Age-dependent decline in stress response capacity revealed by proteins dynamics analysis

Abstract

The aging process is regarded as the progressive loss of physiological integrity, leading to impaired biological functions and the increased vulnerability to death. Among various biological functions, stress response capacity enables cells to alter gene expression patterns and survive when facing internal and external stresses. Here, we explored changes in stress response capacity during the replicative aging of Saccharomyces cerevisiae. To this end, we used a high-throughput microfluidic device to deliver intermittent pulses of osmotic stress and tracked the dynamic changes in the production of downstream stress-responsive proteins, in a large number of individual aging cells. Cells showed a gradual decline in stress response capacity of these osmotic-related downstream proteins during the aging process after the first 5 generations. Among the downstream stress-responsive genes and unrelated genes tested, the residual level of response capacity of Trehalose-6-Phosphate Synthase (TPS2) showed the best correlation with the cell remaining lifespan. By monitor dynamics of the upstream transcription factors and mRNA of Tps2, it was suggested that the decline in downstream stress response capacity was caused by the decline of translational rate of these proteins during aging.

Figure 1

Principle of the microfluidic device and yeast strain construction. (a) (left) Schematic diagram of the overall image of the microfluidic chip. (right) Details of the barriers and cell traps. The chip was fabricated by three-layer structures with different heights for the barriers (2 µm), trapping pillars (4 µm) and the main channels (20 µm). Green and blue ellipses indicate yeast strains with different labels in different channels. (b) Images of yeast culture in a chip. Yeast cells were loaded into the chip, and mother cells were trapped and cultured under the pillar. (c) (left) Schematic representation of the HOG1-MAPK pathway and illustrative schematic of the dual reporter construct. The target genes were tagged with GFP, and the MCM gene associated with the cell cycle was labeled with m-Cherry. (right) Images of phase contrast, GFP and m-Cherry channels. The scale bar represents 2 µm. (d) Microscope images of the m-Cherry channel taken at the indicated time points during a part of the cell’s lifespan (up) and the nuclear m-cherry intensity of the cell as a function of time (down). The yellow contours highlight the cell of interest, and the blue triangles indicate the times at which budding occurred. (e) Kaplan–Meier estimator with right censored cells [orange curve] and cells born earlier than 15 h and died eventually during the observation time (blue curve). The intersections of the purple dotted line and the survival curves represents the median lifespan.

Figure 2

Yeasts undergo an age-induced successive decrease in stress response. (a) Osmotic signals considered in this work. T_on represents the time of the addition of the culture solution, which contained 0.4 M KCl, and T_off represents the time of the addition of the normal culture solution, where T_on = 1 h, and T_off = 5 h. (b) Sequence of green and m-cherry fluorescence images taken at the indicated time points. The yellow circles highlight the cell of interest, and magenta arrows represent the time of the stimuli (left). One representative trace of TPS2 in a single cell in the 0.4 M KCl stress experiment. The shaded area represents the time of the stimuli, and orange solid dots indicate the budding events. A schematic defines the stress response I_r of target proteins and doubling time T in a single-cell time trace (right). (c) Sequential protein level in the process of aging under periodic osmotic stimuli (left); The survival rate (blue dotted line) and osmosis-related stress response (gray dotted line and error bars) during the process of aging (right) (cell number N_TPS2 = 108, N_HSP12 = 42, N_PGM2 = 79, N_GPP1 = 67).

Figure 3

I_r/T instead of I_r shows the better linear correlation with residual lifespan. (a) Two examples of GFP-labeled TPS2 before death in response to KCl stimuli. Vertical dashed lines indicate osmotic shock. (b) Relationship between averaged I_r and T of the TPS2-GFP strain and residual lifespan. The solid blue line represents the linear fitting line. In curve fitting, light data points in the dashed box are excluded. (c) Relationship between averaged I_r/T of TPS2 and residual lifespan. The solid turquoise line represents the linear fitting line. In curve fitting, light data points in the dashed box are excluded.

Figure 4

A weakening response in cellular stress response and TPS2 was identified as a lifespan marker. (a) Periods stimuli, every 6 h with 1-h stimuli of 0.4 M KCl for 72 h and (b) 1 h stimuli of 0.4 M KCl from 23 to 24 h. Response capacity I_r/T of TPS2 and residual lifespan showed a significant positive correlation (two sets of experiments contained 97 and 99 cells, respectively). The lilac column represents the moment of adding osmotic stimulation. (c) Characterization of internal noise during the lifespan of yeast cells. (d) The relationship between TPS2 production (solid line, linear fit) and the internal noise (more than 60 cells).

Figure 5

Transcription factors remain equivalent during the aging process. (a) Transcription factors (HOG1 and MSN2) translocate into the nucleus in response to osmotic stimuli. (b) HOG1-GFP images at the indicated times. The orange dotted box represents the stimulation time. (c) In response to a step stimulus of 0.4 M KCl, dynamic curves of single-cell HOG1 and MSN2 nuclear enrichment. Averaged trace of single-cells is shown in dark (left). The schematic defines the amplitude, duration and integral (oblique line) of the nuclear translocation of transcription factors in a single-cell time trace (right). (d) Integral of TFs corresponding to the datasets described in (c). Boxes extend from the 25th to 75th percentiles [interquartile range (IQR)]; horizontal lines represent the median, and whiskers indicate the lowest and highest datum within 1.5 × IQR from the lower and upper quartiles, respectively.

Figure 6

The weakening stress response during aging is mainly driven by decline of protein translation efficiency. (a) Design and characterization of the Tps2-PP7-PCP system. Schematic depicting the multiple stem-loop sequences (PP7) used to tag Tps2, and RNAs are visualized by the recruitment of iRFP-labeled PCPs. (b) Target protein and depiction of transcriptional bursting. Image of live cells coexpressing Tps2-GFP and RNA-iRFP. (c) Appearance of Tps2 transcription site signals at the indicated times. White arrowheads denote transcription sites. (d) Transcriptional response to a sequence of stimulation pulses (six typical cells). Different colors represent different single cells. The inset is stress response patterns of RNA at different times during the aging process of one representative single cell. The shade represents the duration of stimulus application (left). Integral of mRNA calculated from six cell traces showed that the production of mRNA can maintain an almost stable value during the aging process (right). (e) (left) The change in the parameters during the cellular aging process and curve fitting. A linear function was used to fit k₂, and a Hill function was used to fit d₂. (right) Comparison of model fits to the experimental data sets for periodic 0.4 M KCl in budding yeast. The solid red and blue curves represent the theoretical and experimental results, respectively, and the vertical dashed lines indicate the start and end of the stimuli.