New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining

Abstract

Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4’,6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multiphoton microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.

Sulfurimonas subgroup GD17 cells accumulate polyphosphate under fluctuating redox conditions in the Baltic Sea: possible implications for their ecology

“…Negatively charged polyphosphate is typically associated with positively charged ions, with valences of both +1 and +2 [44][45][46]. Similar EDX results have already been reported for polyphosphate inclusions [47,48]. In addition, GD1 T DAPIstained cells contained bright yellow inclusions and thus differed in their appearance from otherwise blue DAPIstaining cells.…”

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

“…However, in a different binding mode, which involves adenine-uracil dinucleotide-selective intercalation, it also binds RNA. The DAPI/RNA complex exhibits a longer wavelength fluorescence emission, more than the DAPI/dsDNA complex (Hoff, 1988;Gomes et al, 2013). When bound to DNA, it has absorption maxima at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue).…”

Degradation of phosphate polymer polyP enhances lactic fermentation in mice

“…DAPI–lipid fluorescence is weak and fades within a few seconds, while DAPI–polyP fluorescence appears bright yellow, thus allowing semiquantification of polyP with DAPI. Alternatively, a semiquantification method, based on DAPI staining, has been developed (Gomes et al, ). When oocytes were incubated with DAPI, the ECR zona was stained (Figure e).…”