Variations of intracellular density during the cell cycle arise from tip-growth regulation in fission yeast
- Department of Cell and Tissue Biology, University of California, San Francisco, United States
- Department of Bioengineering, Stanford University, United States
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
- MRC Laboratory for Molecular Cell Biology, University College, United Kingdom
- Department of Physics, Massachusetts Institute of Technology, United States
- Brain Science Institute, Korea Institute of Science and Technology, Republic of Korea
- Informatics Institute, University of Amsterdam, Netherlands
- Department of Biological Engineering, Massachusetts Institute of Technology, United States
- Department of Mechanical Engineering, Massachusetts Institute of Technology, United States
- Department of Microbiology and Immunology, Stanford University School of Medicine, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 1 supplement
Precise measurement of intracellular density using QPI based on a z-stack of bright-field images.
(A) QPI method for computing cytoplasmic density from bright-field images. A z-stack of bright-field images of fission yeast cells ±1.5 µm around the mid-plane focal position (top panel) were …
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Figure 1—source data 1
Source Data for Figure 1B on BSA calibration for QPI.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig1-data1-v2.xlsx
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Figure 1—source data 2
Source Data for Figure 1C on density distribution in wildtype cells.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig1-data2-v2.xlsx
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Figure 1—source data 3
Source Data for Figure 1D on density homeostasis behavior.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
Density distribution is robustly maintained.
(A) High-density features in QPI co-localize with lipid droplets. Cells were stained with BODIPY dye to identify lipid droplets. QPI density map (left) and the corresponding fluorescence image of a …
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Figure 1—figure supplement 1—source data 1
Source Data for Figure 1—figure supplement 1C on temperature effects on density.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 4 supplements
Intracellular density varies across the cell cycle.
(A) Wild-type fission yeast cells in exponential phase were imaged in time lapse in a microfluidic chamber and phase-shift maps were extracted by QPI. Shown are images of a representative cell …
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Figure 2—source data 1
Source Data for Figure 2 on density measurements during the cell cycle.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Measurements of buoyant density and buoyant mass using SMR and refractive index measurements using holography and QPI show cell-cycle-dependent density variations.
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Figure 2—figure supplement 1—source data 1
Source Data for Figure 2E and Figure 2—figure supplement 1 on buoyant density measurements.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
The mean density of S. pombe daughter cells was typically lower than the density of the mother cell.
The densities of the mother cell and the associated daughter cells were measured from consecutive QPI density maps (5 min apart) directly before and after cell division, respectively. The daughter …
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Figure 2—figure supplement 2—source data 1
Source Data for Figure 2F and Figure 2—figure supplement 2 on mother/daughter density differences.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
Cellular surface area-to-mass ratio varies less than dry-mass density during the S. pombe cell cycle.
(A) Mass increases exponentially throughout the cell cycle, as evidenced by the constant rate of normalized growth (1/M dM/dt). The dry mass of individual cells was measured as in Figure 2. Larger …
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Figure 2—figure supplement 3—source data 1
Source Data for Figure 2—figure supplement 3B on septum area calculation.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig2-figsupp3-data1-v2.xlsx
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Figure 2—figure supplement 3—source data 2
Source Data for Figure 2 and Figure 2—figure supplement 3 on volume, mass, and area relationships.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig2-figsupp3-data2-v2.xlsx
Figure 2—video 1
QPI density maps of wild-type fission yeast cells.
Wild-type fission yeast cells growing and dividing in exponential phase in rich media were imaged every 5 min in a microfluidic chamber as described in Figure 2A. Phase-shift maps were extracted by …
Figure 3
Extension of the G2 phase of the S. pombe cell cycle results in cell elongation and decreased intracellular density.
(A) Schematic of the fission yeast cell cycle, highlighting the point at which the cdc25 temperature-sensitive mutant delays the G2-M transition. (B) cdc25-22 cells were shifted from the permissive …
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Figure 3—source data 1
Source Data for Figure 3 on cdc25 mutant experiment.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig3-data1-v2.xlsx
Figure 4
Cell-cycle arrests in mitosis and cytokinesis result in increased intracellular density in S. pombe.
(A) Temperature-sensitive mutants cut7-446 (spindle kinesin-5) and cdc16-116 block the cell cycle in mitosis and cytokinesis, respectively. (B) cut7-446 cells were shifted from 25°C to 30°C to delay …
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Figure 4—source data 1
Source Data for Figure 4 on cdc7 and cdc16 mutant experiment.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Cell-cycle-independent growth inhibition by latrunculin A results in increased intracellular density in S. pombe.
(A) Latrunculin A treatment inhibited cell growth and increased intracellular density regardless of cell-cycle stage. Representative QPI density maps of three wild-type cells at different points of …
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Figure 5—source data 1
Source Data for Figure 5 on effect of latrunculin A treatment on intracellular density.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
The increase in intracellular density due to treatment with the actin inhibitor latrunculin A is not dependent on cell size.
Wild-type S. pombe cells were treated with 0.2 mM latrunculin A for 1 hr and density maps were measured using QPI. Density increase over 1 hr normalized to mean density at the first time point was …
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Figure 5—figure supplement 1—source data 1
Source Data for Figure 5—figure supplement 1 on latrunculin A effect on density of cells of different sizes.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 2 supplements
An intracellular density gradient negatively correlates with tip growth in S. pombe.
(A) Top: QPI of a representative cell displaying an intracellular gradient of density. Middle: Density was measured in slices perpendicular to the long axis. Bottom: The new end (non- or slowly …
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Figure 6—source data 1
Source Data for Figure 6 on density relationship to tip growth.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Spatial intracellular density gradients are present in cells with similar widths, and are stably maintained in cells treated with latrunculin A.
(A) The new cell end is wider than the old end. The mean width of the region 1.5–3 µm away from the cell pole was extracted for the new and old ends over time and averaged. The average old end width …
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Figure 6—figure supplement 1—source data 1
Source Data for Figure 6—figure supplement 1A on density gradient.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Total protein and RNA exhibit intracellular spatial gradients.
(A) Cells were fixed and stained with FITC, a dye that stains total protein and RNA (Materials and methods). RNAse treatment removed the RNA staining, revealing the distribution of total protein. …
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Figure 6—figure supplement 2—source data 1
Source Data for Figure 6—figure supplement 2 on FITC staining.
- https://cdn.elifesciences.org/articles/64901/elife-64901-fig6-figsupp2-data1-v2.xlsx
Figure 7 with 1 supplement
Bending of the septum reveals a link between intracellular density and osmotic pressure.
(A) Left: QPI density map of mid2∆ cells that are delayed in cell separation showed bent septa and density differences between two daughter-cell compartments. Note that the septa are bent away from …
Figure 7—figure supplement 1
Septa bend away from the compartment of higher density in mid2 and cdc16 mutant cells.
(A) QPI density maps of mid2∆ cells delayed in cell separation often exhibited differences in density between sister-cell compartments, and in most cases, the septum was bent away from the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Schizosaccharomyces pombe) | Wild-type S. pombe | Other | FC15, h- wild-type strain 972 | Figures 1, 2, 3, 4, 5 and 6; Figure 1—figure supplement 1, Figure 2—figure supplement 1, Figure 2—figure supplement 2, Figure 2—figure supplement 3, Figure 5—figure supplement 1, Figure 6—figure supplement 1 FC lab collection; https://www.uniprot.org/taxonomy/284812 |
| Genetic reagent (S. pombe) | cdc25-22 mutant | Other | FC342, h-cdc25-22 | Figure 3; FC lab collection; https://www.pombase.org/genotype/cdc25-22-C532Y-amino_acid_mutation-expression-not_assayed |
| Genetic reagent (S. pombe) | cut7-446 mutant | Other | FC1455, h-cut7-446 leu1-32 | Figure 4; FC lab collection; https://www.pombase.org/genotype/cut7-446-I954T-amino_acid_mutation-expression-knockdown |
| Genetic reagent (S. pombe) | cdc16-116 mutant | Other | FC13, h-cdc16-116 | Figure 4; FC lab collection; https://www.pombase.org/genotype/cdc16-116-unknown-unknown-expression-not_assayed |
| Genetic reagent (S. pombe) | mid2 mutant | Other | FC881 h-mid2::kanMX ade6 leu1-32 ura4-D18 | Figure 7; FC lab collection; https://www.pombase.org/genotype/mid2delta |
| Genetic reagent (S. pombe) | gpd1 mutant | Other | FC3291, h-gpd1::hphMX6ade6-M216leu1-32 ura4-D18his3-D1 | Figure 6—figure supplement 2 FC lab collection |
| Peptide, recombinant protein | Bovine Serum Albumin | Sigma Aldrich | Cat. #: A3608 | |
| Peptide, recombinant protein | Lectin | Sigma Aldrich | Cat. #: L1395 | |
| Peptide, recombinant protein | RNAse | Thermo Scientific | Cat. #: EN0531 | |
| Chemical compound, drug | Latrunculin A | Abacam | Cat. #: ab144290 | |
| Chemical compound, drug | BODIPY 493/503 | Thermo Fisher | Cat. #: D3922 | |
| Chemical compound, drug | FITC | Sigma Aldrich | Cat. #: F7250 | |
| Chemical compound, drug | OptiPrep | Sigma Aldrich | Cat. #: D1556 | |
| Software, algorithm | Matlab | Mathworks | R2019a | |
| Software, algorithm | FIJI | https://imagej.net/Fiji/Downloads | v. 1.53c | |
| Software, algorithm | Algorithm to retrieve phase information | https://bitbucket.org/kchuanglab/quantitative-phase-imaging/src/master/ Bostan et al., 2016 | ||
| Software, algorithm | Morphometrics | SimTK: Morphometrics: Project Home Ursell et al., 2017 | ||
| Other | CellASIC Onix2 microfluidic control system | Merck | Cat. #: CAX2-S0000 | |
| Other | CellASIC ONIX microfluidic plates | Merck | Cat. #: Y04C-02-5PK | |
| Other | Optical filter | Chroma Technology | Cat. #: D680/3m |
