Metabolic constraints drive self-organization of specialized cell groups
- InStem - Institute for Stem Cell Science and Regenerative Medicine, India
- National Centre for Biological Sciences-Tata Institute of Fundamental Research, India
- Manipal Academy of Higher Education, India
Figures
Figure 1 with 2 supplements
Cells within S.cerevisiae colonies exhibit ordered metabolic specialization.
(A) Low glucose is required for rugose colonies to develop. The panel shows the morphology of mature yeast colonies in rich medium, with supplemented glucose as the sole variable. Scale bar: 2 mm. (B…
Figure 1—figure supplement 1
Gluconeogenesis activity is spatially restricted.
(A) Western blot based detection of proteins involved in gluconeogenesis (Fbp1p and Pck1p), from cells grown in high (2%) and low (0.1%) conditions. The blot is representative of three biological …
Figure 1—figure supplement 2
Gluconeogenesis activity is spatially restricted.
(A) Whole colony fluorescence image of wildtype colony with the gluconeogenesis reporter. Heat map histogram analysis of the fluorescent intensity of the same colony was done using ImageJ. Values …
Figure 2 with 1 supplement
Cells organize into spatially restricted, contrary metabolic states within the colony.
(A) Comparative immediate growth of isolated light cells and dark cells, transferred to a ‘gluconeogenic medium’ (2% ethanol as carbon source), or a ‘glycolytic medium’ (2% glucose as carbon …
Figure 2—figure supplement 1
Light cells exhibit high PPP activity.
(A) Whole colony fluorescence image of wildtype colony with the pentose phosphate pathway (PPP) reporter. Heat map histogram analysis of the fluorescent intensity of the same colony was done using …
Figure 3 with 2 supplements
A mathematical model suggests constraints for the emergence and organization of cells in complimentary metabolic states.
(A) Processes, based on experimental data, incorporated into developing a simple mathematical model to simulate colony development. The dark and light cells are appropriately colored, and the …
Figure 3—figure supplement 1
Effects on the colony as we change individual parameters used in the model.
(A) Changing the diffusion constant (D [L2T−1]) of the resource in the medium. The diffusion constant used to simulate the left panel colony is 100 times smaller than the default colony (middle …
Figure 3—figure supplement 2
Reproducibility of the model, under different scenarios.
Since the colony simulations have elements of stochasticity present in the model, presented are a few replicate colonies from independent simulation. These showcase crucial and reproducible aspects …
Figure 4 with 1 supplement
Trehalose satisfies criteria to be the metabolic resource determining the emergence of light cells.
(A) A schematic illustrating the metabolic intermediates and different end-point metabolites of gluconeogenesis. (B) Extracellular amounts of trehalose measured from developing wild-type colonies. …
Figure 4—figure supplement 1
Quantitation of relative Mal11 and Nth1 protein levels in light and dark cells.
(A) Quantification of the Mal11 protein levels from light and dark cells (Figure 4C). Quantification of the western blot band intensities were done using ImageJ and the intensity values were …
Figure 5 with 1 supplement
Trehalose uptake and utilization determines the existence of light cells.
(A) Estimation of trehalose uptake and breakdown/utilization in light and dark cells. LC-MS/MS based metabolite analysis, using exogenously added 13C Trehalose, to compare breakdown and utilization …
Figure 5—figure supplement 1
Comparative breakdown of labeled trehalose by distinct cells in a colony.
(A) Trehalose breakdown by light cells and dark cells were monitored by isolating light and dark cells from a ~ 5 to 6 day old wild-type colony and incubating them briefly (30 min) with labeled 13C …
Figure 6 with 1 supplement
A resource threshold effect controls cooperative switching of cells to the light state.
(A) Simulation of colony development, based on the default model (which incorporates a resource threshold buildup, followed by consumption, switching to a light state, and expansion), compared to a …
Figure 6—figure supplement 1
Colonies generated using different switching rules.
(A) Wild-type colony with a resource threshold switching rule. In every time step, the algorithm checks if the shared resource levels at the locations of dark cells are more than a threshold value …
Figure 7
Physiological advantages of cells with organized spatial heterogeneity.
(A) Foraging response of wild-type cells (same image used in Figure 1A) and Δnth1 cells measured as a function of their ability to spread on a plate. Colony spreading was quantified by measuring the …
Author response image 1
12 hr old gluconeogenesis reporter colony (Exposure <5 sec).
Author response image 2
A simulation of colony development using the model using different concentrations of gluconeogenic cells A) 95% B) 50% C) 10% during the start of colony development.
Black regions represent dark cells and grey regions represent light cells.
Author response image 3
7 day old wild-type gluconeogenic reporter colonies (PCK1 reporter) grown in a medium containing ethanol+glycerol as the sole carbon source.
Videos
Video 1
Development of the colony.
Simulation video showing the changes in a wild-type model colony. After a small lag, dark cells at the edge start dividing into empty space and due to the threshold switching effect, the center of …
Video 2
Dark cells do not share the metabolic resource with light cells.
Simulation video showing changes in a colony where the dark cells don't share any resource for the light cells to consume. The number of light cells doesn't increase and the final colonies …
Video 3
Dark cells do not switch to light cells.
Simulation video showing changes in a colony where the dark cells don't switch to light cells but continue to produce resource on to the resource grid. In certain cases, there can be light cells at …
Video 4
Light cells share metabolic resource with dark cells (Wrong sharing).
Simulation video showing changes in a colony where the dark cells don't share any resource for the light cells but the light cells provide amino acids for the dark cells to consume (wrong sharing). …
Video 5
Development of colony without a resource threshold.
Simulation video showing changes in a colony where the dark cells switch to light by random chance (probability p=0.5). They don't need the resource levels to reach a certain threshold. Once they …
Tables
Table 1
Mass transitions used for LC-MS/MS experiments.
| Nucleotides | Formula | Parent/Product (positive polarity) | Comment (for 15N experiment) |
|---|---|---|---|
| AMP | C10H14N5O7P | 348/136 | Product has all N |
| 15N_AMP_1 | 349/137 | ||
| 15N_AMP_2 | 350/138 | ||
| 15N_AMP_3 | 351/139 | ||
| 15N_AMP_4 | 352/140 | ||
| 15N_AMP_5 | 353/141 | ||
| GMP | C10H14N5O8P | 364/152 | Product has all N |
| 15N_GMP_1 | 365/153 | ||
| 15N_GMP_2 | 366/154 | ||
| 15N_GMP_3 | 367/155 | ||
| 15N_GMP_4 | 368/156 | ||
| 15N_GMP_5 | 369/157 | ||
| CMP | C9H14N3O8P | 324/112 | Product has all N |
| 15N_CMP_1 | 325/113 | ||
| 15N_CMP_2 | 326/114 | ||
| 15N_CMP_3 | 327/115 | ||
| UMP | C9H13N2O9P | 325/113 | Product has all N |
| 15N_UMP_1 | 326/114 | ||
| 15N_UMP_2 | 327/115 | ||
| Trehalose and sugar phosphates | Formula | Parent/Product (negative polarity) | Comment (for 13C experiment) |
| Trehalose | C12H22O11 | 341.3/179.3 | |
| 13C_Trehalose_12 | 353.3/185.3 | Product has 6 C all of which are labeled | |
| G3P | C3H7O6P | 169/97 | Monitoring the phosphate release |
| 13C_G3P_3 | 172/97 | ||
| 3 PG | C3H7O7P | 185/97 | Monitoring the phosphate release |
| 13C_3 PG_3 | 188/97 | ||
| G6P | C6H13O9P | 259/97 | Monitoring the phosphate release |
| 13C_G6P_6 | 265/97 | ||
| 6 PG | C6H13O10P | 275/97 | Monitoring the phosphate release |
| 13C_6 PG_6 | 281/97 | ||
| R5P | C5H11O8P | 229/97 | Monitoring the phosphate release |
| 13C_R5P_5 | 234/97 | ||
| S7P | C7H15O10P | 289/97 | Monitoring the phosphate release |
| 13C_S7P_5 | 294/97 |
Table 2
Strains and plasmids used in this study.
| Strain/genotype | Information | Source/reference |
|---|---|---|
| Wild-type (WT) | YBC16G1, prototrophic sigma1278b, MAT a | Isolate via Fink Lab |
| WT (pPCK1-mCherry) | Wild-type strain with gluconeogenesis reporter plasmid (mCherry with PCK1 promoter) | this study |
| WT (pHXK1-mCherry) | Wild-type strain with constitutive reporter plasmid (mCherry with HXK1 promoter) | this study |
| WT (pTKL1-mCherry) | Wild-type strain with pentose phosphate pathway reporter plasmid (mCherry with TKL1 promoter) | this study |
| PCK1-flag | MAT a PCK1-3xFLAG::natNT2 | this study |
| FBP1-flag | MAT a FBP1-3xFLAG::natNT2 | this study |
| ICL1-flag | MAT a ICL1-3xFLAG::natNT2 | this study |
| MAL11-flag | MAT a MAL11-3xFLAG::natNT2 | this study |
| NTH1-flag | MAT a NTH1-3xFLAG::natNT2 | this study |
| ∆nth1 | MAT a nth1::kanMX6 | this study |
| ∆mal11 | MAT a mal11::kanMX6 | this study |
| ∆nth1 (pPCK1-mCherry) | ∆nth1 strain with gluconeogenesis reporter plasmid (mCherry with PCK1 promoter) | this study |
| ∆mal11 (pPCK1-mCherry) | ∆mal11 strain with gluconeogenesis reporter plasmid (mCherry with PCK1 promoter) | this study |
| ∆nth1 (pTKL1-mCherry) | ∆nth1 strain with pentose phosphate pathway reporter plasmid (mCherry with TKL1 promoter) | this study |
| ∆mal11 (pTKL1-mCherry) | ∆mal11 strain with pentose phosphate pathway reporter plasmid (mCherry with TKL1 promoter) | this study |
| Plasmid | Information | Source/reference |
| pPCK1-mCherry | mCherry under the PCK1 promoter and CYC1 termin- ator. p417 centromeric plasmid backbone, G418R. | this study |
| pHXK1-mCherry | mCherry under the HXK1 promoter and CYC1 termin- ator. p417 centromeric plasmid backbone, G418R. | this study |
| pTKL1-mCherry | mCherry under the TKL1 promoter and CYC1 termin- ator. p417 centromeric plasmid backbone, G418R. | this study |
Table 3
Parameters of the model for the wild-type case.
| Parameter | Notation | Value |
|---|---|---|
| Growth rate (dark cell block) | gd | 0.01/T |
| Growth rate (light cell block) | gl | 0.04/T |
| Switching threshold | S | 3.0 units |
| Resource produced by each dark cell block | R | 0.07 units/T |
| Resource or amino acids consumed per cell block (light or dark) | C | 0.05 units/T |
| Minimum resource or amino acid reserve needed for division (light or dark) | -- | 1.0 unit |
| Chance to switch to light cell if threshold reached | P | 0.5/T |
| Diffusion constant of the resource | D | 0.24 L2/T |
Additional files
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Source code 1
Simulation codes in Python.
- https://cdn.elifesciences.org/articles/46735/elife-46735-code1-v3.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/46735/elife-46735-transrepform-v3.docx
