Cytosolic Ca2+ measurements by ratiometric fluorescence microscopy in melanoma cells

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca2+-activated K+ (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14–22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca2+ sparks but increased global Ca2+ levels and the magnitude of Ca2+ oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca2+ signals generated by intracellular Ca2+ stores and cytosolic Ca2+ levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

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Involvement of Mitochondrial Mechanisms in the Cytostatic Effect of Desethylamiodarone in B16F10 Melanoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21197346 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7346

Author(s):

Fadi H. J. Ramadan ◽

Aliz Szabo ◽

Dominika Kovacs ◽

Aniko Takatsy ◽

Rita Bognar ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Fluorescent Dyes ◽

Mitochondrial Permeability Transition ◽

Imaging System ◽

Coupling Efficiency ◽

Melanoma Cells ◽

B16f10 Melanoma ◽

Atp Production ◽

B16f10 Melanoma Cells

Previously, we showed that desethylamiodarone (DEA), a major metabolite of the widely used antiarrhythmic drug amiodarone, has direct mitochondrial effects. We hypothesized that these effects account for its observed cytotoxic properties and ability to limit in vivo metastasis. Accordingly, we examined DEA’s rapid (3–12 h) cytotoxicity and its early (3–6 h) effects on various mitochondrial processes in B16F10 melanoma cells. DEA did not affect cellular oxygen radical formation, as determined using two fluorescent dyes. However, it did decrease the mitochondrial transmembrane potential, as assessed by JC-1 dye and fluorescence microscopy. It also induced mitochondrial fragmentation, as visualized by confocal fluorescence microscopy. DEA decreased maximal respiration, ATP production, coupling efficiency, glycolysis, and non-mitochondrial oxygen consumption measured by a Seahorse cellular energy metabolism analyzer. In addition, it induced a cyclosporine A–independent mitochondrial permeability transition, as determined by Co2+-mediated calcein fluorescence quenching measured using a high-content imaging system. DEA also caused outer mitochondrial membrane permeabilization, as assessed by the immunoblot analysis of cytochrome C, apoptosis inducing factor, Akt, phospho-Akt, Bad, and phospho-Bad. All of these data supported our initial hypothesis.

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Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Journal of Visualized Experiments ◽

10.3791/53402 ◽

2015 ◽

Cited By ~ 3

Author(s):

Paula Nunes ◽

Daniele Guido ◽

Nicolas Demaurex

Keyword(s):

Fluorescence Microscopy ◽

Ratiometric Fluorescence

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The uniformity of phagosome maturation in macrophages

The Journal of Cell Biology ◽

10.1083/jcb.200307080 ◽

2004 ◽

Vol 164 (2) ◽

pp. 185-194 ◽

Cited By ~ 112

Author(s):

Rebecca M. Henry ◽

Adam D. Hoppe ◽

Nikhil Joshi ◽

Joel A. Swanson

Keyword(s):

Endoplasmic Reticulum ◽

Quantitative Analysis ◽

Fluorescence Microscopy ◽

Fluorescent Protein ◽

Time Lapse ◽

Raw 264.7 ◽

Phagosome Maturation ◽

Ratiometric Fluorescence ◽

Raw 264.7 Macrophages ◽

Single Path

Many studies of endocytosis and phagocytosis presume that organelles containing a single kind of internalized particle exhibit invariant patterns of protein and phospholipid association as they mature inside cells. To test this presumption, fluorescent protein chimeras were expressed in RAW 264.7 macrophages, and time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics of individual phagosomes containing IgG-opsonized erythrocytes. Quantitative analysis revealed consistent patterns of association for YFP chimeras of β-actin, Rab5a, Rab7, and LAMP-1, and no association of YFP chimeras marking endoplasmic reticulum or Golgi. YFP-2xFYVE, recognizing phosphatidylinositol 3-phosphate (PI(3)P), showed two patterns of phagosome labeling. Some phagosomes increased labeling quickly after phagosome closure and then lost the label within 20 min, whereas others labeled more slowly and retained the label for several hours. The two patterns of PI(3)P on otherwise identical phagosomes indicated that organelle maturation does not necessarily follow a single path and that some features of phagosome maturation are integrated over the entire organelle.

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Illuminate the hidden: in vivo mapping of microscale pH in the mycosphere using a novel whole-cell biosensor

ISME Communications ◽

10.1038/s43705-021-00075-3 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Bi-Jing Xiong ◽

Christian Dusny ◽

Lin Wang ◽

Jens Appel ◽

Kristin Lindstaedt ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Spatial Variability ◽

Ratiometric Fluorescence ◽

Ph Gradients ◽

Fungal Hyphae ◽

Reaction Zones ◽

Whole Cell Biosensor ◽

Over Time ◽

Cell Biosensor

AbstractThe pH of an environment is both a driver and the result of diversity and functioning of microbial habitats such as the area affected by fungal hyphae (mycosphere). Here we used a novel pH-sensitive bioreporter, Synechocystis sp. PCC6803_peripHlu, and ratiometric fluorescence microscopy, to spatially and temporally resolve the mycosphere pH at the micrometre scale. Hyphae of the basidiomycete Coprionopsis cinerea were allowed to overgrow immobilised and homogeneously embedded pH bioreporters in an agarose microcosm. Signals of >700 individual cells in an area of 0.4 × 0.8 mm were observed over time and used to create highly resolved (3 × 3 µm) pH maps using geostatistical approaches. C. cinerea changed the pH of the agarose from 6.9 to ca. 5.0 after 48 h with hyphal tips modifying pH in their vicinity up to 1.8 mm. pH mapping revealed distinct microscale spatial variability and temporally stable gradients between pH 4.4 and 5.8 over distances of ≈20 µm. This is the first in vivo mapping of a mycosphere pH landscape at the microscale. It underpins the previously hypothesised establishment of pH gradients serving to create spatially distinct mycosphere reaction zones.

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The position of lysosomes within the cell determines their luminal pH

The Journal of Cell Biology ◽

10.1083/jcb.201507112 ◽

2016 ◽

Vol 212 (6) ◽

pp. 677-692 ◽

Cited By ~ 220

Author(s):

Danielle E. Johnson ◽

Philip Ostrowski ◽

Valentin Jaumouillé ◽

Sergio Grinstein

Keyword(s):

Fluorescence Microscopy ◽

Atpase Activity ◽

Adenosine Triphosphatase ◽

Biosynthetic Pathway ◽

Buffering Capacity ◽

Ratiometric Fluorescence ◽

Lysosomal Ph ◽

Luminal Ph ◽

Functional Versatility ◽

Lysosomal Protein

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H+–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.

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Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102675 ◽

1988 ◽

Vol 46 ◽

pp. 118-119

Author(s):

K. Jacobson ◽

A. Ishihara ◽

B. Holifield ◽

F. Zhang

Keyword(s):

Fluorescence Microscopy ◽

Cell Biology ◽

Extended Period ◽

Dynamic Structure ◽

Living Cells ◽

Membrane Dynamics ◽

Molecular Cell Biology ◽

Image Averaging ◽

Single Living Cells ◽

Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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Scanning x-ray fluorescence microscopy and principal component analysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100133394 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1752-1753

Author(s):

Brian Cross

Keyword(s):

Principal Component Analysis ◽

Fluorescence Microscopy ◽

Spatial Resolution ◽

High Speed ◽

Image Acquisition ◽

Principal Component ◽

Fluorescence Microscope ◽

Acquisition Rate ◽

X Ray ◽

Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

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