The Proton Sensitivity of Fluorescent Proteins: Towards Intracellular pH Indicators

2011 ◽  
pp. 59-97
Author(s):  
Ranieri Bizzarri
Keyword(s):  
Intracellular Ph ◽  
Fluorescent Proteins ◽  
Ph Indicators

Annexins sense changes in intracellular pH during hypoxia

2007 ◽  
Vol 409 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Katia Monastyrskaya ◽  
Fabian Tschumi ◽  
Eduard B. Babiychuk ◽  
Deborah Stroka ◽  
Annette Draeger
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Fluorescent Proteins ◽  
Annexin A2 ◽  
Transport Systems ◽  
Physiological Parameter ◽  
Membrane Association ◽  
Ph Dependent

The pHi (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pHi. Sensors of pHi include ion transport systems which control intracellular Ca2+ gradients and link changes in pHi to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca2+-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2–S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pHi on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2–S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2–S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2–S100A10 might have consequences for their functions as membrane organizers and channel modulators.

A novel method for absolute calibration of intracellular pH indicators

1989 ◽  
Vol 413 (5) ◽  
pp. 553-558 ◽  
Author(s):  
D. A. Eisner ◽  
N. A. Kenning ◽  
S. C. O'Neill ◽  
G. Pocock ◽  
C. D. Richards ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Absolute Calibration ◽  
Ph Indicators ◽  
Novel Method

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

1019: Visualization of the Neurovascular Bundles and Major Pelvic Ganglion with Fluorescent Proteins after Penile Injection

2006 ◽  
Vol 175 (4S) ◽  
pp. 328-328 ◽  
Author(s):  
Hugo H. Davila ◽  
Maggie Mamcarz ◽  
Irving Nadelhaft ◽  
Raoul Salup ◽  
Jorge Lockhart ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Pelvic Ganglion ◽  
Major Pelvic Ganglion ◽  
Neurovascular Bundles
Sign in / Sign up

Export Citation Format

Share Document