Research

In our lab, we use a quantitative biology approach to answer the fundamental questions about animal cell size regulation: 

(i.e., how do changes in cell size affect cell physiology in health and disease?)

Background

While cell size homeostasis is a century-old observation, only with most recent developments in genome editing, proteomics and quantitative  live-cell imaging it is becoming possible to mechanistically answer the fundamental question of how cells sense and regulate their size. 

Phenomenologically, there are two strategies that cells use for size regulation – they can either adjust their growth rate (A) or adjust their division rate in a size-dependent manner (B). The molecular mechanisms of size-dependent growth rate adjustment are currently unknown, and we are only starting to understand how mammalian cell division is modulated by cell size (see our review: Zatulovskiy and Skotheim, Trends Genet 2020). 

The major breakthrough that Dr. Evgeny Zatulovskiy has made during his work at Stanford was discovering that cell growth in size is coupled to cell division through the size-dependent dilution of a key cell cycle inhibitor, the retinoblastoma protein RB (Zatulovskiy et al., Science 2020). That smaller cells are born with higher concentrations of RB ensures that they have more time to grow and reach the target size before sufficiently diluting RB and progressing through the cell division cycle. 

Dilution of a key cell cycle inhibitor RB couples cell size to G1/S cell cycle progression.

(A) Positive and negative regulators of the cell cycle scale differently with cell size: the amounts of cell cycle activators increase in proportion to cell size, while the amounts of cell cycle inhibitors, including RB, remain constant, therefore their concentrations decrease as cell size increases.

(B) Smaller-born cells have a higher concentration of RB, therefore, they spend more time in G1-phase of the cell cycle and grow more before sufficiently diluting RB and committing to divide (Zatulovskiy et al., Science 2020). This provides a cell-autonomous mechanism for cell size homeostasis.

This finding not only provides a long-sought molecular mechanism for cell size homeostasis, but it also has an important conceptual implication – namely, any process in the cell can be modulated by the cell size as long as its positive and negative regulators differentially scale with cell size. In a follow-up study, we identified dozens of important proteins whose concentrations change with cell size. These proteins are involved in a wide range of important cellular processes, from cytoskeleton organization and metabolism, to DNA repair and cell cycle. For example, we showed that the size-dependent changes to the proteome make larger cells more prone to senescence (Lanz* and Zatulovskiy* et al., Mol Cell 2022; Zatulovskiy* and Lanz* et al., Front Cell Dev Biol 2022). This opens several exciting avenues of research that we are pursuing now in the Zatulovskiy lab, as described below.

How do cells control their size?


We strive to understand the molecular basis of cell size regulation by determining how cell size affect the cell cycle progression and biomass production in animal cells. For this, we use an interdisciplinary approach combining a comprehensive set of quantitative single-cell and high-throughput methods.

a)  Molecular mechanisms underlying the size-dependent growth rate adjustment in somatic cells.

Cellular growth rate is determined by the balance between biosynthesis and degradation. Therefore, to ensure the slowdown of cell growth in larger cells, either the overall protein synthesis rate has to decrease or the protein degradation rate should increase with cell size. Indeed, our proteomics data indicate that the concentrations of ribosomal proteins slightly decrease with cell size, while the concentrations of proteasomal and lysosomal components increase. With chemical and genetic tools, we investigate how global protein synthesis and degradation rates change with cell size. Using proteomics, gene editing and live-cell microscopy, we aim to identify pathways that determine the differential scaling of biosynthesis and degradation machineries.

b)  Molecular mechanisms underlying cell size regulation in embryonic stem cells.

It is completely unknown how pluripotent stem cells control their size. While most somatic cells rely on size-dependent regulation of G1/S cell cycle progression to ensure smaller cells spend more time in G1 phase and grow more before committing to divide, proliferating stem cells often have an abbreviated G1 phase whose modulation would not be sufficient to control cell size. Therefore, they must rely on qualitatively different strategies. To identify these strategies, we perform live-cell imaging of stem cells expressing cell size and cell cycle reporters to determine how their growth and cell cycle progression are affected by cell size. Next, to identify the underlying molecular pathways, we fluorescently tag proteins controlling cell growth and proliferation and measure the size-dependent dynamics of those proteins to test their roles in size regulation – the approach that we successfully used in past to discover a long-sought mechanism of size-dependent cell cycle progression in somatic cells.


Key publications:

E. Zatulovskiy, S. Zhang, D.F. Berenson, B.R. Topacio, J.M. Skotheim. Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division. – 2020 – Science – 369(6502): 466-471.

E. Zatulovskiy, J.M. Skotheim. On the molecular mechanisms regulating animal cell size homeostasis. – 2020 – Trends Genet. – 36(5): 360-372.

Why do cells control their size?


To reveal the effects of cell size on cell physiology, we primarily focus on cellular contexts characterized by remarkable cell size features. One such context is cancer cells, who often have altered and highly variable cell size. By comparing normal and cancer-derived cells, we aim to determine how cell size abnormalities contribute to the key cancer characteristics – unrestricted proliferation, genomic instability, and cell motility leading to metastasis. Another interesting context is pluripotent stem cells, who are remarkably small in size. We therefore investigate how cell size affects stem cell self-renewal, potency and fate choice.

a)  Roles of cell size heterogeneity in tumour progression and drug sensitivity.

While healthy tissues employ dedicated mechanisms to keep cell size within a narrow range that optimal for cell function, pronounced cell size heterogeneity is a common characteristic for many cancers. Increased cell size variation correlates with more aggressive tumour behaviours. This raises the question of whether cell size heterogeneity can promote tumorigenesis by contributing to the key features of cancer cells – their uncontrolled proliferation and enhanced migration, which leads to metastasis. We propose that different-sized cells within the tumour are contributing to different aspects of tumour progression – i.e., smaller cells have a higher proliferative potential, while larger cells are more capable of invading into surrounding tissues. 

To investigate such connections between cell size and tumour progression, we use a CRISPR/Cas9-mediated knock-in technology to fluorescently tag the endogenous genes controlling cell division and migration. This enables us to use live-cell fluorescence microscopy for measuring real-time dynamics of key regulatory proteins in different-sized cells and correlating the concentrations of these proteins with cell proliferation and migration in n ormal and cancer cells.

b)  Effects of cell size on cellular ageing and genomic stability.

Senescence, or cellular ageing, is an irreversible cessation of cell division, which protects animals from unrestricted cell proliferation leading to cancer. Significant increase in cell size is one of the most prominent characteristics of senescent cells. It has been long believed that senescent cells become large because they continue growing while stopping to divide. However, our own data, as well as some recent publications, suggest that the opposite causality also takes place - i.e., large size makes cells more prone to senescence (Lanz* and Zatulovskiy* et al., Mol Cell 2022). This is consistent with the fact that an average cell size in many tissues increases with age. 

The underlying mechanisms connecting cell size with cellular ageing are currently unknown. We aim to mechanistically investigate the causal relationship between cell size and senescence. To do this, we measure the cell cycle dynamics and the levels of senescence markers in different-sized cells from the same culture. We then use proteomics and live-cell microscopy to identify the cell cycle regulators and senescence-associated genes whose expression changes with cell size. 

c)  Effects of cell size on stem cell fate choice and self-renewal.

Most stem cells are remarkably small in size. Existing publications suggest a correlation between the small size and the cell’s potency and proliferative potential. However, the mechanistic links connecting cell size with stem cell potency and proliferation are currently unknown. We aim to determine how cell size affects stem cell potency and fate decisions. Using live-cell microscopy of human embryonic stem cells and induced pluripotent stem cells, we investigate if larger cells are more prone to differentiation, while smaller ones are more likely to self-renew and maintain pluripotency. Next, we investigate how cell size biases cell fate choice during differentiation. To discover the underlying mechanisms, we use proteomics, gene editing, and live-cell microscopy to identify the fate-determining proteins whose concentrations change with cell size.

Effects of cell size on stem cell fate decisions.

(A) During early embryo development, stem cells make numerous decisions to divide, self-renew, or differentiate, and these decisions have to be accurately controlled.

(B) Cell-size-dependent changes in key protein concentrations can bias stem cell fate decisions – e.g., by shifting the balance between pluripotency and differentiation transcription factors.

Key publications:

M.C. Lanz*, E. Zatulovskiy*, M.P. Swaffer, L. Zhang, I. Ilerten, S. Zhang, D.S. You, G. Marinov, P. McAlpine, J.E. Elias, J.M. Skotheim. Increasing cell size remodels the proteome and promotes senescence. – 2022 – Mol Cell. 82(17): 3255-3269.e8.

E. Zatulovskiy*, M.C. Lanz*, S. Zhang, F. McCarthy, J.E. Elias, J.M. Skotheim. Delineation of proteome changes driven by cell size and growth rate. – 2022 – Front Cell Dev Biol. 10: 980721. doi: 10.3389/fcell.2022.980721.