scholarly journals Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

2003 ◽  
Vol 31 (5) ◽  
pp. 1020-1027 ◽  
Author(s):  
D.S. Lidke ◽  
P. Nagy ◽  
B.G. Barisas ◽  
R. Heintzmann ◽  
J.N. Post ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Laser Scanning ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

scholarly journals Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

2010 ◽  
Vol 298 (5) ◽  
pp. L626-L636 ◽  
Author(s):  
Heike Schlenz ◽  
Wolfgang Kummer ◽  
Gitte Jositsch ◽  
Jürgen Wess ◽  
Gabriela Krasteva
Keyword(s):  
Smooth Muscle ◽  
Signaling Pathways ◽  
Laser Scanning ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Caveolin 1

Cholinergic bronchoconstriction is mediated by M2 and M3 muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R−/− and M3R−/− mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

2018 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V. Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

AbstractProper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

scholarly journals Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor

Sensors ◽  
2020 ◽  
Vol 20 (6) ◽  
pp. 1712 ◽  
Author(s):  
De-Ming Yang ◽  
Robeth Viktoria Manurung ◽  
Yu-Syuan Lin ◽  
Tai-Yu Chiu ◽  
Wei-Qun Lai ◽  
...  
Keyword(s):  
Heavy Metal ◽  
High Resolution ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Lead Level ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Blood Lead ◽  
Confocal Laser

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called “safe value” of the BLL seems to unreliably represent the secure threshold for children. In general, lead’s underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood–brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

scholarly journals Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth

2018 ◽  
Vol 2 (2) ◽  
pp. 114-120 ◽  
Author(s):  
Martin Vanek ◽  
Filip Mravec ◽  
Martin Szotkowski ◽  
Dana Byrtusova ◽  
Andrea Haronikova ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Light Exposure ◽  
Laser Scanning Microscopy ◽  
Model Systems ◽  
Confocal Laser ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Red Yeast

AbstractRed yeast Cystofilobasidium capitatum autofluorescence was studied by means of confocal laser scanning microscopy (CLSM) to reveal distribution of carotenoids inside the cells. Yeasts were cultivated in 2L fermentor on glucose medium at permanent light exposure and aeration. Samples were collected at different times for CLSM, gravimetric determination of biomass and HPLC determination of pigments. To compare FLIM (Fluorescence Lifetime Imaging Microscopy) images and coupled data (obtained by CLSM) with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of beta-carotene. Liposomes lecithin:ergosterol:beta-carotene were investigated too. Two different intracellular forms of carotenoids were observed during most of cultivations, with third form appeared at the beginning of stationary phase. Observed behavior is probably due to formation of some kind of carotenoid protective system in membranes of different compartments of yeast cell, especially cytoplasmic membrane.

scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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