(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome [1]

['Samuel Plante', 'Institut De Biologie Intégrative Et Des Systèmes', 'Ibis', 'Université Laval', 'Québec', 'Regroupement Québécois De Recherche Sur La Fonction', 'L Ingénierie Et Les Applications Des Protéines', 'Proteo', 'Département De Biologie', 'Département De Biochimie']

Date: 2023-04

The biophysical properties of the cytoplasm are major determinants of key cellular processes and adaptation. Many yeasts produce dormant spores that can withstand extreme conditions. We show that spores of Saccharomyces cerevisiae exhibit extraordinary biophysical properties, including a highly viscous and acidic cytosol. These conditions alter the solubility of more than 100 proteins such as metabolic enzymes that become more soluble as spores transit to active cell proliferation upon nutrient repletion. A key regulator of this transition is the heat shock protein, Hsp42, which shows transient solubilization and phosphorylation, and is essential for the transformation of the cytoplasm during germination. Germinating spores therefore return to growth through the dissolution of protein assemblies, orchestrated in part by Hsp42 activity. The modulation of spores’ molecular properties are likely key adaptive features of their exceptional survival capacities.

Funding: This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to CRL (RGPIN-2020-04844) and LJF (RGPIN-2022-03022), a Canadian Institutes of Health Research (CIHR) Foundation grant (387697) to CRL, and platform funding from Genome Canada (264PRO) to LJF. CRL holds the Canada Research Chair in Cellular Synthetic and Systems Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Given that budding yeast spores are inherently resistant to stresses that are known to modify many biophysical features of the cytoplasm, we hypothesize that the spore cytoplasm has biophysical properties similar to cells exposed to acute stress and that these will dynamically change during early spore germination. Here, we therefore examine the biophysical properties of dormant budding yeast ascospores and the changes that occur during dormancy breaking to unveil the molecular processes that support this critical life history cell transition. Our results reveal that dormant spore cytosol is highly rigid and acidic and that breaking of dormancy is supported by the neutralization and increased fluidity of the cytoplasm. We used mass spectrometry to examine proteome-wide protein solubility through germination. The measurements of 895 proteins revealed dynamic changes in protein solubility. We uncovered, for instance, the solubilization of several metabolic enzymes during this transition. Our results demonstrate that spores have exceptional biophysical properties and that many of the changes taking place in spores mimic what occurs in yeast experiencing stress relief. One major similarity is the implication in spore germination of a small heat shock protein, Hsp42, which is essential for normal spore activation and whose activity is regulated by its phosphorylation.

Recent studies have shown the potential complex influence of the physical properties and organization of the cytosol in dormancy and stress resistance. Cytosolic viscosity, pH, crowding, and protein phase separation have been linked to global cell adaptation across taxonomic groups. For instance, in tardigrades, desiccation resistance is mediated by intrinsically disordered proteins that form vitrified structures [ 20 ]. Seeds of the plant Arabidopsis thaliana sense hydration as the key trigger for their germination through phase separation of the protein Floe1 [ 21 ]. This process is a highly responsive environmental sensor since the biophysical state of Floe1 changes within minutes when water content is altered [ 22 ]. Examples of the responsiveness of the biophysics of the cell cytoplasm also come from yeast, such as S. cerevisiae, responding to acute stresses. Early heat shock response in yeast includes cytoplasm acidification [ 23 ], viscosity adaptation [ 24 ], and protein phase separation [ 25 – 27 ]. Heat shock response induces the expression of many heat shock proteins composed mainly of molecular chaperones [ 28 ] that act as a dispersal system for the heat-induced phase-separated protein condensates and promotes the rapid recovery from stress [ 29 ].

Fungal life cycles include the production of spores. Although being formed through largely different mechanisms, conidia (asexual spores), and ascospores and basidiospores (sexual spores) have in common to be stable dormant cell types [ 8 , 9 ]. These cells all show a variety of resistance to extreme conditions such as heat, desiccation [ 10 , 11 ], and many harsh complex environments such as insect guts [ 12 ] or immune system assaults [ 13 , 14 ]. Because of the increased resistance of spores to extreme conditions, sporulation is thought to be an adaptive strategy to survive changing environmental conditions [ 15 ]. In ascospores, which are produced by our model the budding yeast Saccharomyces cerevisiae, stress resistance is largely attributed to the thick cell wall of specific composition [ 16 ], and to the accumulation of protective compounds like trehalose or mannitol [ 9 , 17 ]. These protective features develop during sporulation, which is typically induced in vegetative yeast by nutrient stress. When spores are exposed to favorable conditions, germination coordinates the breaking of dormancy and the loss of these protective features, with cell-cycle progression and vegetative growth resumption. This transition involves multiple changes in cellular state [ 18 ], including the reactivation of multiple metabolic reactions. Although the precise nutrient stimuli that drive germination is dependent on ecological contexts, a carbon source such as glucose is typically an essential signal [ 19 ].

Organisms across the tree of life rely on dormancy to withstand hostile conditions. This cellular state implies an arrest of the cell cycle and of cell metabolism and changes in cell properties that favor survival under unfavorable conditions [ 1 , 2 ]. For instance, nematodes, rotifers, and tardigrades produce dormant life stages that allow them to resist acute stresses such as freezing, desiccation, and heat stresses [ 3 – 5 ]. In flowering plants, the embryo develops as a dormant seed, which contributes to its survival over a long period of time by resisting drought and mechanical stress until it reaches favorable conditions to resume growth [ 6 ]. Cell dormancy is also an adaptive strategy in cancer cells, whereby metastatic cells become dormant after dissemination and resume proliferation after treatment has succeeded at eliminating the primary tumors [ 7 ]. As one of the most widespread adaptive survival strategies to extreme conditions, understanding the molecular and cellular bases of cell dormancy is a major goal in cell biology.

Results and discussion

Spores have a dense cytoplasm and display a different ultra architecture that changes during germination Spore germination is the transition of dormant spores toward metabolically active and dividing vegetative yeast cells. Spores and vegetative yeast differ in terms of morphology and this morphology gradually changes through time. Spores are spherical and highly light refractile (Fig 1A) and darken and start growing quickly after the initiation of germination which can be induced by transferring cells to rich media. The hallmark of the completion of germination and the return to vegetative growth is bud emergence, which occurs at about 6 h after induction of germination (Fig 1A). Spores’ transition from high to low refractility correlates with the decrease in optical density at 595 nm (A 595 ) of the pure spore culture [30], with the minimal values reached about 3 h after induction (Fig 1B). Then, subsequent growth leads to an increase of optical density. One of the adaptive features of spores is their resistance to heat. This feature is lost during germination. The quantification of heat shock resistance during germination highlights a drastic cellular transition as early as 1 h after induction, at which point resistance to thermal stress decreases and reaches levels that compare to that of vegetative yeast (Fig 1C). Taken together, these measurements define the time frame and the major time points can be used to examine the underlying cellular and molecular changes. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. The cytoplasm of dormant spores displays high rigidity and density and is an acidic environment. (A) Phase-contrast microscopic images of ascospore (same cell followed through time) at the indicated time after exposure to rich media, which activates germination. The scale bar represents 5 μm. (B) Optical density (A 595 ) and (C) heat resistance of pure spore cultures through time after exposure to rich media. Heat resistance is the ratio of growth after a heat shock at 55°C for 10 min to growth without heat treatment. Experiments were performed in triplicate and values for individual replicates are shown. (D) Representative TEM images of spores at the indicated time after exposure to rich medium and of a vegetatively growing yeast cell (vegetative). Cells were prepared and stained at the same time. Imaging was performed on a single layer. The scale bar represents 1 μm. See S1A Fig for more examples. (E) Mean black level of spore cytosol at the indicated time after exposure to rich medium and of vegetative yeast. Spores at 0, 1-, and 2-h time points are merged into a single category since they are indistinguishable from one another. (F) Top, microscopic images of μNS-GFP particles in vegetative yeasts and spores. Underneath is the corresponding 1-min trajectories of the particles. Color indicates time scale. The scale bar represents 10 μm. Bottom, ensemble MSD of μNS-GFP particles in spores at the indicated time after exposure to rich medium and in vegetative cells. (G) Intracellular pH measured at the indicated time point after germination induction and in exponentially growing cells. Measurements in at least 2,000 cells are shown at each time point. The data underlying this figure can be found in S1 Datasheet. MSD, mean squared displacement; TEM, transmission electron microscopy. https://doi.org/10.1371/journal.pbio.3002042.g001 We obtained a more detailed view of the inner cell during germination using transmission electron microscopy (TEM, Fig 1D). Dormant spores are distinguishable by their small size and the thick spore wall, which is not seen in vegetative cells. The spore cytoplasm appears darker in TEM in comparison to a vegetative yeast, which suggests a denser cytosol (Figs 1D and 1E and S1A). Spores have a different cytoplasmic organization. This is shown by the membranous structures that look highly packed in dormant spores compared to in vegetative yeast (Fig 1D). Cells at 1 and 2 h into germination are still indistinguishable from dormant cells. Visible cytoplasmic organization changes after about 3 h of germination, which correlates with a drop in heat resistance comparable to levels seen in vegetatively growing yeast (Fig 1D). At this time point, there is a rupture of the outer spore wall and the cell starts increasing in size where the spore wall is open (Fig 1D). This size increase is accompanied with a decrease in cytoplasm density (Fig 1E). These observations suggest that transformation of the physical nature of the cytosol environment coincides with germination and return to vegetative growth. To test the cytosol physical properties during germination, we quantified its dynamic through the examination of macromolecular motion. We expressed the reovirus non-structural protein μNS tagged with GFP as a foreign tracer particle, which has shown to be a suited probe for subcellular environment in yeast [31]. μNS self-assembles in 1 or 2 discrete particles in the yeast cytoplasm that we could detect in both spores and vegetative yeast (Figs 1F and S2). The tracking of single particles revealed their lower mobility in dormant spores compared to vegetative cells (Fig 1F). These measurements suggest that dormant spore cytoplasm is highly rigid or dense. Particle motion remained low during the first 2 h of germination, then increased gradually from hatching (3-h time point) until the end of germination (Fig 1F). At bud emergence, the motion of μNS particles is close to that measured in vegetative cells. Other experiments using μNS as tracer particles reported mean squared displacement (MSD) at 1 s lag-time in the order of 10−1 μm2 [31], which is in the range of our results. While energy deprivation in yeast reduces MSD of tracer particles by less than 1 order of magnitude [31,32], we estimated that particles have motion 2 orders of magnitude lower than in vegetative yeast. These results highlight that dormant spores have an exceptionally dense and rigid cytosol. These observations are in agreement with previous work on the fungi Talaromyces macrosporus, where spores were found to be characterized by high viscosity [33]. Stress response in yeast includes cytoplasm acidification that culminates with its rigidification [31], including during heat shock [23]. We therefore hypothesized that the high viscosity of the spore cytoplasm and heat shock resistance would be accompanied by a low pH that would increase during germination. To test this hypothesis, we constitutively expressed the pH biosensor superfold-pHluorin [34], in both vegetative yeast and spores after calibrating pHluorin fluorescence in vivo. We estimated pH to be around 5.9 in dormant spores, confirming previous reports [35,36]. Over the course of germination, the cytosol is gradually neutralized (Fig 1F). As soon as 1 h after exposure to rich media, median intracellular pH rises to 6.2 and it slowly increases until the end of the process (pH i = 7.3). At this point, intracellular pH gets close to that measured in vegetatively growing cells (pH i = 7.4). Previous works showed that acidification and alkylation of yeast cytosol causes reduction and increase of motility of μNS particles respectively, and that this effect happens quickly, in the scale of a few minutes [31]. However, our results show that during germination, the kinetics of change in particle mobility is delayed compared to change of intracellular pH. Although germination involves physicochemical changes related to that seen in vegetative yeast recovering from stress, they are modulated in a germination-specific manner. Our results reveal the contribution of possibly many other factors to changes in viscosity. Altogether, these experiments show that extreme physicochemical conditions prevail in dormant spores compared to vegetative yeast, namely a highly rigid and acidic cytoplasm. These conditions are modulated during germination and return to vegetative growth. These intracellular properties that change during germination can play a critical role in cellular function and organization as they are some of the determinants of protein phase separation [24]. Protein phase separation was shown to underlie heat shock response in yeast and many other forms of stress responses during cell dormancy [37]. We therefore hypothesized that proteins could have a different solubility in spores and that the modification of physicochemical properties during germination affects their solubility in a time-dependent fashion.

Protein solubility changes during germination We adopted a physical separation technique similar to the one used in the context of heat shock to measure biochemical changes in protein solubility proteome-wide in budding yeast [27]. Protein sedimentation was driven by ultracentrifugation, and protein partitioning between the pellet and supernatant fractions was quantified by liquid-chromatography-coupled tandem mass spectrometry (LC-MS/MS, Fig 2A). We measured the proportion of each protein that partitioned in the pellet fraction using P index as a proxy for desolubilization in 3 biological replicates at 4 time points during germination and in vegetative cells. In total, we detected 24,559 unique peptides corresponding to 2,614 proteins across the experiments. We restricted our analysis to the 895 proteins with at least 2 unique peptides that were detected at every time point to measure P index (S3 Table). Values for these 895 proteins range from 0 to 1. Zero indicates that the protein was detected only in the supernatant, and 1 indicates that the protein was detected only in the pellet. Proteins with low P index are referred to as soluble proteins, while proteins with high P index as less soluble ones. Replicated measurements were strongly correlated (S2A Fig). Moreover, the solubility profile of endogenous proteins in the fractionated cell extracts revealed by western blot is similar to their P index trajectories (S3 Fig). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Proteome-wide change in protein solubility during germination. (A) Solubility measurement by LC-MS/MS estimates the proportion of each protein in the pellet (P index ) at each major time point sampled during germination. The experiment was performed in triplicate for all time points. (B) Right, P index values in the course of germination show, from top to bottom, proteins consistently found in the pellet, that transiently solubilize, that gradually solubilize, that gradually accumulate in the pellet, and that are consistently found in the supernatant. Left, individual P index trajectories for each cluster determined by hierarchical clustering. The dotted line is the median trajectory for each cluster. (C) GO term analysis focused on cellular component terms. Terms enriched for each cluster (Mostly in pellet, Changing P index , and Mostly supernatant) are shown as bubble plots. Colors refer to the cluster, position on the x-axis indicates the portion of the proteins in a cluster assigned to a GO term, and size of the bubble is scaled to the -log (p-values). See S2 Fig for additional details. The data underlying this figure can be found in S2 Datasheet. GO, gene ontology; LC-MS/MS, liquid-chromatography-coupled tandem mass spectrometry. https://doi.org/10.1371/journal.pbio.3002042.g002 Five typical P index trajectories were identified using hierarchical clustering (Fig 2B). The 2 largest clusters contain proteins that remain mostly soluble (mostly supernatant, n = 359) and mostly insoluble (mostly in pellet, n = 425). Together, they account for 87% of all proteins we considered in our analysis. This means that most of the proteins do not exhibit detectable changes in physicochemical partition during germination using our approach. However, 111 proteins showed changing P index trajectories divided in 3 clusters. First, 15 proteins showed a transient solubilization early in germination. These proteins predominantly partitioned in the pellet in dormant spores, while 1 h after exposure to rich media, their P index dropped drastically before rising again at the 3-h time point and remained insoluble until the end of germination. Another group of 17 proteins gradually desolubilize in the course of germination. They start with high solubility (low P index ) in dormant spores and gradually reach higher P index value at later time point in the process. Finally, 79 proteins with varying single trajectories gradually gained solubility during germination. Gene ontology (GO) analysis, using all proteins considered for our analysis as a reference set, revealed that clusters are enriched for different cell component terms (Fig 2C). While the mostly supernatant cluster appears to contain essentially cytosolic proteins, the proteins in the mostly pellet and changing P index clusters are assigned to various and more specific cellular components. For instance, the later clusters are both enriched for proteins in non-membrane-bounded organelles, ribonucleoprotein complexes, and cytoskeleton. There is a specific enrichment for nucleolar and ribosomal proteins in the mostly pellet cluster and a specific enrichment for stress granule proteins in the changing P index cluster. These results highlight the level of separation performed by our technique, which seems to separate non-membrane–bounded organelles and macromolecular complexes from the other constituents of the cytosol. We examined the properties of proteins that associate with these changes in solubility. Proteins that change solubility are not more nor less abundant than other proteins (S2B Fig). Principal component analysis (PCA) revealed that of all the protein properties considered, propensity for condensate formation (PSAP, [38]) and score for prion-like domains prediction (PLAAC, [39]) are the ones that contribute the most to the separation of proteins in terms of P index (S2C Fig). Because prion-like domains can contribute to protein phase separation and tune the dynamics of biomolecular condensate [40], these results corroborate the enrichments for non-membrane–bounded organelles and macromolecular complexes we reported in the clusters mostly in pellet and changing P index . However, insolubility does not necessarily reflect phase separation as protein solubility is also influenced by many other factors such as misfolding, formation of protein/RNA granules, or other homogeneous or heterogeneous oligomerization. Nevertheless, because propensity for condensate formation positively correlates with P index , high P index estimates at least partially reflect phase separation of proteins and macromolecular assemblies. In addition, the analysis of known physical interaction among the detected proteins revealed that changes we reported in P index do not correlate with large interaction networks. Out of the 111 changing P index proteins, 6 pairs of physically interacting proteins were found (S4 Fig). This suggests that the changes in protein organization we observed likely reflect bulk changes in cytoplasm properties rather than remodeling of specific interactions.

Many classes of proteins change solubility during germination, including metabolic enzymes To understand the functional significance of change in P index , we searched for GO terms enrichment in 3 clusters that display dynamic change. First, in the transient solubilization cluster, we found significant enrichment for lipid and phospholipid-binding proteins (Fig 3A). This group includes, for instance, the translation initiation factor Cdc33 and the GTP-binding protein Ras2 (S5C Fig). In the gradual desolubilization cluster, which includes for instance the transcription elongation factor Spt5 and the vacuolar carboxypeptidase Cps1, we found enrichment for phosphatidylinositol-3-phosphate (PI3P)-binding proteins (Fig 3A). We suspect that the modulation in solubility we detect in these clusters is a reflection of the gain of activity of many cellular pathways. For instance, gradual insolubility of the SNARE chaperone Sec18 may reflect increasing assembly of membrane-fusion complexes as vesicle transport is resumed to sustain cell growth. Finally, the gradual solubilization cluster is enriched for proteins involved in metabolic process: precisely, amino acid and carbohydrate metabolism, and protein phosphatase activity (Fig 3A), including for instance the ceramide-activated protein phosphatase Sit4 which has roles in the G1/S transition in cell cycle [41]. This may reflect the reentry of the dormant spores in the cell cycle. Among this group, we also identified the stress-related proteins Ola1 and Yef3, which are known to aggregate in response to heat stress and disaggregate during recovery [27]. The behavior of these proteins suggest that dormancy in spores shares features with stress response and that germination would correspond to stress relief. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Solubility changes reflect metabolism activation and mimic stress relief during germination. (A) Enrichment for GO terms in each dynamically changing solubility cluster. Red, transiently solubilizing cluster; green, gradual desolubilization cluster; purple, gradual solubilization cluster. The position on the x-axis indicates the portion of the proteins in a cluster assigned to a GO term, and size of the bubble is scaled to the -log (p-values) from a hypergeometric test. (B) Individual P index trajectories for representative proteins through germination. Proteins are clustered by function; red, stress response proteins; blue, nitrogen metabolism proteins; gray, lipid and carbon metabolism proteins. Error bars represent standard deviation of 3 replicates. (C) Representative fluorescence microscopic images of spores expressing the indicated proteins tagged with GFP during germination. Top to bottom, Acetyl-CoA carboxylase Acc1 (lipid biosynthesis), CTP synthase Ura7 (pyrimidines synthesis), and Glucokinase Glk1 (glycolysis). The Glk1 foci formation and the dissolution of Acc1 and Ura7 foci in course of germination support that dormancy in spores is analogous to a stress state and germination alleviates this state. Dotted lines indicate cell contour determined by brightfield images. Scale bars represent 5 μm. (D) Measure of cellular heterogeneity (coefficient of variation) of the fluorescent proteins in spore at the indicated time after exposure to rich medium or in vegetative cells. Between 20 and 41 cells were analyzed at each time points. (E) Schematics highlighting effects on protein solubility of nutrient starvation and repletion during sporulation and germination, respectively. Pink and blue assemblies represent assemblies of enzymes needed for growth and metabolism during dormancy, which disassemble (pink and blue circles) during germination. The data underlying this figure can be found in S3 Datasheet. GO, gene ontology. https://doi.org/10.1371/journal.pbio.3002042.g003 Within the group of proteins with increasing solubility, we identified enzymes involved in carbohydrate, lipid, and nitrogen metabolisms (Fig 3B). Since nutrient starvation is the key signal that triggers sporulation, the behaviour of these metabolic enzymes that solubilize in the course of germination caught our interest. We investigated 2 of them: the CTP synthase Ura7 and the acetyl-CoA carboxylase Acc1, which are enzymes known to form high molecular weight assemblies in response to nutrient starvation [42,43]. To validate the solubility changes revealed by P index trajectories, we generated cells expressing either Ura7 or Acc1 fused—at their genomic locus—with GFP. Both Ura7 and Acc1 formed cytoplasmic foci in dormant spores (Fig 3C and 3D). Upon germination, Ura7-GFP and Acc1-GFP fluorescence signals changed until they became mostly diffuse in dividing cells. This behavior confirms the dissolution of the protein assemblies observed in the P index trajectories. In addition, we noted the opposite behavior of the glucokinase Glk1. Glk1’s P index trajectory suggests it gains insolubility during germination (Fig 3C and 3D). Correspondingly, we found Glk1-GFP to be diffuse in dormant spores, then appears as dense assemblies in cells as soon as 1 h after exposure to rich media and until the end of germination. Glk1 was found to polymerize and form filaments during the transition from low to high sugar conditions [44]. Its behavior in germinating cells again suggests that spores remain dormant in a starved form and that breaking of dormancy implies changes of enzyme biophysics in response to nutrient repletion. The reverse order of events between spore germination and heat stress and nutrient stress responses for some key proteins suggests a model in which dormancy in spores is analogous to a stress response state and germination corresponds to the relief of the stress state allowing return to metabolic activity and vegetative growth (Fig 3E). Spores therefore most likely borrow stress resistance strategies we observe in vegetative yeast.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002042

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.