Ying Gu’s research while affiliated with The Francis Crick Institute and other places

Cellular metabolism relies on just a few redox cofactors. Selective compartmentalization may prevent competition between metabolic reactions requiring the same cofactor. Is such compartmentalization necessary for optimal cell function? Is there an optimal compartment size? Here we probe these fundamental questions using peroxisomal compartmentalization of the last steps of lysine and histidine biosynthesis in the fission yeast Schizosaccharomyces japonicus. We show that compartmentalization of these NAD⁺ dependent reactions together with a dedicated NADH/NAD⁺ recycling enzyme supports optimal growth when an increased demand for anabolic reactions taxes cellular redox balance. In turn, compartmentalization constrains the size of individual organelles, with larger peroxisomes accumulating all the required enzymes but unable to support both biosynthetic reactions at the same time. Our reengineering and physiological experiments indicate that compartmentalized biosynthetic reactions are sensitive to the size of the compartment, likely due to scaling-dependent changes within the system, such as enzyme packing density.

Most eukaryotes respire oxygen, using it to generate biomass and energy. However, a few organisms have lost the capacity to respire. Understanding how they manage biomass and energy production may illuminate the critical points at which respiration feeds into central carbon metabolism and explain possible routes to its optimization. Here, we use two related fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces japonicus, as a comparative model system. We show that although S. japonicus does not respire oxygen, unlike S. pombe, it is capable of efficient NADH oxidation, amino acid synthesis, and ATP generation. We probe possible optimization strategies through the use of stable isotope tracing metabolomics, mass isotopologue distribution analysis, genetics, and physiological experiments. S. japonicus appears to have optimized cytosolic NADH oxidation via glycerol-3-phosphate synthesis. It runs a fully bifurcated TCA pathway, sustaining amino acid production. Finally, we propose that it has optimized glycolysis to maintain high ATP/ADP ratio, in part by using the pentose phosphate pathway as a glycolytic shunt, reducing allosteric inhibition of glycolysis and supporting biomass generation. By comparing two related organisms with vastly different metabolic strategies, our work highlights the versatility and plasticity of central carbon metabolism in eukaryotes, illuminating critical adaptations supporting the preferential use of glycolysis over oxidative phosphorylation.

Cellular metabolism relies on just a few redox cofactors. Selective compartmentalization may prevent competition between metabolic reactions requiring the same cofactor. Is such compartmentalization necessary for optimal cell function? Is there an optimal compartment size? Here we probe these fundamental questions using peroxisomal compartmentalization of the last steps of lysine and histidine biosynthesis in the fission yeast Schizosaccharomyces japonicus. We show that compartmentalization of these NAD+ dependent reactions together with a dedicated NADH/NAD+ recycling enzyme supports optimal growth when an increased demand for anabolic reactions taxes cellular redox balance. In turn, compartmentalization constrains the size of individual organelles, with larger peroxisomes accumulating all the required enzymes but unable to support both biosynthetic reactions at the same time. We propose that compartmentalized biosynthetic reactions are sensitive to the size of the compartment, likely due to scaling-dependent changes within the system, such as enzyme packing density.

Most eukaryotes respire oxygen, using it to generate biomass and energy. Yet, a few organisms lost the capacity to respire. Understanding how they manage biomass and energy production may illuminate the critical points at which respiration feeds into central carbon metabolism and explain possible routes to its optimization. Here we use two related fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces japonicus, as a comparative model system. We show that although S. japonicus does not respire oxygen, unlike S. pombe, it is capable of efficient NADH oxidation, amino acid synthesis and ATP generation. We probe possible optimization strategies using stable isotope tracing metabolomics, mass isotopologue distribution analysis, genetics, and physiological experiments. S. japonicus appears to have optimized cytosolic NADH oxidation via glycerol-3-phosphate synthesis. It runs a fully bifurcated TCA cycle, supporting higher amino acid production. Finally, it uses the pentose phosphate pathway both to support faster biomass generation and as a shunt to optimize glycolytic flux, thus producing more ATP than the respiro-fermenting S. pombe. By comparing two related organisms with vastly different metabolic strategies, our work highlights the versatility and plasticity of central carbon metabolism in eukaryotes, illuminating critical adaptations supporting the preferential use of glycolysis over oxidative phosphorylation.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

Tropomyosins are structurally conserved α-helical coiled-coil dimeric proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments in non-muscle cells and are essential for calcium regulation of myosin II contractility in the muscle. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live cell imaging tools currently available in many organisms. Here, we report mNeon-Green (mNG) tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin localization and dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8), and Saccharomyces cerevisiae (Tpm1 and Tpm2), in vivo and in isolated S. pombe cell division apparatuses. We extended this approach to also visualize the mammalian TPM2 isoform. Finally, we generated a camelid-nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo without significantly influencing cell growth and dynamics of actin cytoskeleton. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring, and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and actin cables in vivo .

Cellular dimensions profoundly influence cellular physiology. For unicellular organisms, this has direct bearing on their ecology and evolution. The morphology of a cell is governed by scaling rules. As it grows, the ratio of its surface area to volume is expected to decrease. Similarly, if environmental conditions force proliferating cells to settle on different size optima, cells of the same type may exhibit size-dependent variation in cellular processes. In fungi, algae and plants where cells are surrounded by a rigid wall, division at smaller size often produces immediate changes in geometry, decreasing cell fitness. Here, we discuss how cells interpret their size, buffer against changes in shape and, if necessary, scale their polarity to maintain optimal shape at different cell volumes.

Cells of a specific cell type may divide within a certain size range. Yet, functionally optimal cellular organization is typically maintained across different cell sizes, a phenomenon known as scaling. The mechanisms underlying scaling and its physiological significance remain elusive. Here we approach this problem by interfering with scaling in the rod-shaped fission yeast Schizosaccharomyces japonicus that relies on cellular geometry cues to position the division site. We show that S. japonicus uses the Cdc42 polarity module to adjust its geometry to changes in the cell size. When scaling is prevented resulting in abnormal cellular length-to-width aspect ratio, cells exhibit severe division site placement defects. We further show that despite the generally accepted view, a similar scaling phenomenon can occur in the sister species, Schizosaccharomyces pombe. Our results demonstrate that scaling is required for normal cell function and delineate possible rules for cellular geometry maintenance in populations of proliferating cells.