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Abstract Over recent decades, studies of cell-to-cell heterogeneity have shown that meaningful variation in stress responses is an evolved trait, exemplified by bet hedging. Here, we expand upon this work by using a high-throughput single-cell transcriptomics approach inS. cerevisiaeto study cell-to-cell variation in stress responses. We measured individual transcriptional profiles of over ten thousand cells subjected to two types of stress: protein misfolding (an intrinsic stress) and nutrient deprivation (an extrinsic stress). Doing so, we saw substantial heterogeneity in responses to stress across and within different conditions. A previously defined canonical stress response is elevated in all stressful conditions, making it useful as a barometer of stress. However, it does not include the majority of the genes that are the most upregulated in response to any of the stresses we study, suggesting stress responses vary across environments. On the other hand, the genes that were downregulated with stress show a much stronger overlap across conditions. Even more striking is the cell-to-cell heterogeneity we observed in stress responses where some stressed cells exhibit neither a canonical stress response nor a transcriptional response that matches most other cells in the same vessel. Further investigation of these cell-to-cell differences revealed a misconception of population-averaged data: While transposable elements (TEs) and chaperones both appear positively correlated with stress at the population level, single-cell analysis revealed they are actually anticorrelated. In other words, some stressed cells upregulate TEs, while others upregulate chaperones. One possible mechanism for this involves chaperones, such as Hsp90, which are thought to help to silence TEs but are unable to do so effectively when these chaperones become overwhelmed during stress. In sum, we have used a powerful single-cell transcriptomics approach to quantify stress-response heterogeneity and to shed light on the mechanisms underlying cell-to-cell variation in stress responses, both of which are crucial for understanding cell evolution and disease.
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Microfluidic single-cell measurements of oxidative stress as a function of cell cycle position
Single-cell measurements routinely demonstrate high levels of variation between cells, but fewer studies provide insight into the analytical and biological sources of this variation. This is particularly true of chemical cytometry, in which individual cells are lysed and their contents separated, compared to more established single-cell measurements of the genome and transcriptome. To characterize population level variation and its sources, we analyzed oxidative stress levels in 1278 individual Dictyostelium discoideum cells as a function of exogenous stress level and cell cycle position. Cells were exposed to varying levels of oxidative stress via singlet oxygen generation using the photosensitizer Rose Bengal. Single-cell data reproduced the dose-response observed in ensemble measurements by CE-LIF, superimposed with high levels of heterogeneity. Through experiments and data analysis, we explored possible biological sources of this heterogeneity. No trend was observed between population variation and oxidative stress level, but cell cycle position was a major contributor to heterogeneity in oxidative stress. Cells synchronized to the same stage of cell division were less heterogeneous than unsynchronized cells (RSD of 37-51% vs 93%), and mitotic cells had higher levels of reactive oxygen species than interphase cells. While past research has proposed changes in cell size during the cell cycle as a source of biological noise, the measurements presented here use an internal standard to normalize for effects of cell volume, suggesting that a more complex contribution of cell cycle in heterogeneity of oxidative stress.
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- Award ID(s):
- 2126177
- PAR ID:
- 10519977
- Publisher / Repository:
- Springer
- Date Published:
- Journal Name:
- Analytical and Bioanalytical Chemistry
- Volume:
- 415
- Issue:
- 26
- ISSN:
- 1618-2642
- Page Range / eLocation ID:
- 6481 to 6490
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/s00216-023-04924-z
