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How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells.

Ramadass R, Bereiter-Hahn J - Biophys. J. (2008)

Bottom Line: Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents.Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence.Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Cluster of Excellence Macromolecular Complexes, Institute of Cell Biology and Neuroscience, Biocenter, Johann Wolfgang Goethe University, Germany. ramadass@bio.uni-frankfurt.de

ABSTRACT
Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

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Location and orientation of DASPMI in the mitochondrial inner membrane. DASPMI preferentially locates its positively charged methyl pyridinium ring in the aqueous interface facing the outer mitochondrial membrane, and its hydrophobic moiety along the hydrocarbon chain. As for orientation, it aligns itself along the local electric field for a maximum electrochromic response. Under high-membrane-potential conditions, the delocalization of π-electrons leads to a less polar structure, imposing steric constraints on the single bonds neighboring the olefinic double bond.
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fig4: Location and orientation of DASPMI in the mitochondrial inner membrane. DASPMI preferentially locates its positively charged methyl pyridinium ring in the aqueous interface facing the outer mitochondrial membrane, and its hydrophobic moiety along the hydrocarbon chain. As for orientation, it aligns itself along the local electric field for a maximum electrochromic response. Under high-membrane-potential conditions, the delocalization of π-electrons leads to a less polar structure, imposing steric constraints on the single bonds neighboring the olefinic double bond.

Mentions: The direct influence of mitochondrial membrane potential on only the shortest lifetime (τ1) of DASPMI, and its remarkable shortening in energized mitochondria indicate the partial insertion of DASPMI in the outer leaflet of the inner mitochondrial membrane (Fig. 4). This proposition is confirmed by the occurrence of disparate changes between lifetimes pertaining to the LE state (τ1) and charge-shift states of DASPMI-stained energized mitochondria. Furthermore, the >30-fold difference between τ1 and τ3 (in energized mitochondria), excitation maximum of 470 nm in XTH cells (between DMSO and chloroform) and emission maximum of 565 nm (same as chloroform) support our deduction. A blue shift in both excitation and emission spectra is expected for molecules that are only partially inserted into the lipid environment (19–23). The partial insertion of DASPMI in the outer leaflet of the inner mitochondrial membrane ensures a favorable polar environment for the positively charged methylpyridinium moiety and hydrophobic aniline moiety along the nonpolar hydrocarbon chains of lipids. Under this scenario, photoabsorption that is largely confined to the positively charged methylpyridinium moiety can be expected to have an absorption maximum of 470 nm, and emissions predominantly confined to the aniline moiety to have an emission maximum of 565 nm as observed in living cells. This differential solvation has been previously been described for other styryl dyes and has also been observed by us in phospholipids vesicles wherein the absorption maximum was 430 nm and emission maximum of 545 nm (data not shown). In comparison to phospholipid vesicles, the red shift of absorption maxima in mitochondria could be due to partial delocalization of π-electrons under the influence of a local electric field. This assumption of only a partial delocalization of π-electrons, rather than a complete one, is confirmed by the decrease of steady-state anisotropy at longer emission wavelengths (Fig. 5). A complete shift of positive charge to the dimethyl amino group would shift the absorption maxima and anisotropy to higher values, as has been shown to be the case in chloroform (10).


How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells.

Ramadass R, Bereiter-Hahn J - Biophys. J. (2008)

Location and orientation of DASPMI in the mitochondrial inner membrane. DASPMI preferentially locates its positively charged methyl pyridinium ring in the aqueous interface facing the outer mitochondrial membrane, and its hydrophobic moiety along the hydrocarbon chain. As for orientation, it aligns itself along the local electric field for a maximum electrochromic response. Under high-membrane-potential conditions, the delocalization of π-electrons leads to a less polar structure, imposing steric constraints on the single bonds neighboring the olefinic double bond.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553107&req=5

fig4: Location and orientation of DASPMI in the mitochondrial inner membrane. DASPMI preferentially locates its positively charged methyl pyridinium ring in the aqueous interface facing the outer mitochondrial membrane, and its hydrophobic moiety along the hydrocarbon chain. As for orientation, it aligns itself along the local electric field for a maximum electrochromic response. Under high-membrane-potential conditions, the delocalization of π-electrons leads to a less polar structure, imposing steric constraints on the single bonds neighboring the olefinic double bond.
Mentions: The direct influence of mitochondrial membrane potential on only the shortest lifetime (τ1) of DASPMI, and its remarkable shortening in energized mitochondria indicate the partial insertion of DASPMI in the outer leaflet of the inner mitochondrial membrane (Fig. 4). This proposition is confirmed by the occurrence of disparate changes between lifetimes pertaining to the LE state (τ1) and charge-shift states of DASPMI-stained energized mitochondria. Furthermore, the >30-fold difference between τ1 and τ3 (in energized mitochondria), excitation maximum of 470 nm in XTH cells (between DMSO and chloroform) and emission maximum of 565 nm (same as chloroform) support our deduction. A blue shift in both excitation and emission spectra is expected for molecules that are only partially inserted into the lipid environment (19–23). The partial insertion of DASPMI in the outer leaflet of the inner mitochondrial membrane ensures a favorable polar environment for the positively charged methylpyridinium moiety and hydrophobic aniline moiety along the nonpolar hydrocarbon chains of lipids. Under this scenario, photoabsorption that is largely confined to the positively charged methylpyridinium moiety can be expected to have an absorption maximum of 470 nm, and emissions predominantly confined to the aniline moiety to have an emission maximum of 565 nm as observed in living cells. This differential solvation has been previously been described for other styryl dyes and has also been observed by us in phospholipids vesicles wherein the absorption maximum was 430 nm and emission maximum of 545 nm (data not shown). In comparison to phospholipid vesicles, the red shift of absorption maxima in mitochondria could be due to partial delocalization of π-electrons under the influence of a local electric field. This assumption of only a partial delocalization of π-electrons, rather than a complete one, is confirmed by the decrease of steady-state anisotropy at longer emission wavelengths (Fig. 5). A complete shift of positive charge to the dimethyl amino group would shift the absorption maxima and anisotropy to higher values, as has been shown to be the case in chloroform (10).

Bottom Line: Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents.Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence.Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Cluster of Excellence Macromolecular Complexes, Institute of Cell Biology and Neuroscience, Biocenter, Johann Wolfgang Goethe University, Germany. ramadass@bio.uni-frankfurt.de

ABSTRACT
Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

Show MeSH
Related in: MedlinePlus