Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis

Abstract

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 µm size, stainable by a fluorescent dye, 4 ,6-diamidino-2-phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 ± 0.60, 2.52 ± 0.63, 3.32 ± 0.9 and 11.3 ± 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 ± 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

Identification of autophagy genes participating in zinc-induced necrotic cell death inSaccharomyces cerevisiae

“…Although we have not performed definitive chemical analysis on these inclusions, we have determined that they stained with DAPI ( Fig. S1), like polyphosphate inclusions, 19 and were less abundant (6.3 ± 1.5%) in cells with a deletion of VTC4, a gene needed for polyphosphate accumulation in the vacuole. 20 When BY4741 cultures were diluted into medium without supplemental zinc, the number of cells harboring these inclusions dropped to 32.6 ± 1.9% within 2 h and continued falling thereafter.…”

The early stage of polyphosphate accumulation in &lt;i&gt;saccharomyces cerevisiae&lt;/i&gt;: comparative study by extraction and DAPI staining

“…The results show that at the early stage of PolyP biosynthesis after P i starvation the polymer is immediately localized in different cell compartments, first of all in the cytoplasm. The vacuolar localization of PolyP is more typical of depleted (including P i -depleted) media [10,25], while formation of DAPI-staining PolyPcontaining granules in S. cerevisiae is observed at the stationary growth stage [14]. Commercial PolyP with average chain length of 25, 45, 75 phosphate residues from Sigma (USA) and with average chain length of 188 phosphate residues from Monsanto (USA) were used as markers.…”

“…DAPI-lipid fluorescence is white-yellow, week and fades in a few seconds [12]. In living cells mixed fluorescence spectra may be observed [13,14]. The green fluorescence of DAPI appears if PolyP are co-localyzed with DNA, or if a mixed spectrum of DAPI and DAPI-PolyP appears.…”

“…DAPI staining was used for identification of the vacuolar PolyP [10,13,14] and cell surface PolyP [11] in yeast. However, these studies were carried out with stationary growth stage cells without a comparison of DAPI staining results and chemical extraction of PolyP.…”

Concurrent quantification of multiple nanoparticle bound states

“…16 This viscosity simulates conditions that might be experienced in a cellular vacuole. 17 For a third environment, we nonspecifically bound the particles to 2 m polystyrene beads ͑Polysciences 19814-15͒. Binding was confirmed by centrifugal pelleting of the polystyrene beads, which left a clear supernatant with minimal harmonic response.…”