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On the origins of the mitotic shift in proliferating cell layers.

Gibson WT, Rubinstein BY, Meyer EJ, Veldhuis JH, Brodland GW, Nagpal R, Gibson MC - Theor Biol Med Model (2014)

Bottom Line: Using both mathematical and experimental approaches, we show that the mitotic shift derives primarily from passive, non-autonomous effects of mitoses in neighboring cells on each cell's geometry over the course of the cell cycle.Taken together, our results strongly suggest that the mitotic shift reflects a time-dependent accumulation of shared cellular interfaces over the course of the cell cycle.These results uncover fundamental constraints on the relationship between cell shape and cell division that should be general in adherent, polarized cell layers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stowers Institute for Medical Research, 64110 Kansas City, MO, USA. mg2@stowers.org.

ABSTRACT

Background: During plant and animal development, monolayer cell sheets display a stereotyped distribution of polygonal cell shapes. In interphase cells these shapes range from quadrilaterals to decagons, with a robust average of six sides per cell. In contrast, the subset of cells in mitosis exhibits a distinct distribution with an average of seven sides. It remains unclear whether this 'mitotic shift' reflects a causal relationship between increased polygonal sidedness and increased division likelihood, or alternatively, a passive effect of local proliferation on cell shape.

Methods: We use a combination of probabilistic analysis and mathematical modeling to predict the geometry of mitotic polygonal cells in a proliferating cell layer. To test these predictions experimentally, we use Flp-Out stochastic labeling in the Drosophila wing disc to induce single cell clones, and confocal imaging to quantify the polygonal topologies of these clones as a function of cellular age. For a more generic test in an idealized cell layer, we model epithelial sheet proliferation in a finite element framework, which yields a computationally robust, emergent prediction of the mitotic cell shape distribution.

Results: Using both mathematical and experimental approaches, we show that the mitotic shift derives primarily from passive, non-autonomous effects of mitoses in neighboring cells on each cell's geometry over the course of the cell cycle. Computationally, we predict that interphase cells should passively gain sides over time, such that cells at more advanced stages of the cell cycle will tend to have a larger number of neighbors than those at earlier stages. Validating this prediction, experimental analysis of randomly labeled epithelial cells in the Drosophila wing disc demonstrates that labeled cells exhibit an age-dependent increase in polygonal sidedness. Reinforcing these data, finite element simulations of epithelial sheet proliferation demonstrate in a generic framework that passive side-gaining is sufficient to generate a mitotic shift.

Conclusions: Taken together, our results strongly suggest that the mitotic shift reflects a time-dependent accumulation of shared cellular interfaces over the course of the cell cycle. These results uncover fundamental constraints on the relationship between cell shape and cell division that should be general in adherent, polarized cell layers.

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Related in: MedlinePlus

Statistical summary of the aged cell analysis based on Flp-out clone induction. (A-B) Induction of Flp-Out clones in the Drosophila wing disc epithelium produces cell populations marked with GFP (green). Discs Large (DLG; red) marks the septate junctions. (A) Sub-panels i-vi show examples of single cell clones (SCC’s). White stars mark neighboring cells; the labeled cell’s polygonal topology is designated in white. (B) Sub-panels i-vi show examples of two-cell clones (TCC’s). White stars mark neighboring cells; labeled cells’ polygonal topologies are designated in white. (C) The average mitotic cell has approximately seven sides [24], whereas the average non-mitotic cell has approximately six sides [12]. These differences are significant (p < 10-106; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the empirical overall and mitotic distributions are given under the heading “Sample sizes for overall and mitotic cell shape distributions” in the Methods section. (D) The average single-cell clone has approximately 6.66 sides, whereas the average two-cell clone has approximately 6.09 sides. These differences are significant (p < 10-8; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the SCC and TCC distributions are given under the heading “Sample sizes for single cell clone (SCC) analysis and two cell clone (TCC) analysis” in the Methods section. (E) The mitotic cell shape distribution (black) is approximately an integer shift of the overall cell shape distribution (grey). (F) The single cell clone (SCC) distribution (black) is shifted relative to the two-cell clone (TCC) distribution (grey). Panels (E) and (F) display the same data as panels (C) and (D), respectively.
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Figure 4: Statistical summary of the aged cell analysis based on Flp-out clone induction. (A-B) Induction of Flp-Out clones in the Drosophila wing disc epithelium produces cell populations marked with GFP (green). Discs Large (DLG; red) marks the septate junctions. (A) Sub-panels i-vi show examples of single cell clones (SCC’s). White stars mark neighboring cells; the labeled cell’s polygonal topology is designated in white. (B) Sub-panels i-vi show examples of two-cell clones (TCC’s). White stars mark neighboring cells; labeled cells’ polygonal topologies are designated in white. (C) The average mitotic cell has approximately seven sides [24], whereas the average non-mitotic cell has approximately six sides [12]. These differences are significant (p < 10-106; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the empirical overall and mitotic distributions are given under the heading “Sample sizes for overall and mitotic cell shape distributions” in the Methods section. (D) The average single-cell clone has approximately 6.66 sides, whereas the average two-cell clone has approximately 6.09 sides. These differences are significant (p < 10-8; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the SCC and TCC distributions are given under the heading “Sample sizes for single cell clone (SCC) analysis and two cell clone (TCC) analysis” in the Methods section. (E) The mitotic cell shape distribution (black) is approximately an integer shift of the overall cell shape distribution (grey). (F) The single cell clone (SCC) distribution (black) is shifted relative to the two-cell clone (TCC) distribution (grey). Panels (E) and (F) display the same data as panels (C) and (D), respectively.

Mentions: Given that the mitotic shift in Drosophila (and in other organisms) implies a positive correlation between polygon class and cell cycle state, a natural question is whether this relationship is actually causal. Indeed, it is currently debated whether the mitotic shift merely reflects a time-dependent correlation of two independent processes (cell cycle state and passive side-gaining due to neighboring divisions), or whether polygon class may in some way participate in the active induction of cell division [28,34]. To test whether passive side gaining is sufficient to generate the mitotic shift, we directly measured the polygon class in “aged” Drosophila wing disc cells that did not undergo cell division during a twelve-hour time window, and compared this distribution to that observed in cells that underwent a single mitotic event. We stochastically labeled third-instar wing disc epithelial cells with GFP using the FLP-OUT system [37], and permitted the marked cells to grow in vivo for 12 hours, the approximate duration of the cell cycle in the wing disc [38-40]. We focused our analysis on two sub-populations of mitotic clones: single cell clones (SCC’s; Figure 4A, i-vi) and two-cell clones (TCC’s; Figure 4B, i-vi). SCC’s derive from cells that were GFP labeled during Flp-Out induction, but did not undergo mitosis prior to imaging. To control for the possibility that a subset of the SCC’s derived instead from cell sorting, we discarded SCC’s within two cell diameters of each other (this is a conservative approach, as separated cells are almost never observed at the boundaries of even very large clones in this tissue). We quantified the polygonal topologies of the SCC’s using confocal microscopy and a fluorescently labeled antibody against the septate junction-associated protein Discs Large (Figure 4A-B). Previous studies in diverse organisms have shown both experimentally and mathematically that the average polygonal cell must have exactly six sides in a planar tissue (see Figure 4C; interphase cells) [12,20,28,32,41]. By contrast, the population of SCC’s in our sample had on average 6.66 sides 12 hours after clone induction (Figure 4D; single cell clones). For comparison, the experimentally measured average polygonal topology of two cell clones was 6.09 (p < 10-8 ; t-test2 in Matlab; Figure 4D). We conclude that increased cellular age correlates with increased polygonal sidedness in vivo, thus demonstrating that cells experience a net gain in their total number of cell-cell contacts over time.


On the origins of the mitotic shift in proliferating cell layers.

Gibson WT, Rubinstein BY, Meyer EJ, Veldhuis JH, Brodland GW, Nagpal R, Gibson MC - Theor Biol Med Model (2014)

Statistical summary of the aged cell analysis based on Flp-out clone induction. (A-B) Induction of Flp-Out clones in the Drosophila wing disc epithelium produces cell populations marked with GFP (green). Discs Large (DLG; red) marks the septate junctions. (A) Sub-panels i-vi show examples of single cell clones (SCC’s). White stars mark neighboring cells; the labeled cell’s polygonal topology is designated in white. (B) Sub-panels i-vi show examples of two-cell clones (TCC’s). White stars mark neighboring cells; labeled cells’ polygonal topologies are designated in white. (C) The average mitotic cell has approximately seven sides [24], whereas the average non-mitotic cell has approximately six sides [12]. These differences are significant (p < 10-106; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the empirical overall and mitotic distributions are given under the heading “Sample sizes for overall and mitotic cell shape distributions” in the Methods section. (D) The average single-cell clone has approximately 6.66 sides, whereas the average two-cell clone has approximately 6.09 sides. These differences are significant (p < 10-8; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the SCC and TCC distributions are given under the heading “Sample sizes for single cell clone (SCC) analysis and two cell clone (TCC) analysis” in the Methods section. (E) The mitotic cell shape distribution (black) is approximately an integer shift of the overall cell shape distribution (grey). (F) The single cell clone (SCC) distribution (black) is shifted relative to the two-cell clone (TCC) distribution (grey). Panels (E) and (F) display the same data as panels (C) and (D), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4048254&req=5

Figure 4: Statistical summary of the aged cell analysis based on Flp-out clone induction. (A-B) Induction of Flp-Out clones in the Drosophila wing disc epithelium produces cell populations marked with GFP (green). Discs Large (DLG; red) marks the septate junctions. (A) Sub-panels i-vi show examples of single cell clones (SCC’s). White stars mark neighboring cells; the labeled cell’s polygonal topology is designated in white. (B) Sub-panels i-vi show examples of two-cell clones (TCC’s). White stars mark neighboring cells; labeled cells’ polygonal topologies are designated in white. (C) The average mitotic cell has approximately seven sides [24], whereas the average non-mitotic cell has approximately six sides [12]. These differences are significant (p < 10-106; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the empirical overall and mitotic distributions are given under the heading “Sample sizes for overall and mitotic cell shape distributions” in the Methods section. (D) The average single-cell clone has approximately 6.66 sides, whereas the average two-cell clone has approximately 6.09 sides. These differences are significant (p < 10-8; ttest2 in Matlab). Stars represent statistical significance. Sample sizes for the SCC and TCC distributions are given under the heading “Sample sizes for single cell clone (SCC) analysis and two cell clone (TCC) analysis” in the Methods section. (E) The mitotic cell shape distribution (black) is approximately an integer shift of the overall cell shape distribution (grey). (F) The single cell clone (SCC) distribution (black) is shifted relative to the two-cell clone (TCC) distribution (grey). Panels (E) and (F) display the same data as panels (C) and (D), respectively.
Mentions: Given that the mitotic shift in Drosophila (and in other organisms) implies a positive correlation between polygon class and cell cycle state, a natural question is whether this relationship is actually causal. Indeed, it is currently debated whether the mitotic shift merely reflects a time-dependent correlation of two independent processes (cell cycle state and passive side-gaining due to neighboring divisions), or whether polygon class may in some way participate in the active induction of cell division [28,34]. To test whether passive side gaining is sufficient to generate the mitotic shift, we directly measured the polygon class in “aged” Drosophila wing disc cells that did not undergo cell division during a twelve-hour time window, and compared this distribution to that observed in cells that underwent a single mitotic event. We stochastically labeled third-instar wing disc epithelial cells with GFP using the FLP-OUT system [37], and permitted the marked cells to grow in vivo for 12 hours, the approximate duration of the cell cycle in the wing disc [38-40]. We focused our analysis on two sub-populations of mitotic clones: single cell clones (SCC’s; Figure 4A, i-vi) and two-cell clones (TCC’s; Figure 4B, i-vi). SCC’s derive from cells that were GFP labeled during Flp-Out induction, but did not undergo mitosis prior to imaging. To control for the possibility that a subset of the SCC’s derived instead from cell sorting, we discarded SCC’s within two cell diameters of each other (this is a conservative approach, as separated cells are almost never observed at the boundaries of even very large clones in this tissue). We quantified the polygonal topologies of the SCC’s using confocal microscopy and a fluorescently labeled antibody against the septate junction-associated protein Discs Large (Figure 4A-B). Previous studies in diverse organisms have shown both experimentally and mathematically that the average polygonal cell must have exactly six sides in a planar tissue (see Figure 4C; interphase cells) [12,20,28,32,41]. By contrast, the population of SCC’s in our sample had on average 6.66 sides 12 hours after clone induction (Figure 4D; single cell clones). For comparison, the experimentally measured average polygonal topology of two cell clones was 6.09 (p < 10-8 ; t-test2 in Matlab; Figure 4D). We conclude that increased cellular age correlates with increased polygonal sidedness in vivo, thus demonstrating that cells experience a net gain in their total number of cell-cell contacts over time.

Bottom Line: Using both mathematical and experimental approaches, we show that the mitotic shift derives primarily from passive, non-autonomous effects of mitoses in neighboring cells on each cell's geometry over the course of the cell cycle.Taken together, our results strongly suggest that the mitotic shift reflects a time-dependent accumulation of shared cellular interfaces over the course of the cell cycle.These results uncover fundamental constraints on the relationship between cell shape and cell division that should be general in adherent, polarized cell layers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stowers Institute for Medical Research, 64110 Kansas City, MO, USA. mg2@stowers.org.

ABSTRACT

Background: During plant and animal development, monolayer cell sheets display a stereotyped distribution of polygonal cell shapes. In interphase cells these shapes range from quadrilaterals to decagons, with a robust average of six sides per cell. In contrast, the subset of cells in mitosis exhibits a distinct distribution with an average of seven sides. It remains unclear whether this 'mitotic shift' reflects a causal relationship between increased polygonal sidedness and increased division likelihood, or alternatively, a passive effect of local proliferation on cell shape.

Methods: We use a combination of probabilistic analysis and mathematical modeling to predict the geometry of mitotic polygonal cells in a proliferating cell layer. To test these predictions experimentally, we use Flp-Out stochastic labeling in the Drosophila wing disc to induce single cell clones, and confocal imaging to quantify the polygonal topologies of these clones as a function of cellular age. For a more generic test in an idealized cell layer, we model epithelial sheet proliferation in a finite element framework, which yields a computationally robust, emergent prediction of the mitotic cell shape distribution.

Results: Using both mathematical and experimental approaches, we show that the mitotic shift derives primarily from passive, non-autonomous effects of mitoses in neighboring cells on each cell's geometry over the course of the cell cycle. Computationally, we predict that interphase cells should passively gain sides over time, such that cells at more advanced stages of the cell cycle will tend to have a larger number of neighbors than those at earlier stages. Validating this prediction, experimental analysis of randomly labeled epithelial cells in the Drosophila wing disc demonstrates that labeled cells exhibit an age-dependent increase in polygonal sidedness. Reinforcing these data, finite element simulations of epithelial sheet proliferation demonstrate in a generic framework that passive side-gaining is sufficient to generate a mitotic shift.

Conclusions: Taken together, our results strongly suggest that the mitotic shift reflects a time-dependent accumulation of shared cellular interfaces over the course of the cell cycle. These results uncover fundamental constraints on the relationship between cell shape and cell division that should be general in adherent, polarized cell layers.

Show MeSH
Related in: MedlinePlus