Maintenance of an optimal redox environment is critical for appropriate functioning of cellular p... more Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for mainten...
Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sul... more Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sulphuric acid to control the bacterial population. This treatment can lead to loss of cell viability, with consequences on the fermentation yield. Thus, the aim of this study was to define the functional cellular responses to inorganic acid stress. Saccharomyces cerevisiae strains with mutation in several signalling pathways, as well as cells expressing pH-sensitive GFP derivative ratiometric pHluorin, were tested for cell survival and cytosolic pH (pHc) variation during exposure to low external pH (pHex). Mutants in calcium signalling and proton extrusion were transiently sensitive to low pHex, while the CWI slt2Δ mutant lost viability. Rescue of this mutant was observed when cells were exposed to extreme low pHex or glucose starvation and was dependent on the induced reduction of pHc. Therefore, a lowered pHc leads to a complete growth arrest, which protects the cells from lethal stress ...
Cells are extremely crowded, and a central question in biology is how this affects the intracellu... more Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, ...
Maintenance of an optimal redox environment is critical for appropriate functioning of cellular p... more Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for mainten...
Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sul... more Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sulphuric acid to control the bacterial population. This treatment can lead to loss of cell viability, with consequences on the fermentation yield. Thus, the aim of this study was to define the functional cellular responses to inorganic acid stress. Saccharomyces cerevisiae strains with mutation in several signalling pathways, as well as cells expressing pH-sensitive GFP derivative ratiometric pHluorin, were tested for cell survival and cytosolic pH (pHc) variation during exposure to low external pH (pHex). Mutants in calcium signalling and proton extrusion were transiently sensitive to low pHex, while the CWI slt2Δ mutant lost viability. Rescue of this mutant was observed when cells were exposed to extreme low pHex or glucose starvation and was dependent on the induced reduction of pHc. Therefore, a lowered pHc leads to a complete growth arrest, which protects the cells from lethal stress ...
Cells are extremely crowded, and a central question in biology is how this affects the intracellu... more Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, ...
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Papers by Gertien Smits