A dual reporter system for intracellular and extracellular amino acid sensing in budding yeast

INTRODUCTION

Amino acids are essential building blocks with key roles in cellular growth, metabolism, and signaling, requiring regulatory mechanisms to ensure the maintenance of amino acid homeostasis (Bröer and Bröer, 2017; González and Hall, 2017; Takagi, 2019). Budding yeast (Saccharomyces cerevisiae) cells sense and respond to intracellular amino acid availability through the General Amino Acid Control (GAAC), the Target of Rapamycin Complex 1 (TORC1), and to extracellular amino acids via the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathways (Hinnebusch, 2005; Ljungdahl, 2009; Ljungdahl and Daignan-Fornier, 2012; González and Hall, 2017). These well-characterized systems play a crucial role in maintaining cellular amino acid homeostasis.

The GAAC pathway is a conserved signaling pathway that regulates gene expression and cellular metabolism in response to changes in amino acid availability (González and Hall, 2017). It allows yeast cells to sense amino acid depletion and mount a transcriptional response through Gcn4, leading to elevated expression of enzymes necessary for amino acid biosynthesis and adaptation to nutrient stress conditions (Hinnebusch, 2005). Gcn2 kinase is an upstream sensor of amino acid availability that binds to uncharged tRNAs that accumulate when amino acids are scarce. This triggers its autophosphorylation and activation leading to phosphorylation of a translation initiation factor eIF2α to inhibit global protein synthesis (Dong et al., 2000). When eIF2α is phosphorylated, it promotes the preferential translation of GCN4 mRNA (Hinnebusch, 2005). In the presence of amino acids, Gcn4 is rapidly degraded by the ubiquitin-proteasome system (Kornitzer et al., 1994; Chi et al., 2001; Irniger and Braus, 2003). Gcn4 promotes the expression of over 500 genes, including more than 30 amino acid biosynthetic genes (Jia et al., 2000; Natarajan et al., 2001; Rawal et al., 2018). As a result, the expression of these genes leads to an increased production of amino acids, compensating for the amino acid deprivation and restoring cellular homeostasis.

The second pathway responding to intracellular amino acid availability occurs through TORC1, which is composed of Tor1 or Tor2, Kog1, and Lst8 anchored in the vacuolar membrane (González and Hall, 2017). Unlike the GAAC pathway, TORC1 is activated in the presence of intracellular amino acids, particularly leucine and glutamine. Leucine stimulates TORC1 activity by binding to leucyl-tRNA synthetase Cdc60, which then interacts with Gtr1 and blocks GTP hydrolysis, thus locking the Gtr complex in the TORC1-activating state (Sancak et al., 2008, 2010; Binda et al., 2009; Bonfils et al., 2012). Glutamine activates TORC1 via parallel pathway independent of the Gtr complex by binding to the vacuolar membrane protein Pib2, (Stracka et al., 2014; Tanigawa et al., 2021). There are qualitative differences in TORC1 activation in response to nutrients; the Gtr-based mechanism is required for rapid, transient activation, while a glutamine-associated mechanism induces a delayed, sustained activation (Stracka et al., 2014). In both cases, TORC1 activation leads to the phosphorylation of effector kinases, such as Sch9 and Tap42, which in turn stimulate anabolic processes, including protein synthesis and ribosome biogenesis, while simultaneously suppressing catabolic processes like autophagy.

The GAAC and TORC1 pathways for amino acid biosynthesis are interconnected through regulatory feedback mechanisms. TORC1 acts as a negative regulator of the GAAC pathway by partially restricting the activity of Gcn2 (Cherkasova and Hinnebusch, 2003). When TORC1 is active, it binds to Tap42, preventing Gcn2 dephosphorylation at Ser577, which is crucial for uncharged tRNA sensing. Conversely, rapamycin induced TORC1 inhibition results in Gcn2 dephosphorylation and activation, thereby promoting eIF2α phosphorylation. Intriguingly, an opposite regulatory effect also exists, where Gcn2 down-regulates TORC1 though Kog1 phosphorylation in response to amino acid deprivation (Yuan et al., 2017). However, understanding how TORC1 and GAAC pathways are coordinated in different conditions requires further investigation.

In addition to monitoring their internal amino acid state, yeast cells utilize the SPS pathway to sense and respond to the presence of external amino acids. Ssy1 functions as a sensor of the relative concentrations of intracellular and extracellular amino acids (Wu et al., 2006), interacting with scaffold protein Ptr3 and the endoprotease Ssy5 through its cytoplasmic N-terminal domain. Binding to extracellular amino acids induces a conformational change in Ssy1, which promotes the hyperphosphorylation of Ptr3 and Ssy5. This leads to the proteasomal degradation of the inhibitory prodomain of Ssy5, thereby activating its chymotrypsin-like catalytic domain (Pfirrmann et al., 2010; Omnus et al., 2011). The proteolytic targets of Ssy5 are two homologous zinc-finger transcription factors, Stp1 and Stp2 (Andréasson and Ljungdahl, 2002). The proteolytic cleavage of Stp1 and Stp2 facilitates their nuclear targeting, which in turn triggers the transcriptional activation of amino acid permease genes. This activation enhances the uptake of extracellular amino acids (Andréasson and Ljungdahl, 2002; Andréasson et al., 2006; Boban and Ljungdahl, 2007).

While there is a good understanding of how the amino acid homeostatic pathways function, less is known about their engagement and interconnection under varying conditions and the extent of variation in their function between individual cells. Separate LacZ-based reporters have been used to monitor the activity of the GAAC (Hinnebusch, 1985; Albrecht et al., 1998; Yang et al., 2000; Staschke et al., 2010) and SPS pathways (Regenberg et al., 1999; Abdel-Sater et al., 2004), while luciferase-based reporter was developed to assess TORC1 pathway (Kessi-Pérez et al., 2019). However, these reporters are limited to monitoring one pathway at a time in a population of cells and thus lack the ability to provide dynamic information on amino acid homeostasis at the single-cell level. A FRET-based reporter for TORC1 activity allows for the evaluation of its engagement in single cells, providing high sensitivity and spatial resolution for detecting TORC1 activation within cells (Zhou et al., 2015). However, the limited multiplexing capabilities of FRET-based reporters, which have more stringent spectral requirements, prevent the simultaneous assessment of multiple amino acid sensing pathways.

To overcome these limitations, we describe here the development of a novel system for simultaneously monitoring amino acid biosynthesis and uptake pathway engagement in yeast. This system uses fluorescent reporters controlled by promoters of genes induced by these pathways thereby reporting on the downstream signaling of these pathways. Specifically, we generated constructs where green fluorescent protein (GFP) is placed under a Gcn4 and TORC1 responsive promoter, and red fluorescent protein mKate2 is placed under a Stp1/Stp2-responsive promoter. We show that these constructs report on the engagement of the amino acid biosynthesis and uptake pathways, respectively. These constructs exhibit distinct responses to varying amino acid levels and enable simultaneous monitoring of these amino acid homeostatic responses at the single-cell level under different conditions. Our temporal analysis of yeast colony development indicates that cells undergo specialization into amino acid biosynthesis or uptake, which is important for colony viability.

RESULTS

Constructing fluorescent amino acid biosynthesis and uptake reporters for intracellular and extracellular amino acid sensing

As cells respond to amino acid limitations through distinct transcriptional mechanisms, we aimed to develop fluorescent reporter genes that could concurrently inform on the engagement of pathways regulating intracellular and extracellular amino acid sensing. For monitoring intracellular amino acid sensing pathway engagement, we generated a construct where GFP was placed under the control of a 1 kb region of the ARG8 promoter. ARG8 is a transcriptional target of Gcn4 (Messenguy, 1987; Coey and Clark, 2022), which is translated in response to intracellular amino acid limitation (Figure 1A), promoting the transcription of GFP as an indicator of GAAC pathway engagement (Figure 1B). We expected that this reporter also integrates the other intracellular amino acid sensing pathway, TORC1, as previous transcriptomics analysis identified significant reduction in ARG8 gene expression when cells were treated with TORC1 inhibitor, rapamycin (Gowans et al., 2018). We subsequently refer to this construct as the amino acid biosynthesis reporter.

For simultaneous monitoring of SPS pathway status, which responds to the presence of external amino acids by initiating a transcriptional response to up-regulate amino acid transporter genes for enhanced uptake (Figure 1C), we inserted red fluorescent protein mKate2 downstream of the GNP1 promoter. GNP1 responds to Stp1/Stp2 transcriptional activation, thereby reflecting SPS pathway engagement (Klasson et al., 1999; Forsberg et al., 2001a). It is subsequently referred as the amino acid uptake reporter (Figure 1D).

The promoter regions of the biosynthesis and uptake sensor constructs were designed to include various regulatory elements for a comprehensive physiological representation of the activities within these pathways. For instance, both the ARG8 and GNP1 promoters contain putative stress response elements, along with other regulatory features such as the arginine control element in the ARG8 construct (Supplemental Figure S1, A and B). These constructs were designed to include distinct selection markers to facilitate their dual genomic integration into safe harbor loci X-2 (Pr_ARG8-GFP) and XII-1 (Pr_GNP1-mKate2) (Stovicek et al., 2015), where foreign DNA can be inserted with minimal risk of disrupting essential genes or regulatory element. Construction of this strain enabled simultaneous monitoring of gene expression reflecting amino acid biosynthetic and uptake capacities.

To assess how these reporters respond to different amino acid levels, we cultured the prototrophic reporter cell line in three different media: yeast nitrogen base media (YNB; medium without amino acids), supplemented minimal media (MM; medium with low concentrations of Leu, Met, His, and Ura), and synthetic complete media (SC; medium with high concentrations of a broad range of amino acids: Arg, Asp, Leu, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, and Tyr). Cells were harvested in exponential phase, and we subsequently measured the GFP and mKate2 fluorescence intensity using flow cytometry. The amino acid biosynthesis pathway sensing reporter exhibited the lowest expression in SC media, characterized by a high amino acid concentration (Figure 1E). Under these conditions, the TORC1 pathway is active (Alfatah et al., 2021), while the GAAC pathway is inactive (Natarajan et al., 2001). Conversely, in amino acid–deprived YNB medium, where GAAC pathway is activated (Gulias et al., 2023), the expression was ∼ 2-fold higher compared with SC media (Figure 1E). Notably, the highest expression was observed in supplemented MM, which contains low levels of Leu, Met, and His (Figure 1E).

We reasoned that the high reporter activity in the MM media could derive from the simultaneous activity of both TORC1 and GAAC pathways, due to presence of Leu (Bonfils et al., 2012) and incomplete and misbalanced supplementation of amino acids (Niederberger et al., 1981). To test this, we examined reporter expression in YNB media supplemented with individual amino acids (His, Leu, or Met), both with and without the TORC1 inhibitor rapamycin (Figure 1F). We found that adding Leu and Met, but not His, significantly increased reporter expression compared with its regulation by the GAAC pathway alone (Figure 1F). Adding rapamycin to the media with Leu reduced the reporter signal to the level seen in YNB medium, confirming that Leu in MM medium activates the biosynthesis reporter in a TORC1-dependent manner (Figure 1F). However, in the medium with Met, the reporter signal remained elevated even in the presence of rapamycin, indicating that Met activated the reporter in a TORC1-independent manner.

To check whether Met increased GAAC pathway activity, we measured endogenously tagged Gcn4-GFP protein levels using flow cytometry in the same conditions. We found that addition of Met significantly increased Gcn4 protein levels, indicating higher Gcn4 translation due to increased GAAC activity (Hinnebusch, 1985) (Figure 1G). This aligns with prior work, showing that an excess of methionine relative to other amino acids depletes valine, triggering the accumulation of specific uncharged tRNAs that activate the GAAC (Niederberger et al., 1981). In line with these results, reporter expression in MM media was significantly lower under rapamycin treatment, but the GFP signal remained higher than the basal level of reporter activity in YNB media, confirming that reporter activation in MM media is mediated through the activation of both the GAAC and TORC1 pathways (Figure 1F). Collectively, our findings show that the biosynthesis reporter indicates the engagement of both the GAAC and TORC1 pathways.

We then examined the expression of the SPS reporter responsible for extracellular amino acid sensing under the same conditions. As expected, the uptake reporter showed the highest expression in conditions where amino acids were present in the media (MM and SC), whereas, in the absence of amino acids (YNB), the reporter expression was 7–8 folds lower, indicating pathway inactivity (Figure 1H). Notably, higher amino acid concentrations in SC did not lead to a further increase in the expression of mKate2 compared with MM. This suggests that the pathway is fully activated even in the presence of small amounts of amino acids, such as leucine (Klasson et al., 1999; Gaber et al., 2003), while higher concentrations in SC medium raise intracellular leucine levels and blunt the SPS signaling response by reducing the relative difference between extracellular and intracellular levels sensed by Ssy1 (Wu et al., 2006) (Figure 1H). To test whether this reporter is selective sensitive to specific amino acids, we measured the activity of the uptake reporter in YNB media supplemented with individual amino acids found in the MM media (His, Met, Leu). This showed that Leu and Met alone, but not His, effectively stimulate SPS pathway activity (Figure 1I). These data indicate that the amino acid uptake reporter can effectively report on the presence of specific external amino acids.

BY4741 is a commonly used strain that carries auxotrophic mutations that prevent the biosynthesis of histidine (his3Δ1), leucine (leu2Δ), methionine (met15Δ), and uracil (ura3Δ), and can be employed as selection markers for strain construction. Because we utilized prototrophic BY4741 strains for our experiments, which lack these metabolic deficiencies (Mülleder et al., 2012), we sought to evaluate whether the auxotrophies in the BY4741 strain would influence the biosynthesis or uptake reporter expression. We compared the reporter expression levels between the prototrophic and auxotrophic parental strains in MM and SC media where the reporter genes were expressed. The results obtained through flow cytometry showed no significant differences in the expression levels of either reporter genes, either in minimal or synthetic full media (Figure 1, J and K). This indicates that reporter the BY4741 auxotrophies do not impact the amino acid biosynthesis and uptake pathway activities and affirm that these reporter constructs can be utilized in both prototrophic and auxotrophic strains. However, in auxotrophic strains, the SPS sensing system is likely always active due to the presence of supplemented amino acids in MM and SC media that cannot be synthesized. To fully utilize these reporter constructs for monitoring differences in diverse media conditions, we conducted further experiments with prototrophic strains in varying nutrient environments.

Deletion of key GAAC and SPS components reveals interplay between the amino acid sensing pathways

To examine potential cross-talk between the amino acid sensing pathways and further validate the functionality of the two reporters, we individually deleted essential components of the GAAC and SPS pathways and measured their impact on the reporter expression. Specifically, we deleted the responsible transcription factor GCN4 for GAAC and the serine protease SSY5 responsible for the nuclear translocation of the transcription factors Stp1/Stp2 in the SPS pathway (see Figure 1, A and C). First, we evaluated the effect of these mutations on cellular growth under varying amino acid conditions (YNB, MM, and SC media) using automated growth curve measurements (Supplemental Figure S2). We found that SSY5 deletion does not impact cellular growth in YNB, MM, or SC media in prototropic strain, while it is important to note that SSY5 deletion impairs leucine uptake and is therefore lethal in leucine auxotrophic strains (Forsberg et al., 2001b) (Supplemental Figure S2, B–D). However, gcn4∆ cells grew poorly in YNB media without amino acids, indicating that the activation of the GAAC pathway is essential for growth (Supplemental Figure S2B). In MM, where cells rely on both GAAC and TORC1 pathways for amino acid biosynthesis, gcn4∆ cells grew, but at a substantially slower rate than wild-type (WT) (Supplemental Figure S2C). In SC media, which contains most amino acids and where the GAAC pathway is inactive, gcn4∆ cells showed growth similar to WT (Supplemental Figure S2D). Using flow cytometry, we analyzed the effects of these mutations on the amino acid biosynthesis and uptake pathways under varying amino acid conditions. To mitigate the potential effect of growth arrest on reporter expression, we analyzed the effect of GCN4 deletion only in MM and SC media, where cells actively divided (Supplemental Figure S2, B–D). As expected, the amino acid synthesis reporter activity significantly decreased in gcn4∆ cells growing in low amino acid conditions in MM medium, where the GAAC pathway is active, but had no significant impact on the reporter activity in SC media where the GAAC pathway is inactive (Figure 2A). Interestingly, the biosynthesis reporter values remained higher in the gcn4Δ strain in MM media compared with SC media, which can be explained by Leu-mediated activation of the TORC1 pathway (Figure 1F). Importantly, the deletion of GCN4 had no effect on the SPS pathway engagement in any of the tested conditions (Figure 2B). These results confirm the amino acid biosynthesis reporter as a valid readout for pathway engagement and demonstrate that Gcn4-mediated transcription does not significantly affect SPS pathway activity.

Next, we investigated the impact of SSY5 deletion on the SPS pathway reporter signal in high and low amino acid conditions. In YNB, where the SPS pathway is inactive (Iraqui et al., 1999), the SSY5 deletion had no impact on the reporter expression (Figure 2C). However, in conditions where amino acids were present in the media, activating the SPS pathway, the deletion of SSY5 efficiently suppressed the reporter expression. This indicates that the absence of SSY5 efficiently blocks Stp1/Stp2 transcription on the GNP1 promoter (Figure 2C).

When we assessed the activity of biosynthesis pathways in ssy5Δ strains, we noted a significant decrease in the reporter signal in both MM and SC media. This suggests that the SPS pathway plays a role in activating amino acid biosynthesis pathways when amino acids are present in the media (Figure 2D). To further investigate this, we measured the levels of endogenously tagged Gcn4-GFP protein as a proxy for GAAC activation in WT and ssy5Δ strains using flow cytometry. As anticipated, in WT cells, the Gcn4-GFP levels were significantly higher in media lacking or having limited amino acids compared with SC medium (Figure 2E). Interestingly, in the ssy5Δ cells, both in MM and SC conditions, Gcn4 protein levels were elevated, indicating an increase in GAAC activity (Figure 2E). Therefore, our findings in Figure 2D likely reflect a scenario where GAAC is active and TORC1 is inactive. In ssy5Δ cells, the absence of a functional SPS system prevents high-affinity leucine uptake (Forsberg et al., 2001b), which suppresses TORC1 activity. This can promote GAAC pathway activity through Gcn2 dephosphorylation (Cherkasova and Hinnebusch, 2003), with GAAC activity further promoted by the imbalance in intracellular amino acids caused by the absence of SSY5 (Zaborske et al., 2009). Notably, in YNB media lacking amino acids, Gcn4-GFP protein levels in ssy5Δ strains were slightly reduced (Figure 2E). This reduction could be due to failures in importing external amino acids, potentially released into the media through secretion or from lysed cells (Correia-Melo et al., 2023). Overall, the deletion of SSY5 demonstrates that the activities of the TORC1 and GAAC pathways are influenced by the amino acids imported via the SPS pathway.

These findings collectively validate that our reporters serve as proxies for the GAAC, TORC1, and SPS pathway activities and indicate a cross-talk between these separate amino acid sensing pathways.

The dual reporter allows for the simultaneous monitoring of internal and external amino acid sensing in single cells

We then sought to evaluate whether our dual amino acid sensing reporter could be utilized for microscopic applications to analyze the amino acid biosynthesis and uptake pathway activities at the single-cell level. Under the same conditions (YNB, MM, and SC), we visualized live cells using a wide-field fluorescent microscopy (Figure 3A). Consistent with our flow cytometry analysis (Figure 1, E and H), we observed the highest amino acid biosynthesis reporter activity in low amino acid conditions (YNB and MM) (Figure 3B). In contrast, the uptake reporter intensity was greatest in conditions where amino acids were present in the media (MM and SC), and lowest in the absence of amino acids (YNB) (Figure 3C).

To gain a better understanding of the relationship between the two reporters, we analyzed the correlation of single-cell fluorescence intensities of the amino acid biosynthesis and uptake reporters. Despite displaying varying activities in different media, the reporters consistently showed a significant correlation between GFP and mKate2 signals in each analyzed condition (Figure 3, D–F). The Pearson's correlation coefficient was highest in media containing amino acids, including MM (R = 0.4656, p < 0.0001) and SC (R = 0.4984, p < 0.0001) (Figure 3, E and F), where SPS pathway activity had the most pronounced impact on the amino acid synthesis reporter (Figure 2D). Even in the absence of amino acids in YNB media, the correlation remained lower but still significant (R = 0.3771, p < 0.0001). This validates the interconnected nature of the amino acid biosynthesis and uptake pathways, with the SPS pathway influencing TORC1 and GAAC pathway activities (Figure 2, D and E). These results collectively demonstrate that the dual reporters can be employed to investigate internal and external amino acid sensing activities in single cells.

Growth phase and culture conditions impact amino acid sensing systems

Yeast cells are typically cultivated in liquid or solid (agar) media, undergoing different growth phases determined by nutrient availability. In solid media, yeast cells form colonies—interactive cell communities where resources, such as amino acids, are shared between cells (Palková et al., 1997; Zikánová et al., 2002; Campbell et al., 2015, 2016; Kamrad et al., 2023).

We aimed to investigate whether the reporter system could be used to assess the influence of different culture conditions and growth phases on amino acid biosynthesis and uptake pathways. For this purpose, we utilized supplemented minimal medium in which both reporters were active. We cultured cells in liquid culture, collecting them at either the exponential or stationary growth phase (after 2 d of growth), or grew them on solid agar media for 2 d, leading to the formation of colonies (Figure 4A).

Fluorescent cytometry analysis revealed that both reporters exhibited the highest expression levels in exponential liquid culture (Figure 4, B and C) corresponding to high availability of amino acids. However, expression of the reporters was lowest in stationary liquid culture where nutrients start to become limiting (Figure 4, B and C). Interestingly, both the amino acid biosynthesis and uptake pathways displayed significantly higher activity in cells in 2-d cultures grown as colonies on solid media as compared with liquid media, suggesting that more amino acids are available for cells under these conditions. Microscopic analysis further supported this finding, showing that amino acid biosynthesis and uptake pathway activities correlate in exponential (R = 0.5538, p<0.0001) and 2-d colony growth (R = 0.5070, p<0.0001) (Figure 4D). In contrast, during the stationary phase, cells exhibited two distinct clusters (Figure 4E), one where both reporters are active and one where they are suppressed with high overall correlation between the reporter signals (R = 0.7841, p<0.0001) (Figure 4E). This subset of cells with reduced reporter activity likely represents nondividing quiescent cells resulting from glucose depletion in the stationary phase conditions. Cellular quiescence results in down-regulation of protein translation (de Virgilio, 2012), which could potentially affect reporter signal. Together, these results demonstrate that the dual reporter system provides a single-cell readout for studying the impact of growth phases, culture conditions, and cell-cell interactions in amino acid sensing.

Metabolic differentiation though amino acid sensing pathways benefits colony survival

To further investigate whether these reporters can shed light on the metabolic programming of cells within colonies, we analyzed amino acid sensing pathways across 10-d colony growth using fluorescence imaging and flow cytometry. First, we assessed spatial gross variations in amino acid sensing pathway activities within the colonies using a fluorescence biomolecular scanner. By day 4 of colony development, both biosynthesis and uptake reporters showed elevated expression in cells positioned on the outer rim of the colony (Figure 5A), suggesting utilization of amino acid sensing pathways for colony growth. Interestingly, cells in the center exhibited enhanced amino acid uptake but not biosynthesis reporter activity, indicating up-regulation of amino acid uptake in upper layer of the colonies where extracellular amino acid levels decrease (Čáp et al., 2012). Such spatial metabolic specialization became even more pronounced on day 8, where cells on the outer rim of the colony exclusively expressed biosynthesis reporter, while both reporters were expressed in the center of the colony (Figure 5A). Moreover, colonies displayed sectored patterns with heightened reporter activities. Formation of metabolically specialized segments in the colony indicates decoupling of amino acid biosynthesis and uptake, potentially stemming from local variations in nutrient availability and/or cellular growth (Giometto et al., 2018; Banwarth-Kuhn et al., 2020).

To gain cell-level resolution on these populations, we performed a flow cytometry analysis during different stages of colony growth. Most cells on days 2 and 4 expressed both reporters (81% and 63%, respectively) (Figure 5, B and C). However, by day 4, 30% of cells specialized solely in amino acid uptake (Figure 5C), while a population of cells specializing solely in biosynthesis emerged by day 8 (9.63%) (Figure 5D). To further examine these populations, we conducted a microscopy analysis. This corroborated the flow cytometry findings by demonstrating the presence of cells expressing either of the two reporters (Figure 5E). Correlation analysis between the two reporters indicated their decoupling in cells at day 8 of colony growth (R = 0.01, p = 0.778) (Figure 5F). Overall, these results demonstrate that a subset of cells undergo metabolic differentiation in colonies over 8 d where they specialize in either amino acid synthesis or import.

Flow cytometry analysis indicated an increase in the proportion of nonfluorescent cells over time, potentially representing a fraction of dead cells within the colony (Figure 5, B–D). To verify this, we assessed cell viability using calcofluor staining in microscopy images. Calcofluor selectively stains the cell wall of living cells, while dead cells exhibit dye internalization into the cytoplasm (Fischer et al., 1985). Our results confirmed a gradual decline in colony survival over time (Figure 5G). Notably, a smaller subset of colonies exhibited a death rate exceeding 50% by day 8 (Figure 5G). Flow cytometry analysis of these less viable colonies revealed that only ∼ 25% of cells expressed both reporters. Importantly, no specialized cells in amino acid biosynthesis or uptake were detected (Figure 5H). This suggests that the metabolic specialization of cells into either amino acid biosynthesis or uptake promotes the overall survival of colonies.

To test this, we reasoned that by interfering with the extracellular amino acid sensing module, we might disrupt the ability of cells to specialize. Thus, we deleted SSY5 and analyzed the death rate of cells grown in colonies over a 14-d period. Our findings revealed that, although SSY5 deletion no initial effect on cell viability, after the emergence of specialization into uptake and biosynthesis (at day 8, see Figure 5, D–F), ssy5∆ cells exhibited a significantly higher death rate compared with WT cells (Figure 5I). Together these results imply that specialization in amino acid uptake or biosynthesis is benefits overall cell survival in the late stages of colony development. Overall, these results demonstrate that the dual reporter system enables investigating spatial organization of metabolic differentiation in cellular communities.

DISCUSSION

Maintaining amino acid homeostasis is critical for cellular functions such as growth, metabolism, and signaling. Conversely, imbalances in amino acid levels can have detrimental effects on cellular function and contribute to various diseases and disorders (Bröer and Bröer, 2017; González and Hall, 2017; Takagi, 2019). Therefore, it is important to have tools that enable visualizing how cells sense their internal and external amino acid status. In this study, we generated fluorescent reporters that provide a cellular proxy for the transcriptional activity of the GAAC, TORC1, and SPS pathways. These constructs are made available for the research community through Addgene.

Consistent with previous observations with LacZ-based Stp1/Stp2 activity reporters, our SPS-pathway reporter exhibited augmented expression in medium with high amino acid concentration and composition (Iraqui et al., 1999). Also, similar to what has been shown with LacZ-based construct reporting Gcn4 activity, our reporter exhibited highest expression levels in medium with low concentration and composition of amino acids (Albrecht et al., 1998). In addition to GAAC, our biosynthesis reporter responds to TORC1 activity. Importantly, the biosynthesis reporter enables independent assessment of these two amino acid biosynthesis pathways, for instance through growing cells in media where only one pathway is active (e.g., YNB vs. SC) or by using pathway specific inhibitors such as rapamycin. Overall, our reporter constructs offer advantages over previously developed systems by enabling simultaneous, quantitative assessments of amino acid uptake and biosynthesis pathway engagement at single-cell resolution. While FRET-based reporters also enable sensitive detection of TORC1 engagement in single cells (Zhou et al., 2015), they lack the flexibility to simultaneously monitor other amino acid sensing pathways due to the multiplexing constraints of FRET fluorophores. Another key benefit of our system is its compatibility with high-throughput platforms, such as flow cytometry-based single-cell sorting, which allows for further analysis of differentiated cells using omics approaches. Thus, this dual-fluorescent reporter system is a powerful tool for large-scale studies of amino acid homeostasis and related cellular processes at the single-cell level.

The reporters facilitate the study of context-dependent coregulation in amino acid–sensing pathways. Prior studies have demonstrated a mutual inhibition between the GAAC and TORC1 pathways in amino acid biosynthesis (Cherkasova and Hinnebusch, 2003; Yuan et al., 2017). Our data present novel insights into the dynamic interplay of biosynthesis and uptake pathways under specific conditions. In MM medium where both GAAC and TORC1 are active (Figures 1E and 3B), leucine induces the biosynthesis reporter in a TORC1-dependent manner, while methionine promotes GAAC pathway activity (Figure 1, F and G). This suggests that the TORC1-dependent inhibitory effect of Ser577 phosphorylation on Gcn2 and subsequent translation of GCN4 can be overridden by elevated levels of uncharged tRNAs (Dong et al., 2000) in media with incomplete amino acid supplementation (Niederberger et al., 1981). Our results also indicate that the previously reported down-regulation of TORC1 activity by Gcn2 (Yuan et al., 2017) does not occur in the presence of leucine. Furthermore, through an analysis of the response of two reporters in relation to silencing either the GAAC or SPS pathway, we observed an interplay between amino acid biosynthesis and uptake pathways. Deleting GCN4 had no impact on uptake reporter expression, whereas deleting SSY5 affected the biosynthesis reporter. The SSY5 deletion reduced biosynthesis reporter expression in media where the amino acid uptake pathway is typically activated. These effects are likely a consequence of the diminished leucine uptake in the ssy5∆ strain, which reduces TORC1 pathway activity and enhanced GAAC activity through the accumulation of uncharged tRNAs, caused by failure to import amino acids (Zaborske et al., 2009). The heightened GAAC activity may also be contributed to by reduced TORC1-mediated inhibition of Gcn2 (Cherkasova and Hinnebusch, 2003).

We provided evidence of the effectiveness of these reporters in analyzing amino acid biosynthesis and uptake pathways at the single-cell level across various culture conditions. Our findings, using prototrophic yeast grown in MM, align with previous studies indicating that cells grown in colonies exhibit distinct metabolic characteristics compared with those in liquid culture (Čáp et al., 2012; Campbell et al., 2015, 2016). In colonies, yeast cells can engage in metabolic exchange, leading to distinct phenotypes emerging over time (Čáp et al., 2012). In our study, the reporters identified cellular metabolic differentiation within 8-d-old colonies. At this stage, subset of cells became specialized in either amino acid biosynthesis or uptake pathways (Figure 5, A and D–F). Notably, this metabolic differentiation status depends on the spatial location of cells within the colony (Figure 5A), suggesting that peripheral cells may act as “producers,” synthesizing amino acids that nourish the “consumer” cells located in the interior of the colony. The sectorial localization of specialized cells within the colony align with previous mathematical models, suggesting that sectorial colony populations arise from cells exhibiting differential growth rates due to nutrient limitation (Giometto et al., 2018; Banwarth-Kuhn et al., 2020). Only in the middle of the colony, cells showed a strong correlation between both reporter signals, consistent with previous findings of up-regulated ARG8 and GNP1 genes in the upper layer of the colony (Čáp et al., 2012). Importantly, we provide evidence that this specialization in either amino acid synthesis or uptake may be important for the overall cell survival in aged colonies where nutrients become limiting (Figure 5, G and I), consistent with prior work showing that SPS pathway components promote colony survival by affecting a specific subset of differentiated cells (Čáp et al., 2012). Hence, our reporters offer insights into regulating cooperative interactions and phenotypic transitions among colony-embedded cells in both spatial and temporal dimensions. For instance, colony differentiation has also been observed in filamentous fungi (Masai et al., 2006; Levin et al., 2007; Daly et al., 2020), which are frequently utilized in various biotechnological applications, including enzyme production and food fermentation. However, controlling growth conditions and achieving optimal cell states pose challenges due to spatial and temporal heterogeneity within colonies, as well as complex interactions with environmental factors (Masai et al., 2006; Daly et al., 2020). Our reporters could enable real-time, single-cell monitoring of changes in amino acid metabolism during colony growth, providing a deeper understanding of how fungi adapt to and utilize nutrients. This could contribute to the development of more efficient strains with enhanced growth, protein production, and secretion capabilities. Therefore, these reporter constructs can be valuable tools for biotechnological applications that aim to manipulate amino acid biosynthesis or uptake in S. cerevisiae and other industrially relevant filamentous fungi.

Last, our reporter constructs serve as valuable tools for studying cellular specialization and colony development beyond biotechnological applications. By capturing amino acid sensing at the single-cell level, they provide insights into cellular heterogeneity and how differentiated cells contribute to colony function (Campbell et al., 2016). They also enable the study of cell-to-cell communication, a process that declines with aging (Váchová and Palková, 2011), and help uncover metabolic diversity in wild yeast strains (Kaya et al., 2021). Furthermore, by integrating multilevel measurements, these reporters allow differentiated cells to be sorted and analyzed using metabolomics, proteomics, and transcriptomics, providing a comprehensive view of how amino acid sensing influences gene and protein expression.

Limitations

Our reporters, while powerful, have limitations worth considering. The constructs are designed to include a broad promoter region (1000 base pairs upstream of the open reading frames of ARG8 and GNP1) to encompass all regulatory elements for efficient transcription. Consequently, these regions contain additional promoter elements, such as stress-related ones that may affect transcription under specific conditions (Beck and Hall, 1999). In future work, we envision it will be possible to tailor more specific promoter elements that may also allow the building separate response elements for GAAC and TORC1 with different fluorophore reporter genes. Additionally, our dual reporter has some general limitations inherent to promoter-based approaches. First, it might not accurately reflect amino acid metabolism in a quiescent state where protein translation is suppressed. Second, it does not provide the spatial resolution of FRET-based pathway activity reporters (Zhou et al., 2015) or temporal resolution of nuclear translocation–based reporters (Guerra et al., 2022). Last, the interpretation of Pearson's correlation coefficients for cytosolic reporters should be approached with caution, as some degree of correlation may be inherently expected due to colocalization (Adler and Parmryd, 2010). While our observed correlations across different growth phases suggest biologically meaningful relationships, future studies could benefit from including a control reporter unresponsive to amino acid availability to distinguish pathway-specific correlations.

MATERIALS AND METHODS

Yeast strains

All strains were derivatives of S. cerevisiae S288C BY4741 (Brachmann et al., 1998) (listed in Supplemental Table S1). Strains expressing genomically integrated amino acid reporters were generated using the EasyClone 2.0 kit (Stovicek et al., 2015). Briefly, the ARG8 and GNP1 promoter regions were paired with GFP and mKate2 encoding genes and incorporated into integrative yeast plasmids pCfB2188 or pCfB2197 by employing USER cloning (Stovicek et al., 2015). For promoters, the DNA sequences 1000 base pairs upstream of ARG8 and GNP1 open reading frames were amplified from yeast genomic DNA using PCR. Fluorescent proteins were amplified from tagging plasmids pYM25 (GFP) (Janke et al., 2004) and pYM25-mKate2 (Paukštytė et al., 2023) (see Supplemental Table S1). All PCR products contained overhangs that made them compatible with USER cloning based on instructions provided by the manufacturer (Stovicek et al., 2015). Vectors were linearized and nicked with SfaAI and Nb.BsmI to comply with the USER reaction. Via the USER reaction, PCR products were integrated into nicked plasmids, generating pCfB2188-Pr_ARG8-GFP::URA3 and pCfB2197-Pr_GNP1-mKate2::NatMX, which were then transformed into Escherichia coli DH5α (Zymo research). After confirming the correct plasmid construction via PCR and sequencing, the plasmids were linearized with the restriction enzyme NotI and transformed into yeast for genomic integration in safe harbor chromosomal loci X-2 (pCfB2188-Pr_ARG8-yeGFP-URA3) and XII-1 (pCfB2197-Pr_GNP1-mK2-NatMX) (see Yeast transformation).

Prototrophic strains were generated by inserting pHLUM plasmid (Mülleder et al., 2012) into strains expressing amino acid reporters. Genes GCN4 and SSY5 were deleted by homologous recombination. In short, deletion cassettes based on plasmid pFA6a-kanMX6 for strains expressing the dual reporters or pFA6a-NatMX for cells expressing Gcn4-GFP reporter (Janke et al., 2004) were generated by PCR using primers homologous to the sequence upstream and downstream of genes GCN4 and SSY5. Homologous recombination resulted in the replacement of genomic versions of these genes with the deletion cassette.

Growth media and growth conditions

Yeast strains were grown at 30°C in YPD medium (1% yeast extract, 2% Bacto peptone, 2% glucose), YNB medium (YNB) (6.7g/l YNB without amino acids (BD Difco 291920), 2% glucose), supplemented minimal medium (MM) (0.02 g/l of Ura, Met, Leu, His, 6.7 g/l YNB without amino acids (BD Difco 291920), 2% glucose), SC medium (0.013 g/l L-Ade, 0.35 g/l L-Arg, L-inositol, 0.26 g/l L-Asp, L-Leu, 0.057 g/l L-His, 0.52 g/l L-Ile, 0.09 g/l L-Lys, 0.19 g/l L-Met, 0.082 g/l L-Phe, L-Trp, 0.01 g/l L-Ser, 0.12 g/l L-Thr, L-Val, 0.018 g/l L-Tyr, 0.02 g/l Ura, 6.7 g/l YNB without amino acids [BD Difco 291920], 2% glucose). In addition to these components, solid media plates contained 2% of agar.

Yeast transformation

Yeast cell culture, grown overnight, was diluted to OD₆₀₀ of 0.1 in liquid YPD media and cultivated until it reached OD₆₀₀ of 0.6. Subsequently, cells were collected by centrifugation at 500 × g for 5 min, washed twice with transformation buffer (100 mM LiOAc, 10 mM Tris, 1 mM EDTA, pH 8), and resuspended in 72 µl of transformation buffer. Then, 2 µl of plasmid DNA or 10 µl of PCR product, along with 8 µl of 100 mg/ml salmon sperm DNA (previously boiled for 10 min at 100°C and cooled on ice), were added to the cell suspension, followed by 500 µl of PEG buffer (40% PEG-3350 (m/V), 100 mM LiOAc, 10 mM Tris, 1 mM EDTA, pH 8). The mixture was incubated at room temperature for 30 min. Then 65 µl of DMSO were added, and yeast cells were exposed to heat shock at 42°C for 15 min. Cells were then spun down for 5 min at 300 × g. If an auxotrophic marker was used for selection, yeast pellets were resuspended in 100 µl of the selective SC (-ura) or SC (-his) media and plated on agar plates with the same media. If antibiotics were used for selection, samples were incubated in 3 ml of YPD media for 2–3 h before being plated on YPD plates containing antibiotics.

Flow cytometry

Cells were grown in exponential phase in SC(-his) or (-ura) liquid media for 48 h before the experiment. For measuring reporter activity, cells were washed with PBS, resuspended into YNB, MM, or SC media and grown for 12 h maintaining them in exponential phase. After the incubation, ∼ 2 × 10⁶ cells per sample were diluted in 2 ml of media and analyzed by flow cytometry.

Flow cytometry analysis was performed using the BD LSRFortessa Flow Cytometer at the Viikki Flow Cytometry Unit in the University of Helsinki. GFP fluorophore was excited with a 488-nm laser and an optical filter of 530/30 (515–545 nm), while mKate2 fluorophore was excited with a 561-nm laser and an optical filter of 610/620 (600–620 nm). Samples were collected by gating cell population emitting fluorescent signal, which was distinguished from reporter-free WT cell population. For each of the three biological replicate samples, 10,000 cells were analyzed. FlowJo v10 software was used for the analysis of flow cytometry data.

Cell growth measurements

Cells were grown in SC(-his) or SC(-ura) liquid media in the exponential phase for 48 h before the experiment. Approximately 2 × 10⁶ cells per sample were harvested by centrifugation and washed twice with PBS. The cells were then resuspended in 1 ml of YNB, MM, or SC media at a concentration of 1.5 × 10⁵ cells/ml (OD_600nm 0.01), and 400 µl were loaded into a sterile honeycomb microplate (Growth Curves, Finland). Three biological replicates for each genetic background and two technical replicates for each sample were used. Growth rate was measured as absorbance at OD_600nm in a Bioscreen C device (Growth Curves, Finland) every 10 min for 24 h at 30°C with continuous shaking.

Live-cell imaging

Cells were grown in the exponential phase in SC(-his) or (-ura) liquid media for 7–8 h before the experiment. For measuring reporter activity, cells derived from three biological replicates were washed with PBS, resuspended into YNB, MM, or SC media and grown for 16 h maintaining them in exponential phase. After incubation, 2.55 × 10⁶ cells were stained with calcofluor (0.1 mg/ml) for 5 min. Then, cells were washed twice with PBS and resuspended in 1 ml of their corresponding media. A total of 50 µl of the cell suspension was placed into precoated well of a 96-well thin glass-bottom imaging plate with 200 µl of the respective medium. The wells were precoated by incubating with 100 µl of concanavalin A solution (2 mg/ml) for 30 min at room temperature and washing them once with 150 µl of PBS.

Live imaging was conducted using a customized Olympus IX-73 inverted widefield fluorescence microscope DeltaVision Ultra (GE Healthcare) equipped with a Pco edge 4.2ge sCMOS camera and running the CentOS 7 Linux operating system. A 100x oil objective and Blue (ex. 390 nm - em. 435 nm), Green (ex. 475 nm - em. 525 nm) and Red (ex. 575 nm - em. 625 nm) filter settings were used based on the fluorophore characteristics.

Analyzing cells in different growth phases

Cells were cultured in the exponential phase in MM(-ura) liquid media for 12 h before the experiment. Subsequently, exponentially growing cells were consistently diluted to maintain a cell density of no more than 1.8 × 10⁷ cells/ml or OD660 < 1 for 48 h. Conversely, stationary phase cells were diluted to ∼ 1.5 × 10⁵ cells/ml and grown over 48 h without further dilutions, resulting in a cell density above 1.3 × 10⁸ cells/ml. Simultaneously, around 500 cells were plated onto solid MM(-ura) agar medium plates and grown for 48 h, during which colonies formed from individual cells.

After 2 d, cells at every growth phase were analyzed using flow cytometry and microscopy (for details, see sections Preparation of Cells for Flow Cytometry and Preparation of Cells for Live Cell Imaging). For this, ∼ 2 × 10⁶ cells growing in the liquid media were diluted in 2 ml of media, while 3–6 individual colonies were collected with a loop and resuspended in 0.5 ml of media.

Fluorescent imaging of colonies

Colonies growing in MM(-ura) solid agar medium over 10 d were scanned by placing the plates upside down onto the Sapphire FL biomolecular imager (Azure Biosystems). Images of various horizontal planes were obtained using 488 nm laser and 513/17 nm emission filter to detect biosynthesis reporter signal and using 638 nm laser and 676/37 nm emission filter to detect the uptake reporter signal.

Image analyses

Imaging data were analyzed using the FIJI ImageJ software. Signal intensity of individual cells expressing amino acid reporters were measured using segmentation. For this, images were preprocessed by converting them to 8-bit files and manually adjusting the threshold on one of the channels so that all cells would be included. Based on this threshold, a manipulative mask was created where overlapping objects were split with Watershed function. Individual cells were then selected automatically with Analyze Particles tool and manually checked to exclude noncell particles and premature daughter cell buds. Readymade mask was then transferred to the original image and fluorescence intensity and particle area was measured for all selected particles on Green and Red channels.

Statistical analyses

Statistical significance of comparisons presented in this study was assessed using GraphPad software. Two-tailed unpaired t test was applied to determine statistical significance between two groups. Ordinary one-way ANOVA with Tukey's multiple comparisons test was used to assess statistical significance in reporter expression in the WT prototrophic strain under different conditions. Two-way ANOVA with Tukey's multiple comparisons test was used to assess statistical significance of reporter expression in auxotrophic and prototrophic strains under different conditions, as well as comparisons between deletion mutant strains. In Figures 3, D–F and 4, D–F outliers were removed using ROUT-method with the Q-value of 1% and correlation coefficient (r) was calculated using the conventional Pearson's correlation test.

FOOTNOTES

DATA AVAILABILITY

All raw data are available through Harvard Dataverse (Accession link: https://doi.org/10.7910/DVN/LTCSXP). Reporter plasmids are available from Addgene #216512 and #216513.

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