Cytoplasmic fluidization triggers breaking spore dormancy in fission yeast

Abstract

The cytoplasm is a complex, crowded environment that influences myriad cellular processes including protein folding and metabolic reactions. Recent studies have suggested that changes in the biophysical properties of the cytoplasm play a key role in cellular homeostasis and adaptation. However, it still remains unclear how cells control their cytoplasmic properties in response to environmental cues. Here, we used fission yeast spores as a model system of dormant cells to elucidate the mechanisms underlying regulation of the cytoplasmic properties. By tracking fluorescent tracer particles, we found that particle mobility decreased in spores compared to vegetative cells, and rapidly increased at the onset of dormancy breaking upon glucose addition. This cytoplasmic fluidization depended on glucose sensing via the cAMP-PKA pathway. PKA activation led to trehalose degradation through trehalase Ntp1, thereby increasing particle mobility as the amount of trehalose decreased. In contrast, the rapid cytoplasmic fluidization did not requirede novoprotein synthesis, cytoskeletal dynamics, or cell volume increase. Furthermore, the measurement of diffusion coefficients with tracer particles of different sizes suggests that the spore cytoplasm impedes the movement of larger protein complexes (40–150 nm) such as ribosomes, while allowing free diffusion of smaller molecules (∼3 nm) such as second messengers and signaling proteins. Our experiments have thus uncovered a series of signaling events that enable cells to quickly fluidize the cytoplasm at the onset of dormancy breaking.Significance statementCellular processes are influenced by the biophysical properties of the cytoplasm such as crowding and viscoelasticity. Although it has been suggested that cells tune the cytoplasmic properties in response to environmental changes, the molecular mechanisms remain unclear. Here, we used the dormant fission yeast spores and uncovered signaling pathways that facilitate cytoplasmic fluidization during dormancy breaking. Furthermore, we tracked the mobility of intracellular tracer particles, and found that the spore cytoplasm impedes the mobility of larger protein complexes, while allowing free diffusion of smaller molecules. These results suggest that small signaling proteins can diffuse relatively freely in the spore cytoplasm and have the ability to transmit dormancy breaking signals, while the motion of large complexes, such as ribosomes, is restricted.

Live‐cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe

“…In contrast, a cAMP phosphodiesterase, Cgs2, negatively regulates the PKA activity by degrading cAMP (DeVoti et al, 1991;. The cAMP-PKA pathway plays a crucial role in divergent cellular processes in fission yeast such as stress resistance (Matsuo & Kawamukai, 2017), sexual differentiation (Maeda et al, 1994), spore germination (Hatanaka & Shimoda, 2001;Sakai et al, 2023), gluconeogenesis (Hoffman & Winston, 1991), cell size control (Kelkar & Martin, 2015;Uysal Özdemir et al, 2024), heterochromatin formation (Bao et al, 2022), and autophagy (Pérez-Díaz et al, 2023). Among these, it is well known that cAMP-PKA pathway represses the expression of Ste11, a key transcription factor for sexual differentiation, by inactivating the zinc finger transcription factor Rst2 (Otsubo & Yamamoto, 2012).…”

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeastSchizosaccharomyces pombe

“…In contrast, a cAMP phosphodiesterase, Cgs2, negatively regulates the PKA activity by degrading cAMP (DeVoti et al, 1991; Mochizuki & Yamamoto, 1992). The cAMP-PKA pathway plays a crucial role in divergent cellular processes in fission yeast such as stress resistance (Matsuo & Kawamukai, 2017), sexual differentiation (Maeda et al, 1994), spore germination (Hatanaka & Shimoda, 2001; Sakai et al, 2023), gluconeogenesis (Hoffman & Winston, 1991), cell size control (Kelkar & Martin, 2015; Uysal Özdemir et al, 2024), heterochromatin formation (Bao et al, 2022), and autophagy (Pérez-Díaz et al, 2023). Among these, it is well known that cAMP-PKA pathway represses the expression of Ste11, a key transcription factor for sexual differentiation, by inactivating the zinc finger transcription factor Rst2 (Otsubo & Yamamoto, 2012).…”

Measuring single-cell density with high throughput enables dynamic profiling of immune cell and drug response from patient samples

“…The coupling between crowding level and cell physiology makes cell density a key proxy for characterizing fundamental cellular processes such as proliferation, apoptosis, metabolic shifts, and differentiation 1,3 , indicating its potential as a biomarker for cellular fitness and drug response. Studies on single-cell organisms such as bacteria and yeast have reported that molecular crowding levels significantly change during cell state transitions between proliferation and dormancy, and density is thought to acutely reflect these transitions 5–8 . Whether such connections between density and proliferation exist in primary mammalian cells remains unclear, in part due to limitations in existing density measurement methods.…”