, Bufo marinus. Erythrocytes were lysed and their cytoskeletons disassembled by sonication and high salt extraction. The solubilized proteins were recovered and fractionated using Sephadex G-200 column chromatography. The protein fractions from the column were analysed by SDS-PAGE and pooled into three groups: high molecular weight (HMW) proteins that eluted from the column in the void volume and had a protein composition that included HMW polypeptides; intermediate MW proteins that were shown by SDS-PAGE to contain polypeptides smaller than 120,000 D; and low MW (LMW) proteins that contained polypeptides smaller than 70,000 D. Each group was further fractionated by phosphocellulose (PC) chromatography. The flow-through was recovered, and bound proteins were then eluted by a step gradient of salt (0.2, 0.4, 0.6 and 0.8 M KCl). To assay for microtubule cross-bridging activity, column fractions were incubated with taxol-stabilized microtubules, formed from PC-purified brain tubulin (PC microtubules). Negatively stained samples were examined in the electron microscope for the reconstitution of microtubule bundles with interconnecting cross-bridges. The HMW protein fraction from the G-200 column contained the cross-bridging factor. When these proteins were further fractionated by PC chromatography only the fraction eluted by 0.2 M KCl induced the formation of microtubule bundles with cross-bridges. No other protein fraction isolated by the described method revealed cross-bridges between microtubules in vitro.

Animals↗