Localization and phosphorylation in the Snf1 network is controlled by two independent pathways

AMPK/SNF1 is the master regulator of energy homeostasis in eukaryotic cells and has a key role in glucose de-repression. If glucose becomes depleted, Snf1 is phosphorylated and activated. Activation of Snf1 is required but is not sufficient for mediating glucose de-repression indicating a second glucose-regulated step that adjusts the Snf1 pathway. To elucidate this regulation, we further explore the spatial dynamics of Snf1 and Mig1 and how they are controlled by concentrations of hexose sugars. We utilize fluorescence recovery after photobleaching (FRAP) to study the movement of Snf1 and how it responds to external glucose concentrations. We show that the Snf1 pathway reacts both to the presence and to the absolute concentration of glucose. Furthermore, we identify a negative feedback loop regulating Snf1 activity. We also show that Mig1 localization correlates with the Snf1 phosphorylation pattern and not with the Mig1 phosphorylation pattern, suggesting that inactivation of Snf1 has a more pronounced effect on the localization of Mig1 than on the phosphorylation of Mig1. Our data offer insight into the true complexity of regulation of this central signaling pathway by one signal (glucose depletion) interpreted by the cell in different ways.

External Carbon Source Facilitates Indirect Cr (VI) Bioreduction Process by Anaerobic Sludge Produced from Kitchen Waste

This study presented the investigation on indirect Cr (VI) bioreduction process by anaerobic sludge produced from kitchen waste (ASKW) using an external source of glucose and sulfate to favor the reducing environment. These compounds were added at the beginning of the experiment along with 500 mg·L−1 Cr (VI). The system containing 1 g of glucose and 2 g of sulfate attained a higher reduction, which was 10% higher than that of the control experiment. This study indicated that a neutral environment (pH ~7), along with a high release of polysaccharides (PS), improved the removal efficiency by Cr (VI) bioreduction process. Desulfovibrio and Sulfurospirillum (genus level), which accounted for 3% and 1% of the whole microorganism, respectively, were responsible for the sulfidogenic reaction. Additionally, Thermovirga (genus level) reduced from 14% to 11% and 10%. These microorganisms contributed to dominating the indirect Cr (VI) bioreduction process. SEM and FTIR analysis of the sludges obtaining from the indirect Cr (VI) bioreduction systems indicated that the external glucose could facilitate the formation of looser porous structures and richer functional groups of sludges, thus adsorbing more Cr (III) to reduce its toxicity. Meanwhile, the intensity of the hydroxyl bond, which possesses strong reducibility, was much higher after adding external glucose. Chromate reductase gene (chrR) and sulfite reductase gene (dsrA) contributed to the indirect Cr (VI) bioreduction process. These might be the main mechanisms of the external glucose acting on indirect Cr (VI) bioreduction by ASKW.

Yeast Nuak1 phosphorylates histone H3 threonine 11 in low glucose stress by the cooperation of AMPK and CK2 signaling

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress-responsive genes. Understanding how this histone modification ‘senses’ external glucose changes remains elusive. Here, we show that Tda1, the yeast ortholog of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMP-activated protein kinase (AMPK) directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.

Yeast Nuak1 phosphorylates histone H3 threonine 11 in low glucose stress conditions by the cooperation of AMPK and CK2 signaling

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress responsive genes. Understanding how this histone modification ‘senses’ external glucose changes remains elusive. Here, we show that Tda1, the yeast orthologue of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMPK directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.

A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labeled hexose transporters and quantified the spatial correlations with intra-colony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, for example amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labelled hexose transporters and quantified the spatial correlations with intracolony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, e.g., amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

A voltage-dependent depolarization induced by low external glucose in neurons of the nucleus of the tractus solitarius of rats: interaction with KATP channels regulated by external glucose.

The brainstem nucleus of the tractus solitarius (NTS) is an integrative center for autonomic counterregulatory responses to hypoglycemia, NTS neurons can also sense fluctuations in extracellular glucose levels altering their membrane potential. KATP channels links the metabolic status of the neuron to its excitability, but the role of KATP channels in controlling NTS neurons excitability and in sensing extracellular glucose changes is not clear. Here we investigated using in vitro electrophysiological recordings in brainstem slices the influence of KATP channels on the membrane potential of NTS neurons in normoglicemic and hyperglycemic external glucose concentrations, and after switching the external glucose to a hypoglycemic level. We found that in normoglicemic (5 mM) external glucose application of tolbutamide, a KATP blocker, induced a substantial depolarization of most NTS neurons, while application of diazoxide, a KATP opener, hyperpolarized the membrane of all NTS neurons. Interestingly, neurons not responsive to tolbutamide were more depolarized than responsive neurons. In a hyperglycemic solution (10 mM glucose) few neurons depolarized in response to tolbutamide. We found that these neurons were more depolarized than neurons in 5 mM glucose and only the more hyperpolarized responded to tolbutamide. The non-responsive neurons did not respond to tolbutamide even when hyperpolarized. Interestingly application of a low-gucose solution (0.5 mM) did not hyperpolarized the RMP but produced a depolarization in most neurons. This effect was voltage-dependent not seen in neurons more depolarized, but could be observed when the neurons were hyperpolarized. Depolarization by tolbutamide avoided further depolarization by low glucose, unless the membrane was hyperpolarized. Application of 0.5 mM glucose solution in neurons incubated in 10 mM glucose depolarized the membrane only in more hyperpolarized neurons, which responded to tolbutamide, or after membrane hyperpolarization. The effect of glucose was caused by activation of a cationic current with a reversal potential around the potential were the neurons were non-responsive to low glucose. We conclude that NTS neurons present KATP channels open at rest in normoglicemic conditions, and that their state is affected by extracellular glucose. Moreover, NTS neurons depolarize the membrane in response to the application of a low-glucose solution, but this effect is occluded by membrane depolarization triggered by KATP blockage. This suggests a homeostatic regulation of the membrane potential by glucose and a possible mechanism related to hypoglycemia-associated autonomic failure.