Intracellular diffusion in the cytoplasm increases with cell size in fission yeast

Background:

Cell size, though varying dramatically across different cell types, remains tightly regulated within a specific cell population. When this regulation breaks down, aberrant cell sizes are often associated with ageing, senescence, and various pathological conditions including cancer. However, the fundamental mechanisms by which cell size impacts cellular physiology represent a critical gap in our understanding.

The cytoplasm, far from being a simple cellular compartment, emerges as a key player in this size-dependent regulation. Acting as the matrix in which cell physiology occurs, it influences virtually all biochemical reactions through its effects on viscosity, macromolecular crowding, and phase separation. Previous studies in yeast have revealed that aberrantly sized cells exhibit various physiological defects, including decreased cytoplasmic density. Could the cytoplasm be the link between cell size and cell physiology?

One compelling hypothesis centres on the DNA-to-cytoplasm ratio. As cells grow larger without a corresponding increase in DNA content, they may lack sufficient gene copies or biosynthetic machinery to support their increased volume. This theory is supported by observations that cells with increased ploidy can achieve larger sizes without exhibiting typical size-related defects.

Despite these advances, numerous questions remain unanswered. How do changes in cell size precisely alter the biophysical properties of the cytoplasm? Which cellular components are responsible for fluctuations in cytoplasmic density? What are the molecular mechanisms that link DNA-to-cytoplasm ratio to cellular physiology? This preprint attempts to answer these questions.

Key Findings:

Nanoparticles Diffuse More in Larger Cells

By utilizing temperature-sensitive mutants (wee1-50 and cdc25-22) that significantly alter cell size, the authors demonstrated that 40 nm cytGEM nanoparticles—genetically encoded fluorescent particles designed to mimic large macromolecules—diffuse faster in larger cells and slower in smaller ones. Within each mutant strain, smaller cells exhibited reduced nanoparticle diffusion compared to their larger counterparts. Additionally, the authors employed the cdc2-asM17 strain, which can be specifically inhibited by the ATP analogue 1-NM-PP1 to arrest cells in the G2 phase and induce cell enlargement. Treatment with 1-NM-PP1 for 3 and 6 hours resulted in progressively larger cells compared to DMSO-treated controls, accompanied by increased nanoparticle diffusion. These findings reinforce the conclusion that nanoparticle diffusion and thereby possibly cytoplasmic density positively correlates with cell size.

The DNA-to-Cytoplasm Ratio is Crucial for Maintaining Nanoparticle Diffusion

To assess whether the DNA-to-cytoplasm ratio, rather than cell size alone, governs nanoparticle diffusion in the cytoplasm, the authors utilized the sid2-as and cdc11-119 mutants, which are defective in cytokinesis resulting in multinucleate cells. These mutants maintain the same DNA-to-cell length ratio as smaller, mononucleate cells. Specifically, sid2-as cells treated with the ATP analogue 1-NM-PP1 for 3 and 6 hours formed progressively larger cells with multiple nuclei. Despite the increase in cell size, the diffusion coefficients of nanoparticles in these multinucleate cells remained comparable to those in smaller, control cells. This finding indicates that maintaining the DNA-to-cytoplasm ratio is essential for regulating nanoparticle diffusion, independent of cell size.

Increased Diffusion in Larger Cells Can Be Attributed to Lower Protein Concentration

To investigate why nanoparticles diffuse more rapidly in larger cells, the authors measured protein concentrations using two probes: the Rps2-GFP marker for ribosomal proteins and FITC staining for overall protein levels. Both measurements indicated that larger cells had lower concentrations of ribosomal proteins and total proteins compared to medium-sized cells. Specifically, Rps2-GFP fluorescence was significantly reduced in cells larger than 18 µm, and FITC staining showed a modest decrease in overall protein concentration in these larger cells. These reductions in protein concentration likely decrease cytoplasmic crowding, thereby facilitating the faster diffusion of nanoparticles in larger cells.

Proteome Composition Changes with Cell Size

To investigate the molecular basis for altered nanoparticle diffusion in cells of different sizes, the authors performed SILAC-based mass spectrometry to compare the proteomes of small and large cells. They identified 3,353 proteins and found that in larger cells, proteins associated with the nucleus and ribosomes were underrepresented (sub-scaled), whereas proteins related to the endoplasmic reticulum, mitochondria, and vacuoles were overrepresented (super-scaled). Gene ontology analysis revealed that sub-scaled proteins are involved in nuclear functions and gene expression, while super-scaled proteins participate in metabolic pathways and are linked to membrane-bound organelles. Importantly, similar proteomic changes were observed in human cells, suggesting that size-dependent proteome remodelling is a conserved feature in eukaryotes. These findings demonstrate that cell size significantly influences the composition of the cytoplasmic proteome, thereby affecting the biophysical properties of the cytoplasm.

Conclusion

Collectively, these results highlight the pivotal role of cell size in modulating the biophysical environment of the cytoplasm. Larger cells exhibit increased nanoparticle diffusion primarily due to reduced protein concentrations and specific proteomic adjustments that maintain the DNA-to-cytoplasm ratio. This study underscores the intricate relationship between cell size, protein composition, and intracellular dynamics, providing valuable insights into how cell size regulation can impact overall cellular physiology and function.

What we liked about this preprint:

Leeba: At this year’s Plant and Microbial Cytoskeleton Gordon Research Conference, I met Fred Chang and was captivated by his research on how modulating cytoplasmic properties can influence cellular dynamics within S. pombe. I was also intrigued by his work on how other fungi adjust their cytoplasmic characteristics to interact with multicellular organisms. Inspired by his insights, I was excited to discover this preprint, which shows how cell length can modulate cytoplasmic properties.

Sameer: I have been actively following Fred Chang and Jan Skotheim’s groups work. I appreciate their contributions to our understanding of long-standing fundamental questions in cell scaling through the application of novel cell biology approaches. Particularly, this work contributes to the growing body of literature suggesting that DNA-to-cell size ratio governs cytoplasmic density and thereby controls cytoplasmic diffusion (this study), nuclear size in yeast (Joël Lemière et. al) and across metazoans (Biswas et.al).

Questions for the authors: 

1. Since ribosomal proteins subscales in larger fission yeast cells, can one engineer extra copies of ribosomal proteins to rescue the cytoplasmic dilution? Similarly, can overexpressing ectopic large proteins in the large cells rescue the phenotype? Conversely, would simply inhibiting protein translation lead to diluted cytoplasm in WT cells?

2. Why doesn’t the correlation between cell size and nanoparticle diffusion appear in wild-type cells, even though their cell length range is broader than that of wee1-50 mutants? Additionally, how do these results align with the observed decrease in cellular density as wild-type cells grow during the cell cycle (Odermatt et al)?

3. Have the authors confirmed that protein concentrations are similar between smaller mononucleate cells and larger multinucleate cells using Rps2-GFP or FITC probes? Alternatively, could the presence of multiple nuclei affect cytoplasmic viscosity and thereby influence diffusion measurements?

4. In Figure S2A, do DMSO-treated sid2-as cells have multiple nuclei while maintaining cell lengths comparable to wild-type cells? If not, is there a way to generate small multinucleate cells to determine if their diffusion coefficients are reduced similarly to wee1-50 mutants?

References:

• Pascal D Odermatt, Teemu P Miettinen, Joël Lemière, Joon Ho Kang, Emrah Bostan, Scott R Manalis, Kerwyn Casey Huang, Fred Chang (2021) Variations of intracellular density during the cell cycle arise from tip-growth regulation in fission yeast eLife 10:e64901
• Joël Lemière, Paula Real-Calderon, Liam J Holt, Thomas G Fai, Fred Chang (2022) Control of nuclear size by osmotic forces in Schizosaccharomyces pombe eLife 11:e76075
• Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes. Abin Biswas, Omar Muñoz, Kyoohyun Kim, Carsten Hoege, Benjamin M. Lorton, David Shechter, Jochen Guck, Vasily Zaburdaev, Simone Reber bioRxiv 2023.09.05.556409; doi: https://doi.org/10.1101/2023.09.05.556409

Tags: cell biology, cell size, diffusion, fission yeast, s.pombe

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