A physicochemical roadmap of yeast replicative aging

Abstract

Author ContributionsConceptualization: S.N.M., A.J.B and L.M.V.; Investigation, Formal analysis: S.N.M. and D.J.T.; Methodology and Resources: M.M.C., I.L.R., and A.S.; Writing: S.N.M., A.J.B, and L.M.V.; Supervision: M.K., C.P.L., A.J.B, L.M.V. AbstractCellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding, are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show the macromolecular crowding changes less in longer-lived cells in contrast to shorter-lived cells. While the average pH and crowding levels change only modestly with aging, we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively-aged yeast cells.

Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

“…We verified our strains and crowding sensor by initially quantifying molecular crowding and area changes in cells experiencing osmotic stress (Figure 1c and d). While vacuoles are clearly visible in fluorescence micrographs (see Figure 1 and Supplementary Figure 2) and expected to be an excluded volume [8], their presence in the whole-cell analysis does not significantly change the values or distribution measured and thus continuing analysis on a whole-cell level is justified (Figure 1b). At 0 M NaCl cells are in a low stress condition, but under exposure to 1 M NaCl crowding increases, evidenced by NFRET increasing by 9.6% ( Figure 1c) while measurable cell area is reduced by around 21% (Figure 1d) and thus volume by around half for a spherical cell, consistent with previously found values [14], due to water being mechanically forced from the cell by osmotic pressure [39].…”

“…Yeast cultures were grown in rich (YPD: 2% glucose, 2% peptone, 1% yeast extract) or synthetic (2% glucose, 1x yeast nitrogen base; including appropriate amino acids and bases) media. The mCerulean3/mCitrine FRET crGE probe [7,8] under control of the TEF1 promoter was sub-cloned into yeast integration vector pRS303, linearized with NsiI, then stably integrated into at the HIS3 locus of wild-type BY4742 yeast by homologous recombination. Successful integrations were isolated on synthetic drop-out media (2% glucose, 1x yeast nitrogen base; 1x amino acid and base drop-out compositions lacking Histidine (SD -His, Formedium Ltd, UK).…”

“…The intensities were corrected for autofluorescence by subtraction of the mean autofluorescence values found by imaging BY4742 wild type cells in experimental conditions. As in previous work [8,28], the normalized ratiometric FRET was defined as = √ ⁄…”

The more the merrier: effects of macromolecular crowding on the structure and dynamics of biological membranes

“…The protein probe has the crucial advantage that it is entirely genetically encoded, allowing expression in many different hosts, genetic fusions with localization tags or other proteins, and manipulation of its structure through genetic engineering. The majority of applications involve this proteinbased class of probes, which function in bacteria [208][209][210][211], yeast [210,212] and mammalian cell lines [208,213,214], as well as their compartments [210,215]. It allowed crowding determination under stress conditions, such as osmotic stress [211,213] and ageing [212].…”

“…Substituting the cyan/yellow (mCerulean3 or mTur-quoise2/mCitrine or mVenus) FPs for green/red (Clover/Ruby, GFP/mCherry or EGFP/mScarlet-I) provides probes that can be used under less autofluorescence, with less pH sensitivity and allows for more straightforward normalization (N FRET ) due to a lower bleed through [212][213][214]. The probes were applied to study adaptation of mammalian cells to osmotic stress and under the very challenging conditions of yeast replicative ageing [212,213]. The latter experiments tracked the ageing of an individual cell over~2 days, with drifting cell physiology and pH.…”