Stress defense and cell growth are inversely related in bulk culture analyses; however, these studies miss substantial cell-to-cell heterogeneity, thus obscuring true phenotypic relationships. Here, we devised a microfluidics system to characterize multiple phenotypes in single yeast cells over time before, during, and after salt stress. The system measured cell and colony size, growth rate, and cell-cycle phase along with nuclear trans-localization of two transcription factors: stress-activated Msn2 that regulates defense genes and Dot6 that represses ribosome biogenesis genes during an active stress response. By tracking cells dynamically, we discovered unexpected discordance between Msn2 and Dot6 behavior that revealed subpopulations of cells with distinct growth properties. Surprisingly, post-stress growth recovery was positively corelated with activation of the Dot6 repressor. In contrast, cells lacking Dot6 displayed slower growth acclimation, even though they grow normally in the absence of stress. We show that wild-type cells with a larger Dot6 response display faster production of Msn2-regulated Ctt1 protein, separable from the contribution of Msn2. These results are consistent with the model that transcriptional repression during acute stress in yeast provides a protective response, likely by redirecting translational capacity to induced transcripts.
All data generated and analyzed during this study are included in the manuscript and supporting files. Code used to analyze image files and generate data for cellular phenotypes is included in Source Code Files. Supplementary Data Files containing cells and associated phenotypic information are included. Source Data Files have been provided for Figure 4, and Figure 4 - supplement 1.
- Audrey P Gasch
- Megan N McClean
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Naama Barkai, Weizmann Institute of Science, Israel
© 2022, Bergen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.
Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated towards osteoblast lineage cells, but also expressed higher levels of osteogenic related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases.