Single-cell imaging in Saccharomyces cerevisiae uncovers a critical role for the G1/S transition activator Bck2 in cell size adaptation

Chadha, Yagya (2025): Single-cell imaging in Saccharomyces cerevisiae uncovers a critical role for the G1/S transition activator Bck2 in cell size adaptation. Dissertation, LMU München: Fakultät für Biologie

Abstract

In constant environments, cell size control maintains a constant mean cell size with a low variation in a process known as size homeostasis. In changing environments, cell size control must also adjust the mean cell size to the optimum for the new environment before implementing size homeostasis, in a process known as size adaptation. A wealth of data is available on eukaryotic size homeostasis, while comparatively little is known about size adaptation. This is partially because investigation of cell size control mechanisms requires single-cell information obtained through complex and time-intensive analysis of time-lapse microscopy data. This image analysis becomes even more complex in changing nutrient conditions as it additionally requires complete pedigrees and cell categorization. This work uses recent advances in machine-learning-assisted image analysis to characterize size adaptation to changing nutrients at the single cell level. Importantly, it reveals that in the eukaryotic unicellular fungus Saccharomyces cerevisiae (budding yeast), size regulators that appear redundant in size homeostasis serve unique functions in size adaptation. Three major regulators of size homeostasis in budding yeast are the G1/S transition activator proteins Cln3 and Bck2, and the G1/S transition inhibitor, Whi5. Surprisingly, deletions of WHI5, BCK2 or CLN3 affect cell size but do not strongly disrupt size homeostasis efficiency. This work finds that a double deletion of WHI5 and BCK2 results in a strain that is surprisingly similar to wild-type cells in cell size and does not show a loss of size homeostasis efficiency during fermentative growth. Thus, there is an unexplained redundancy in size homeostasis, both in the sense that size homeostasis is robust to deletions of major size regulators and that regulators like Bck2 and Cln3, which are synthetically lethal, serve the same function of G1/S transition activation. Given that Whi5, Cln3 and Bck2 have all previously been linked to nutrient sensing or nutrient response, this work tested if changing nutrient conditions could reveal a cost of the whi5Δbck2Δ double deletion to size homeostasis. Nutrient-switch experiments from glucose to glycerol-ethanol media revealed that immediately after a nutrient switch, the whi5Δbck2Δ strain indeed had a worse size homeostasis efficiency than wild-type cells. Moreover, size homeostasis efficiency had not been systematically studied in changing environments before and this work reported a strong but temporary increase in cell size and decrease in size homeostasis efficiency after the nutrient switch. Live cell microscopy coupled to a nutrient switch and followed by machine learning-assisted image analysis allowed tracking and categorization of individual cells as well as their progeny. This complex categorization revealed strong heterogeneous phenotypes within the cell population. All cells arrested in the ongoing cell cycle stage immediately after the switch. Cells that faced the switch in S/G2/M phase of the cell cycle, either as mother or bud, also arrested in the G1 phase of the next cell cycle, indicating that a memory of the nutrient switch persists in these cells. Strains that had a BCK2 deletion experienced longer G1 arrests and more cellular overgrowth during those arrests than strains with a CLN3 deletion, indicating a more critical role for Bck2 in exit from post-switch G1 arrests. Cln3 has previously been shown to be depleted upon a nutrient switch from rich to poor growth medium. Therefore, this work hypothesizes that a temporary nutrient-dependent depletion of Cln3 after the nutrient switch makes Bck2 the only available G1/S activator. Altogether, this work demonstrates that mimicking traits of natural environments in the laboratory can help resolve redundancies reported in steady-state studies.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Budding yeast, Cell size, BCK2, Cell cycle, G1/S transition, Nutrient switch, Microscopy, Single-cell Imaging, Image analysis
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 13. Oktober 2025
• 1. Bericht­erstatter:in: Falter-Braun, Pascal