Large organellar changes occur during mild heat shock in yeast

Abstract

When the temperature is increased, the heat-shock response is activated to protect the cellular environment. The transcriptomics and proteomics of this process are intensively studied, while information about how the cell responds structurally to heat stress is mostly lacking. Here, Saccharomyces cerevisiae were subjected to a mild continuous heat shock (38°C) and intermittently cryo-immobilised for electron microscopy. Through measuring changes in all distinguishable organelle numbers, sizes and morphologies in over 2100 electron micrographs, a major restructuring of the internal architecture of the cell during the progressive heat shock was revealed. The cell grew larger but most organelles within it expanded even more, shrinking the volume of the cytoplasm. Organelles responded to heat shock at different times, both in terms of size and number, and adaptations of the morphology of some organelles (such as the vacuole) were observed. Multivesicular bodies grew by almost 70%, indicating a previously unknown involvement in the heat-shock response. A previously undescribed electron-translucent structure accumulated close to the plasma membrane. This all-encompassing approach provides a detailed chronological progression of organelle adaptation throughout the cellular heat-stress response.

Keywords: Budding yeast; Electron microscopy; Heat shock; Organelles; Ultrastructure.

Fig. 1.

Revealing ultrastructural changes during a mild heat shock using a transmission electron microscopy screening method. (A) Cartoon of the experimental procedure: cells were grown at 30°C and heat shocked at 38°C for 0, 5, 15, 30, 45 and 90 min. Cells were then high-pressure frozen, freeze-substituted and sectioned at 70 nm for electron microscopy. Each heat-shock experiment was carried out in triplicate and at least 100 images were collected of each sample, resulting in a total of 2143 images analysed. Size and morphology of all clearly visible organelles were modeled (coloured circles in rightmost image) and quantified to reveal known and unknown effects of exposing yeast cells to heat shock. (B) Example images show that large morphological changes and cellular reorganisation occurred during heat shock. Furthermore, a new phenotype that we call electron translucent clusters (ETCs) appear during heat shock. n, nucleus; v, vacuole; m, mitochondrion (label next to organelle); mvb, multivesicular bodies (label next to organelle); ld, lipid droplet. Scale bar: 500 nm.

Fig. 2.

Cells and vacuoles increase in size. (A) Cells with vacuoles (arrow) before and after 30 min of heat shock. Scale bar: 500 nm. (B) Cell area in electron micrographs of thin sections. On average, the cell size increases by 19% over the course of 90 min. The pink, yellow and blue shapes represent the three separate replicates. The width represents the number of measurements within a certain range of values, one point represents one measurement. Solid horizontal line and inference bands are median±i.q.r. Grey bars represent the median of all data points within one timepoint. (C) Vacuoles in electron micrographs of thin sections. On average, the vacuole size increases by 48% over the course of 90 min. (D) Cells expressing Vph1-GFP heat shocked at indicated time points. Scale bar: 5 µm. (E) Quantification of vacuole number per cell from cells in D, n>200 per replicate and time point. Data are mean±s.e.m. (F) Quantification of vacuole size from cells in D, n>200 per replicate and time point. Data are mean±s.e.m. All timepoints were statistically analysed individually. *P<0.05, **P≤0.01.

Fig. 3.

Heat shock affects vacuolar morphology and acidity. (A) Gallery of different vacuolar electron densities. Scale bar: 500 nm. (B) Quantification of vacuolar electron density during heat shock. More than 100 vacuoles per time point were analysed from three biological replicates. (C) Electron microscopy (EM) images of dividing wild-type cells grown at 30°C. Arrows point to vacuoles. Scale bars: 1 µm. (D) Quantification of electron density of vacuoles in mothers (Ms) and their daughters (Ds) during cell division, n=53 dividing cells. (E) EM images of wild type, prb1Δ and vma2Δ grown at 30°C, and quantification of vacuolar electron density, n=181 (wild type), 150 (prb1Δ) and 274 (vma2Δ) vacuoles from a minimum of 100 cells. Greyscale for bottom right panel as in B. Arrows point to vacuoles. Scale bar: 500 nm. (F) EM and fluorescence microscopy (FM) of indicated strains. Heat shock (HS) is 45 min at 38°C. The fluorescent pH-sensitive probe BCECF accumulates in yeast vacuoles and shows vacuolar pH. The more fluorescence, the more basic the vacuolar pH. Scale bars: 1 µm for EM; 5 µm for FM. (G) Quantification of vacuolar BCECF fluorescence mean intensity. More than 30 vacuoles were analysed from three biological replicates. Graph shows mean±s.d. fluorescence intensity relative to wild type. (H) Fluorescence microscopy of indicated strains. The fluorescent pH-sensitive probe quinacrine accumulates in yeast vacuoles and shows vacuolar pH. The more fluorescence, the more acidic the vacuolar pH. Scale bar: 5 µm. (I) Quantification of vacuolar quinacrine fluorescence mean intensity. 100 vacuoles were analysed per strain.

Fig. 4.

Nucleus and mitochondria change in size and morphology. (A) Nucleus morphology before and after 30 min heat shock, as electron-dense content (EDC, arrow) appears next to the nucleolus (arrowhead). Scale bar: 250 nm. (B) Nucleus area in electron micrographs of thin sections. The pink, yellow and blue shapes represent the three separate biological replicates. The width represents the number of measurements within a certain range of values; one point represents one measurement. Solid horizontal line and inference bands are median±i.q.r. Grey bars represent the median of all data points within one timepoint. (C) Proportion of nuclei with EDC throughout heat shock. n values are the same as those in B. (D) Mitochondria morphology before and after 30 min heat shock, as an EDC (arrow) appears. Scale bar: 250 nm. (E) Mitochondria area in electron micrographs of thin sections. On average, the mitochondria increase in size by 52% over the course of 90 min. (F) Proportion of mitochondria with EDCs throughout heat shock. n values are the same as those in E. All timepoints were statistically analysed individually. *P<0.05, **P≤0.01.

Fig. 5.

MVBs and LDs increase in size and a new phenotype appears. (A) MVB morphology before and after 30 min of heat shock. Scale bar: 100 nm. (B) Number of MVBs (mean±s.e.m.). (C) Area of MVBs increased by 73%, on average. The pink, yellow and blue shapes represent the three separate biological replicates. The width represents the number of measurements within a certain range of values; one point represents one measurement. Solid horizontal line and inference bands are median±i.q.r. Grey bars represent the median of all data points within one timepoint. (D) LD morphology before and after 30 min of heat shock. Scale bar: 200 nm. (E) Number of LDs, increased by factor 2.4, averaged over three sets with error bars corresponding to s.e.m. (F) Area of LDs increased by 85%, on average. Graph description as in C. (G) Electron translucent clusters (ETCs) (arrowhead) next to an LD (arrow). Scale bar: 500 nm. (H) Area of the cell section that was covered with electron-translucent clusters over time in heat shock, n=2143 cells. (I) Maximum projections of fluorescent images of cells stained with BODIPY for visualisation of LDs. Scale bars: 2 µm. All timepoints were statistically analysed individually. *P<0.05, **P≤0.01.

Fig. 6.

Membrane contact sites are influenced by heat shock. (A) Contact site between nucleus (n) and vacuole (v). Scale bar: 50 nm. (B) Percentage of sections containing both nucleus and at least one vacuole with a contact site between the two (NV). Data are mean±s.e.m. of three biological replicates, n=1033. (C) Length of contact site in relation to circumference of nucleus and vacuole (median±i.q.r.), n=496 MCS, pooled from three biological replicates. (D) Contact site between vacuole (v) and mitochondrion (m). Scale bar: 50 nm. (E) Percentage of sections containing minimum one vacuole and minimum one mitochondrion with a contact site between the two (VM). Data are mean±s.e.m. of three biological replicates, n=1347 sections. (F) Length of contact site in relation to circumference of vacuole and mitochondria (median±i.q.r.), n=140 MCS, pooled from three biological replicates. All timepoints were statistically analysed individually. **P≤0.01, ***P≤0.001.

Fig. 7.

Relative and comparative changes in organelle sizes. (A) Change in individual organelle area over heat shock with starting size normalised to 1. Data are mean±s.e.m. of the three biological replicates. (B) Change in organelle area in relation to cell area at the respective time point with starting sizes normalised to 1. Data are mean±s.e.m. of the three biological replicates calculated by dividing average organelle size by average cell size at the respective timepoint.

Fig. 8.

Model of structural changes occurring during mild heat shock. Model of structural changes occurring during a 90 min mild heat shock.