Luminescence Digital Imaging Microscopy

Jovin, Thomas M.; Arndt‐Jovin, Donna J.

doi:10.1146/annurev.bb.18.060189.001415

Cited by 208 publications

(87 citation statements)

References 45 publications

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“…The main changes from the beginning t o the end of the S phase concern the size of spots (small at the beginning, large at the end), the spot number (maximum in the middle), location close to the nuclear, and nucleolar boundaries (absent at the beginning and in the middle, perinuclear and perinucleolar thereafter, and perinuclear at the end). These results are in agreement with previous findings (20,25,45,47).…”

Section: Biological Relevance Of the Replicationsupporting

confidence: 94%

“…Two main characteristics of the late replication patterns are the perinuclear and perinucleolar labelling and the large spot size. This suggests that the late replication involves heterochromatin, as it is generally accepted (20,351. The fact that BrdUrd spots appear condensed is not surprising since replication could occur on condensed material, such as mitotic figures (16), and in vitro even across the nucleosome without histone displacement (4).…”

Section: Biological Relevance Of the Replicationmentioning

confidence: 84%

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Eukaryotic DNA replication is a topographically ordered process

Humbert

Usson

1992

Cytometry

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This paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd-labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature. o 1992 Wiley-Liss, Inc. Key terms: Anti-BrdUrd monoclonal antibodies, fluorescence, topographical parameters, image analysisThere is a body of evidence to support the hypothesis that eukaryotic DNA replication occurs as a nonrandom process in a reproducible temporal order (see 35,40 for review); the best known example is the late replication of the inactive X chromosome of mammalian female (5,211.The mechanisms responsible for this fixed replication sequence are not known, but factors such as chromatin condensation, DNA functional activity, or intranuclear arrangement may be related to the S phase ordering on the basis of many observations. It is generally conceded that euchromatin replicates early and heterochromatin replicates late. Chromosomal band analysis reveals that early DNA synthesis corresponds to the Giemsa R-band, while late DNA synthesis corresponds to the G-band (14,21). Holmquist suggested that late replication, which gradually appears during developmental process of embryonic cells along with facultative heterochromatinization, may actively determine gene repression (13).The DNA synthesized in late S might be less important for cell survival than that synthesized in early S. This hypothesis is supported by the data reviewed by Laird et al. (26) indicating that fragile sites in chromosomes of humans, Drosophila, and Microtus represent regions where DNA replicates late. Furthermore, a number of potentially active genes has been shown to replicate early (12). The relationship between gene activity, intra-nuclear arrangement, and replication timing remains unclear, but alteration of genes, as in translocation, is accompanied by changes in their replication time sequence (21). Iqbal et al. explored the question of "the relationship between the temporal replication of a proto-oncogene and its genomic organization" (18). The existence of a relationship between gene location, involving the nuclear matrix arrangement, and DNA replication has been the subject of a number of biochemical studies (8,19,33,41). However, no definitive answer is available yet, due to the variety of nuclear matrix isolation procedures used (8,381.To learn more of the DNA replication process as a function of gene ac...

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Section: Biological Relevance Of the Replicationsupporting

confidence: 94%

Section: Biological Relevance Of the Replicationmentioning

confidence: 84%

Eukaryotic DNA replication is a topographically ordered process

Humbert

Usson

1992

Cytometry

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“…The fluorescence lifetime and quantum yield of the donor decreases resulting in a decrease of the donor intensity (donor quenching), whereas the intensity of the acceptor is enhanced (sensitized emission). E can also be determined by selectively bleaching the donor (7,8) or the acceptor. In acceptor photobleaching, the enhancement of the donor intensity is measured after photodestruction of the acceptor dye (9,10).…”

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confidence: 99%

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

et al. 2013

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Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. V C 2013 International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; FRET; laser scanning cytometry; high throughput; protein-protein interactions FLUORESCENCE or F€ orster resonance energy transfer (FRET) is a nonradiative process, in which energy is transferred from an excited fluorescent donor dye to a neighboring acceptor within its 2-10 nm vicinity by dipole-dipole coupling (1). In order for FRET to take place, the emission spectrum of the donor should overlap with the absorption spectrum of the acceptor, and the two dyes should have a proper relative orientation (2,3). The process is characterized by the FRET efficiency, E, which is the probability that a donor in the excited state transfers its energy to a nearby acceptor. E depends on the 6th power of the separation distance, R, between the donor and the acceptor:where R 0 is the F€ orster radius, at which E 5 0.5. Because of its sensitive distance dependence, FRET can be used as a molecular ruler to assess intra-or inter-molecular distances (4), molecular conformation or association state. FRET has several effects ...

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“…Microscopic FRET increases the resolution of conventional fluorescence microscopy to the molecular level, allowing quantitative examination of proteinprotein interactions or molecular clustering based on the nonradiative energy transfer between donor and acceptor molecules (17). The rate of energy transfer decays with the sixth power of the distance, so FRET assesses molecular proximity on a length scale of 1-10 nm (11,(18)(19)(20). To examine GalNAc-T͞Gal-T2 interactions using FRET, a set of plasmids encoding chimeric proteins bearing the EGFP derivatives with spectral overlap (ECFP and EYFP) fused at the C terminus of the N-terminal domains of GalNAc-T, Gal-T2, ␤1,4 Gal-T, and ManII was constructed.…”

Section: Egfp Derivatives Fused At the C Terminus Of N-terminal Domaimentioning

confidence: 99%

From the Cover: Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Giraudo¹

2001

Proceedings of the National Academy of Sciences

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Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc: lactosylceramide͞GM3͞GD3 ␤-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2͞GM2͞GD2 ␤-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the fulllength enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.

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Luminescence Digital Imaging Microscopy

Abstract: The field of optical microscopy is undergoing a technological revolution. The developments underlying this phenomenon are the advent of electro optical detectors and digitizing electronics, which afford great sensitivity,

Cited by 208 publications

References 45 publications

Eukaryotic DNA replication is a topographically ordered process

Eukaryotic DNA replication is a topographically ordered process

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

From the Cover: Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

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