Biology subjects

Botman, D.

Publications and source records attributed to Botman, D..

8 recordsLinked to original sources

bioRxiv2026

Glutamine-derived NADH fuels lactate production to maximize ATP yield under oxygen consumption constraints in head and neck cancer

Metabolic reprogramming is one of the hallmarks of cancer. A well-known change is the Warburg effect: glucose is consumed at increased rates and fermented to lactate instead of being respired to carbon dioxide, despite the presence of sufficient oxygen (O2). We show that head and neck squamous cell carcinoma (HNSCC) cells excrete more carbon in the form of lactate than is consumed as glucose, indicating the presence of additional carbon sources for lactate. Amino acid uptake and [U-13C]-glutamine labelling data show that glutamine is highly consumed and oxidized in the tricarboxylic acid (TCA) cycle, but remarkably does not contribute to lactate carbon. To explain these observations, we generated a cell-line specific metabolic model to understand the link between lactate production and glutamine metabolism. In the model, we show how low cellular O2 consumption capacity can cause increased lactate production with glutamine as NADH source. The model suggests that ATP production under limited O2 consumption is augmented by increased glutamine oxidation which yields ATP, FADH2 and NADH. FADH2 can only be respired, despite yielding less ATP than NADH respiration. NADH can be exported from the mitochondrion to be reoxidized by lactate dehydrogenase, permitting more cycles of glutamine oxidation, thereby increasing the total ATP yield. By experimentally inhibiting mitochondrial respiration, we confirmed that non-glucose carbon sources are used for lactate production under decreased O2 consumption capacity. This unexpected feature of O2-constrained ATP production provides a credible mechanism for the increased lactate production via a novel redox cycle in HNSCC cells.

systems biology↗

bioRxiv2025

Side-by-side systematic characterization of novel FPs in budding yeast

Fluorescent proteins (FPs) have become indispensable for biological research. Since the discovery of the first FP, Aequorea victoria green fluorescent protein (avGFP), new fluorescent proteins are continuously being developed. To achieve optimal experimental results, selecting an FP based on specific characteristics--such as brightness, photostability, photochromicity, monomericity, pH robustness, and fluorescence lifetime--is essential. However, a thorough side-by-side comparison of these characteristics is missing for the latest generation of FPs. Here, we provide a comprehensive characterization in yeast of the most recently developed FPs, including FPs that were codon-optimized for yeast. We believe this provides an excellent compendium for choosing the most suitable FP for research purposes.

molecular biology↗

bioRxiv2024

An mTurquoise2-based glucose biosensor

Glucose is an important substrate for organisms to acquire energy needed for cellular growth. Despite the importance of this metabolite, single-cell information at a fast time-scale about the dynamics of intracellular glucose levels is difficult to obtain as the current available sensors have drawbacks in terms of pH sensitivity or unmatched glucose affinity. To address this, we developed a convenient method to create and screen biosensor libraries using yeast as workhorse. This resulted in TINGL (Turquoise INdicator for GLucose), a robust and specific biosensor with an affinity that is compatible with intracellular glucose detection. We calibrated the sensor in vivo through equilibration of internal and external glucose in a yeast mutant unable to phosphorylate glucose. Using this method, we measured dynamic glucose levels in budding yeast during transitions to glucose. We found that glucose concentrations reached levels up to 1 mM as previously determined biochemically. Furthermore, the sensor showed that intracellular glucose dynamics differ based on whether cells are glucose-repressed or not. Finally, the human codon-optimized version (THINGL, Turquoise Human INdicator for GLucose), also showed a robust response after glucose addition to starved human cells, showing the versatility of the sensors. We believe that this sensor can aid researchers interested in cellular carbohydrate metabolism.

biochemistry↗

bioRxiv2023

cAMP in budding yeast: a messenger for sucrose metabolism?

S. cerevisiae (or budding yeast) is an important micro-organism for sucrose-based fermentation in biotechnology. Yet, it is largely unknown how budding yeast adapts to sucrose transitions. Sucrose can only be metabolized when the invertase or the maltose machinery are expressed and we propose that the Gpr1p receptor signals extracellular sucrose availability via the cAMP peak to adapt cells accordingly. A transition to sucrose or glucose gave a transient cAMP peak which was maximally induced for sucrose. When transitioned to sucrose, cAMP signalling mutants showed an impaired cAMP peak together with a lower growth rate, a longer lag phase and a higher yield compared to a glucose transition. These effects were not caused by altered activity or expression of enzymes involved in sucrose metabolism and imply a more general metabolic adaptation defect. Basal cAMP levels were comparable among the mutant strains, suggesting that the transient cAMP peak is required to adapt cells correctly to sucrose. We propose that the short-term dynamics of the cAMP signalling cascade detects long-term extracellular sucrose availability and speculate that its function is to maintain a fermentative phenotype at continuously low glucose and fructose concentrations.

microbiology↗

bioRxiv2022

Using the AKAR3-EV biosensor to assess Sch9- & PKA-signalling in budding yeast

Budding yeast uses the well-conserved TORC1-Sch9 and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these two cascades will improve our understanding of cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9 and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV robustly measures the Sch9- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose and fructose, but highly heterogeneous for mannose. The Sch9 and PKA pathways have a relatively high affinity for glucose (K0.5 of 0.24 mM) under glucose derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that the Sch9- and PKA-dependent phosphorylation activity are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.

cell biology↗

bioRxiv2019

An improved ATP FRET sensor for yeast shows heterogeneity during nutrient transitions

Adenosine 5-triphosphate (ATP) is the main free energy carrier in metabolism. In budding yeast, shifts to glucose-rich conditions cause dynamic changes in ATP levels, but it is unclear how heterogeneous these dynamics are at the single-cell level. Furthermore, pH also changes and affects readout of fluorescence-based biosensors for single-cell measurements. To measure ATP changes reliably in single yeast cells, we developed yAT1.03, an adapted version of the AT1.03 ATP biosensor, that is pH-insensitive. We show that pregrowth conditions largely affect ATP dynamics during transitions. Moreover, single-cell analyses showed a large variety in ATP responses, which implies large differences of glycolytic startup between individual cells. We found three clusters of dynamic responses, and show that a small subpopulation of wild type cells reached an imbalanced state during glycolytic startup, characterised by low ATP levels. These results confirm the need for new tools to study dynamic responses of individual cells in dynamic environments.

molecular biology↗

bioRxiv2019

A new FRET biosensor enlightens cAMP signalling in budding yeast

The cAMP-PKA signalling cascade in budding yeast regulates adaptation to changing environments. We developed yEPAC, a FRET-based biosensor for cAMP measurements in yeast. We used this sensor with flow cytometry for high-throughput single cell-level quantification during dynamic changes in response to sudden nutrient transitions. We found that the characteristic cAMP peak differentiates between different carbon source transitions, and is rather homogenous among single-cells, especially for transitions to glucose. The peaks are mediated by a combination of extracellular sensing and intracellular metabolism. Moreover, the cAMP peak follows Webers law; its height scales with the relative, and not the absolute, change in glucose. Lastly, our results suggest that the cAMP peak height conveys information about prospective growth rates. In conclusion, our yEPAC-sensor makes possible new avenues for understanding yeast physiology, signalling and metabolic adaptation.

microbiology↗

bioRxiv2018

In vivo characterisation of fluorescent proteins in budding yeast.

Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.

molecular biology↗