Cell-autonomous size homeostasis in mammals

Why study cell size?

Size is one of the most fundamental parameters of a cell. Yet, we do not understand what sets a cell’s size or how they maintain that size over time.

This mechanism is especially important in cell types in our bodies that are constantly proliferating – that is, our stem cells. Both growth and division can change cell size, and must be coordinated to keep cell size stability.

And why does cell size stability matter? For a single cell, its size determines the concentration of its genome, which limits how much machinery cells have access to to grow and perform their functions. In multicellular tissues, cell size dictates how tissues are organized, how cells move, how cells touch each other, and other physical forces driving cell behaviors.

Despite this, cell size has not yet been systematically studied in multicellular tissues.

What we knew (about yeast) and didn’t know (about mammals)

Budding yeast cells coordinate their G1-to-S cell cycle transition to cell size. This way, two cells that were born at different sizes end up more similar in size as they enter S phase.

However, despite more than a decade of research in cell culture models, it was unclear whether mammalian cells coupled their cell cycle progression to their cell growth in quite the same way. Different cell lines seemed to have different cell size behaviors, and it was argued that for metazoan cells, the coordination between cell growth and cell cycle mainly occurred at the signaling level, and was not autonomous to the cell.

We knew next to nothing about cells in vivo, however. Therefore, I developed an experimental system experimental system in which I can study cell size homeostasis in an in vivo animal, using intravital imaging to directly observe how single cells grow and divide in the living mouse skin, in 4D.

The G1/S transition in vivo is sensitive to cell size

By analyzing how single cells grow in vivo, I found that cells that are born smaller spend longer in G1 phase compared to larger born cells. This longer G1 time means they are allowed to grow more compared to their larger-born cousins. In comparison, the rest of the cell cycle combined (S, G2, and M phases), was insensitive to cell size. (Xie & Skotheim, 2020)

This coordination between cell growth and the G1/S progression is very similar to what was observed in budding yeast, and contrary to the majority of cell culture models.

The G1/S transition happens at an autonomously-encoded cell size threshold

Clearly, cell size is influencing whether skin stem cells in vivo enter S phase or not at any given time. However, it was unclear how much influence cell size has on this key decision, compared to the slew of other environmental signals and changes these stem cells experience every day.

Using quantitative image analysis, I analyzed how the dividing cell’s morphology as well as the tissue microenvironment surrounding it changed over time. Then, using this rich set of information of cell and microenvrionment morphometrics, I built statistical models to isolate which feature can predict whether cells will enter the G1/S transition. (Xie et al., 2024)

Single cell size threshold predicts G1/S

My models predict G1/S transition with ~90% accuracy. Surprisingly, these models reveal that only cell volume has any significant predictive power. In fact, a simplified model using cell volume alone performed nearly as well as the full model, meaning that G1 and post-G1-phases are separable by a single number: a cell size threshold.

Perturbing the microenvironment using laser cell ablations did not change this cell size threshold in neighboring cells, despite driving faster growth rates in those neighbors. Therefore, this threshold is autonomous to the dividing cell.

G1/S cell size threshold accounts for ~65% of stem cell cycle heterogeneity

The in vivo skin stem cell cycle is highly heterogeneous. For the mouse hindpaw, it could be as short as 1 day or longer than 1 week. G1 phase is by far the most variable cell cycle phase in vivo, accounting for 96% of the total variation in cell cycle. I showed that a combination of two factors, cell size at birth (how close to the threshold a cell starts at) and cell growth rate (how fast a cell approaches the threshold), can accurately predict the total duration of G1.

Conclusion

The high heterogeneity cell cycles of adult stem cells in vivo is due mainly to cell size homeostasis mechanisms. That is to say, the cell cycle progression in vivo is stringently coupled to cell size, and variation in cell cycle length serves to maintain uniformity in cell size. Why cell size uniformity needs to be actively maintained, however, is still a mystery.

Cell growth and division must be coordinated to maintain a stable cell size, but how this coordination is implemented in multicellular tissues remains unclear. In unicellular eukaryotes, autonomous cell size control mechanisms couple cell growth and division with little extracellular input. However, in multicellular tissues we do not know if autonomous cell size control mechanisms operate the same way or whether cell growth and cell cycle progression are separately controlled by cell-extrinsic signals. Here, we address this question by tracking single epidermal stem cells growing in adult mice. We find that a cell-autonomous size control mechanism, dependent on the RB pathway, sets the timing of S phase entry based on the cell’s current size. Cell-extrinsic variations in the cellular microenvironment affect cell growth rates but not this autonomous coupling. Our work reassesses long-standing models of cell cycle regulation within complex metazoan tissues and identifies cell-autonomous size control as a critical mechanism regulating cell divisions in vivo and thereby a major contributor to stem cell heterogeneity.

Cell size homeostasis is often achieved by coupling cell-cycle progression to cell growth. Growth has been shown to drive cell-cycle progression in bacteria and yeast through "sizers," wherein cells of varying birth size divide at similar final sizes [1-3], and "adders," wherein cells increase in size a fixed amount per cell cycle [4-6]. Intermediate control phenomena are also observed, and even the same organism can exhibit different control phenomena depending on growth conditions [2, 7, 8]. Although studying unicellular organisms in laboratory conditions may give insight into their growth control in the wild, this is less apparent for studies of mammalian cells growing outside the organism. Sizers, adders, and intermediate phenomena have been observed in vitro [9-12], but it is unclear how this relates to mammalian cell proliferation in vivo. To address this question, we analyzed time-lapse images of the mouse epidermis taken over 1 week during normal tissue turnover [13]. We quantified the 3D volume growth and cell-cycle progression of single cells within the mouse skin. In dividing epidermal stem cells, we found that cell growth is coupled to division through a sizer operating largely in the G1 phase of the cell cycle. Thus, although the majority of tissue culture studies have identified adders, our analysis demonstrates that sizers are important in vivo and highlights the need to determine their underlying molecular origin.

Why study cell size?

What we knew (about yeast) and didn’t know (about mammals)

The G1/S transition in vivo is sensitive to cell size

The G1/S transition happens at an autonomously-encoded cell size threshold

G1/S cell size threshold accounts for ~65% of stem cell cycle heterogeneity

Conclusion

References

2024

2020