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Single-particle tracking (SPT) is a powerful technique for probing the diverse physical properties of the cytoplasm. Genetically encoded nanoparticles provide an especially convenient tool for such investigations, as they can be expressed and tracked in cells via fluorescence. Among these, 40-nm genetically encoded multimerics (GEMs) provide a unique opportunity to explore the cytoplasm. Their size corresponds to that of ribosomes and big protein complexes, allowing us to investigate the effects of the cytoplasm on the diffusivity of these objects while excluding the influence of chemical interactions during stressful events and pathological conditions. However, the effects of GEM expression levels on the measured cytoplasmic diffusivity remain largely uncharacterized in mammalian cells. To optimize the GEMs tracking and assess expression level effects, we developed a doxycycline-inducible GEM expression system and compared it with a previously reported constitutive expression system. The inducible GEM expression system reduced the number of GEM particles from 2000 to as low as 5–500 per average 2D cell cytoplasmic area, depending on doxycycline concentration and incubation time. This optimization enabled adjustment of particle density for imaging and improved homogeneity across the cell population. Moreover, we enhanced the analysis of GEM diffusivity by incorporating an effective diffusion coefficient that accounts for the type of motion and by quantifying motion heterogeneity through standard deviations of particle displacements within and between cells.
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In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density
Cells control the properties of the cytoplasm to ensure proper functioning of biochemical processes. Recent studies showed that cytoplasmic density varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here, we study how a cell cycle oscillator comprising cyclin-dependent kinase (Cdk1) responds to changes in cytoplasmic density by systematically diluting or concentrating cycling Xenopus egg extracts in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density: as low as 0.2× to more than 1.22× relative cytoplasmic density (RCD). A further dilution or concentration from these values arrested the system in a low or high steady state of Cdk1 activity, respectively. Interestingly, diluting an arrested cytoplasm of 1.22× RCD recovers oscillations at lower than 1× RCD. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of tuning, forming a hysteresis loop. We propose a mathematical model which recapitulates these observations and predicts that the Cdk1/Wee1/Cdc25 positive feedback loops do not contribute to the observed robustness, supported by experiments. Our system can be applied to study how cytoplasmic density affects other cellular processes.
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- Award ID(s):
- 1817909
- PAR ID:
- 10346555
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 6
- ISSN:
- 0027-8424
- 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/10.1073/pnas.2109547119
