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Tissue confinement regulates cell growth and size in epithelia

John Devany, Martin J. Falk, Liam J. Holt, Arvind Murugan, Margaret Gardel

Preprint posted on 5 July 2022 https://www.biorxiv.org/content/10.1101/2022.07.04.498508v1

Too crowded for comfort: cells in confinement stop proliferating to maintain proper tissue architecture, but how? Devany and colleagues present a quantitative framework to address this question.

Selected by Nicolaes Hyun-Kee Min

Background

In healthy tissue, cell division is limited by spatial confinement through contact inhibition of proliferation1. Loss of contact inhibition leads to abnormal tissue architecture and can spark the initiation of solid tumors2. Mechanical stress activation of YAP by cell density3,4 and E-cadherin cell adhesions5 have been shown to be necessary for contact inhibition, but a holistic model of how tissue confinement regulates cell size and cell division to maintain proper tissue architecture is lacking.

In isolated systems, cells maintain their size by feedback regulation governing growth and division. For instance, p38 MAPK is activated in cells below a threshold size, promoting their growth. Once the cells reach their target size, CDK4 mediates their progression through the cell cycle6. Similar homeostatic regulation of cell growth and division is observed in vivo, where mouse epidermal cells use a “G1 sizer”, where they vary their G1 durations to divide at consistent volumes regardless of their birth size7. There are clearly single-cell and tissue-level regulations of cell cycle and growth. However, how these different levels of regulation interplay to drive contact inhibition in a maturing tissue is unknown.

Devany and colleagues sought to understand this interplay by utilizing an in vitro model of epithelia maturation. Epithelial cells cultured on collagen gels were observed at different stages: (1) as subconfluent colonies where they are free to grow without contact inhibition; (2) at the onset of confluence; and (3) as mature epithelium, where cell division and growth are arrested by confinement. They found that cells are smaller in the mature epithelium and that cell growth and division are uncoupled during maturation. They simulated confinement and demonstrated that confinement regulates Cyclin D1 degradation and cell growth. Using their experimental paradigm, the authors built a quantitative framework of tissue confinement as cells arrest their growth and division.

Key findings

Uncoupling of cell growth and division drives cell size reduction in maturing epithelium.

The authors first demonstrated that cell volume is reduced in tissues and mature epithelial culture compared to unconfined colonies or single cells. To determine the contributions of cell growth and division to this reduction in cell size, the authors used CellTrace, an intracellular dye that dilutes in signal as cells grow in size, to assay growth and time-lapse cell counting to measure division. They observed an uncoupling of growth and division, where growth halts abruptly at onset of confluence whereas division continues until maturation. To demonstrate that this uncoupling drives cell volume reduction in mature epithelium, they conditionally expressed cell cycle inhibitor p27, or its non-functional mutant p27ck, at onset of confluence. They observed larger cell volumes when cell cycle was inhibited by p27 expression, demonstrating that division of growth-halted cells is what likely drives cell volume reduction.

Figure 1. Model depicting how uncoupling of growth arrest and cell cycle arrest result in cell volume reduction. SC: subconfluent, OC: onset of confluence, ME: mature epithelium. Adapted from Devany et al. 2022. Figure 2J.

Tissue confinement limits cell growth, but not cell cycle length.

To quantitatively define tissue confinement, the authors developed a framework in which they modelled tissue growth as a collection of growth of the constituent single cells (hypothetical maximum exponential rate) and the observed growth rate. Tissue confinement, C, was defined as the degree of deviation of the observed tissue growth from the hypothetical maximum exponential rate, where C=0 when they are identical and C=1 when there is no observed tissue growth. They then put this framework to the test by observing cell growth and cell cycle length, using the fluorescent cell cycle stage marker FUCCI. These results again showed that as tissue confinement increases, cell growth sharply declines, but cell cycle length remains constant.

Figure 2. Mathematical and experimental framework of tissue confinement reveals uncoupling of cell growth arrest and cell cycle length.  Adapted from Devany et al. 2022. Figure 4F.

Cyclin D1 depletion mediates cell cycle arrest of confined cells in a “G1 sizer” model.

The authors then sought to determine the mechanism behind this uncoupling of cell growth and the cell cycle. First, the authors corroborated the “G1 sizer” model7 of tissue growth in confinement, where smaller cells are arrested at G1 until they reach a threshold volume to proceed into the cell cycle. The authors then performed immunofluorescence for cell cycle regulator Cyclin D1 in p27 expressing larger cells in mature epithelium, or in smaller cells with p27ck expression or delayed p27 expression. By measuring nuclear area as a proxy for cell size, the authors found a profound correlation between Cyclin D1 expression and cell size, demonstrating that in growth-halted cells, subsequent cell cycle halting may occur through Cyclin D1 degradation. To test this, the authors overexpressed cyclin D1 or degradation resistant mutant Cyclin D1 (CyDAA) at the onset of confluence. They observed further cell volume reduction to the point of near-nuclear size as well as DNA damage, indicating that in growth-halted small cells, Cyclin D1 degradation is required for cell cycle arrest. If Cyclin D1 persists, cell size decreases to the point where it’s as small as the genome which then leads to DNA damage.

Why I chose this study

I remember attending Dr. Liam Holt’s talk at the SickKids Research Institute in 2019 and being fascinated by how cancer cells adapt to increased pressure in confinement to survive and thrive. I was initially drawn to this study by Dr. Liam Holt’s co-authorship. However, as I finished reading the preprint, I decided to highlight this study because of the wide applicability and utility of the mathematical and experimental framework of tissue confinement presented here. As a demonstration, the authors used this framework to show how tissue confinement correlates with cell growth and cell cycle length in maturing epithelium. I could see it being used in different developmental and disease models. For example, it could be used to describe how regulation of cell growth and the cell cycle by tissue confinement are abrogated in solid tumor models. In my area of research of brain tumors, it could help model the confinement of brain tumor and surrounding neural tissue by the skull to describe the degrees of compression.

Questions to the authors

  • A parameter required for modeling tissue confinement is the hypothetical maximum exponential rate, which is well characterized for MDCK cells. Could this framework be applied to other contexts where this exponential rate is not so well characterized? How could it be determined in these contexts?
  • Cyclin D1 is an oncogene in multiple solid tumor types. Could Cyclin D1 overexpression-induced cell shrinking and DNA damage described in this study be an important mechanism of oncogenesis by Cyclin D1?

References

  1. McClatchey, A., & Yap, A. (2012). Contact inhibition (of proliferation) redux. Current Opinion In Cell Biology, 24(5), 685-694.
  2. Pavel, M., Renna, M., Park, S., Menzies, F., Ricketts, T., & Füllgrabe, J. et al. (2018). Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis. Nature Communications, 9(1).
  3. Pan, Y., Heemskerk, I., Ibar, C., Shraiman, B., & Irvine, K. (2016). Differential growth triggers mechanical feedback that elevates Hippo signaling. Proceedings Of The National Academy Of Sciences, 113(45).
  4. Ibar, C., Kirichenko, E., Keepers, B., Enners, E., Fleisch, K., & Irvine, K. (2018). Tension-dependent regulation of mammalian Hippo signaling through LIMD1. Journal Of Cell Science.
  5. Kim, N., Koh, E., Chen, X., & Gumbiner, B. (2011). E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components. Proceedings Of The National Academy Of Sciences108(29), 11930-11935.
  6. Tan, C., Ginzberg, M., Webster, R., Iyengar, S., Liu, S., & Papadopoli, D. et al. (2021). Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPK. Developmental Cell56(12), 1756-1769.e7. doi: 10.1016/j.devcel.2021.04.030
  7. Xie, S., & Skotheim, J. (2020). A G1 Sizer Coordinates Growth and Division in the Mouse Epidermis. Current Biology30(5), 916-924.e2. doi: 10.1016/j.cub.2019.12.062

Tags: cells, confinement, growth

Posted on: 10 August 2022

doi: https://doi.org/10.1242/prelights.32485

Read preprint (1 votes)

Authors' response

John Devany and Margaret Gardel shared

Thanks for your interest in our work and the great questions.

  • A parameter required for modeling tissue confinement is the hypothetical maximum exponential rate, which is well characterized for MDCK cells. Could this framework be applied to other contexts where this exponential rate is not so well characterized? How could it be determined in these contexts?

As you pointed out, we measure the proliferation of MDCK cells in both the single cell and tissue context to determine tissue confinement but in vivo the single cell behavior can’t be characterized. There are several possible ways to address this issue so that tissue confinement can be determined in vivo. We’ve done simulations of confined tissues and found that confinement changes the cell size distribution at steady state. Therefore, confinement could be estimated from the size distribution. However, this assumes a tissue where all cells are dividing as G1 sizers and are diploid so additional corrections or more complex models may be required for a specific tissue. Mechanically stretching the tissue may provide a means to perturb the confinement in vivo to determine the normal confinement, however this has yet to be tested in vitro using our framework. It would also be an interesting direction to explore the signaling pathways responsible for suppressing growth in response to tissue confinement. For example, we saw a relationship between confinement and YAP signaling in MDCK cells. If such a measurement can be made in different biological systems and shows a consistent correlation with confinement, then YAP activity (or a different pathway) could be used to estimate the tissue confinement in uncharacterized systems in vivo.

  • Cyclin D1 is an oncogene in multiple solid tumor types. Could Cyclin D1 overexpression-induced cell shrinking and DNA damage described in this study be an important mechanism of oncogenesis by Cyclin D1?

The connection of Cyclin D1 overexpression and DNA damage to cancer is also an interesting direction we’ve been thinking about. We couldn’t find any published cell size measurements in tumors to see if there is a correlation between Cyclin D1 overexpression and loss of cell size regulation in cancer. However, there may be a connection in small cell cancers which, qualitatively, show abnormally small cell size and high nuclear to cytoplasmic volume ratios in histology. These cancers almost always have Rb1 mutations which should cause loss of G1/S regulation similar to Cyclin D1 overexpression. It may be possible that an early step in the development of small cell cancers involves loss of cell size regulation and DNA damage causing further mutations and cell transformation.

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