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NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells.
Full text linkα-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating aS both in vitro and in cells. These data suggest that NADH binds to aggregated aS. NMR experiments in vitro substantiate such binding, which occurs during aggregation. NADH fluorescence is thus useful to detect aS aggregation and by extension the associated oxidative stress.-Plotegher, N., Stringari, C., Jahid, S., Veronesi, M., Girotto, S., Gratton, E., and Bubacco, L. NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells
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Hydration and protein dynamics: frequency domain fluorescence spectroscopy on proteins in reverse micelles
Full text linkLysozyme, human serum albumin (HSA), and liver alcohol dehydrogenase (LADH) have been studied in reverse micellesby frequency domain fluorescence spectroscopy. The emission of the tryptophanyl residues of the proteins was monitored.Fluorescence and anisotropy decays were measured from 2 to 350 MHz for each protein in reverse micelles and in aqueoussolutions. The wide range of modulation frequencies available allowed direct monitoring of the internal motions of tryptophanresidues, occurring in the subnanosecond time range, together with the whole protein rotational dynamics in the micelles.The results indicate that the rotational correlation times for the internal motions and the overall protein rotation in reversemicelles decrease with increasing water concentration. Lysozymes showed peculiar rotational dynamics which reflect denaturationoccurring as the protein increases its water content in the reverse micelle. This effect was not observed for the other proteins.Dynamic measurements appear useful in understanding structural changes arising from the interactions between proteinsand micellar systems
Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells.
Full text linkBACKGROUND:
Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease.
METHODS:
We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging.
RESULTS:
Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional.
CONCLUSIONS:
We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells.
GENERAL SIGNIFICANCE:
The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinson's disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models
Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin
No full textErythrocyte membrane microheterogeneity studied by DPH lifetime decay using multifrequency fluorometry.
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Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions.
Full text linkThe effect of cholesterol on microheterogeneity of liposomes obtained from saturated and unsaturated phospholipids was studied by measuring the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Data obtained by frequency domain fluorometry have been analyzed either by discrete exponential or continuous lifetime distribution approaches. In egg phosphatidylcholine liposomes, the addition of cholesterol increases the lifetime value or the centre of the lifetime distribution. At high cholesterol concentration, good fits are obtained using a monomodal distribution analysis or single exponential component. At low cholesterol concentration an additional short component of low fractional intensity must be included to obtain a good fit. In dipalmitoylphosphatidylcholine, the addition of cholesterol decreases the long lifetime component centre value both in the gel and in the liquid-crystalline state. The DPH lifetime value is sensitive to the dielectric constant of the probe microenvironment, and cholesterol has been shown to modify water penetration in the bilayer. Using this information our data indicate that cholesterol affects the polarity of the microenvironment in liposomes of unsaturated phosphatidylcholine and saturated phosphatidylcholine in different ways. Although the major conclusions of this paper are obtained using changes of the distribution centre upon cholesterol addition, there are also preliminary indications that the lifetime distribution width decreases as cholesterol is added. We have interpreted this observation as being due to the homogenizing effect of cholesterol
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Erythrocyte membrane heterogeneity studied using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distribution.
Full text linkThe fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene has been used to characterize the structural organization of erythrocyte membranes. At 37 degrees C a large fraction of the decay (0.96) is associated with a lifetime value of 11.31 ns, while a minor fraction has a short lifetime of 2.63 ns. The distribution analysis approach has shown that the 11 ns component can be described using a Lorentzian distribution function having a full width at half maximum of 0.27 ns. The width of this component is associated with the membrane structural organization since liposomes from erythrocyte total lipid extract exhibit a narrower width. Moreover the distribution width is sensitive to different treatments of erythrocyte membrane
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