Genome concentration limits cell growth and modulates proteome composition in .

Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in cells arrested for DNA replication limits total RNA polymerase activity within physiological cell sizes across tested nutrient conditions.

THE FAM53C/DYRK1A axis regulates the G1/S transition of the cell cycle.

A growing number of therapies are being developed to target the cell cycle machinery for the treatment of cancer and other human diseases. Consequently, a greater understanding of the factors regulating cell cycle progression becomes essential to help enhance the response to these new therapies. Here, using data from the Cancer Dependency Map, we identified the poorly-studied factor FAM53C as a new regulator of cell cycle progression.

Somatic polyploidy supports biosynthesis and tissue function by increasing transcriptional output.

Cell size and biosynthetic capacity generally increase with increased DNA content. Somatic polyploidy has therefore been proposed to be an adaptive strategy to increase cell size in specialized tissues with high biosynthetic demands. However, if and how DNA concentration limits cellular biosynthesis in vivo is not well understood.

The G-S transition is promoted by Rb degradation via the E3 ligase UBR5.

Mammalian cells make the decision to divide at the G-S transition in response to diverse signals impinging on the retinoblastoma protein Rb, a cell cycle inhibitor and tumor suppressor. Passage through the G-S transition is initially driven by Rb inactivation via phosphorylation and by Rb's decreasing concentration in G. While many studies have identified the mechanisms of Rb phosphorylation, the mechanism underlying Rb's decreasing concentration in G was unknown.

Whi5 hypo- and hyper-phosphorylation dynamics control cell-cycle entry and progression.

Progression through the cell cycle depends on the phosphorylation of key substrates by cyclin-dependent kinases. In budding yeast, these substrates include the transcriptional inhibitor Whi5 that regulates G1/S transition. In early G1 phase, Whi5 is hypo-phosphorylated and inhibits the Swi4/Swi6 (SBF) complex that promotes transcription of the cyclins CLN1 and CLN2.

The G1/S transition in mammalian stem cells is autonomously regulated by cell size.

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.

Somatic polyploidy supports biosynthesis and tissue function by increasing transcriptional output.

Cell size and biosynthetic capacity generally increase with increased DNA content. Polyploidy has therefore been proposed to be an adaptive strategy to increase cell size in specialized tissues with high biosynthetic demands. However, if and how DNA concentration limits cellular biosynthesis is not well understood, and the impacts of polyploidy in non-disease states is not well studied.

Whi5 hypo- and hyper-phosphorylation dynamics control cell cycle entry and progression.

Progression through the cell cycle depends on the phosphorylation of key substrates by cyclin-dependent kinases. In budding yeast, these substrates include the transcriptional inhibitor Whi5 that regulates the G1/S transition. In early G1 phase, Whi5 is hypo-phosphorylated and inhibits the SBF complex that promotes transcription of the cyclins and .

Cell Size Contributes to Single-Cell Proteome Variation.

Accurate measurements of the molecular composition of single cells will be necessary for understanding the relationship between gene expression and function in diverse cell types. One of the most important phenotypes that differs between cells is their size, which was recently shown to be an important determinant of proteome composition in populations of similarly sized cells. We, therefore, sought to test if the effects of the cell size on protein concentrations were also evident in single-cell proteomics data.

Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells.

Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how size influences cell physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be biochemically different. Here, we corroborate these results in mouse hepatocytes and extend our analysis using yeast.

The G1/S transition is promoted by Rb degradation via the E3 ligase UBR5.

Mammalian cells make the decision to divide at the G1/S transition in response to diverse signals impinging on the retinoblastoma protein Rb, a cell cycle inhibitor and tumor suppressor. Rb is inhibited by two parallel pathways. In the canonical pathway, Cyclin D-Cdk4/6 kinase complexes phosphorylate and inactivate Rb.

Evolution of cell size control is canalized towards adders or sizers by cell cycle structure and selective pressures.

Cell size is controlled to be within a specific range to support physiological function. To control their size, cells use diverse mechanisms ranging from 'sizers', in which differences in cell size are compensated for in a single cell division cycle, to 'adders', in which a constant amount of cell growth occurs in each cell cycle. This diversity raises the question why a particular cell would implement one rather than another mechanism? To address this question, we performed a series of simulations evolving cell size control networks.

Delineation of proteome changes driven by cell size and growth rate.

Increasing cell size drives changes to the proteome, which affects cell physiology. As cell size increases, some proteins become more concentrated while others are diluted. As a result, the state of the cell changes continuously with increasing size.

The cell cycle inhibitor RB is diluted in G1 and contributes to controlling cell size in the mouse liver.

Every type of cell in an animal maintains a specific size, which likely contributes to its ability to perform its physiological functions. While some cell size control mechanisms are beginning to be elucidated through studies of cultured cells, it is unclear if and how such mechanisms control cell size in an animal. For example, it was recently shown that RB, the retinoblastoma protein, was diluted by cell growth in G1 to promote size-dependence of the G1/S transition.

Increasing cell size remodels the proteome and promotes senescence.

Cell size is tightly controlled in healthy tissues, but it is unclear how deviations in cell size affect cell physiology. To address this, we measured how the cell's proteome changes with increasing cell size. Size-dependent protein concentration changes are widespread and predicted by subcellular localization, size-dependent mRNA concentrations, and protein turnover.

Eukaryotic Cell Size Control and Its Relation to Biosynthesis and Senescence.

The most fundamental feature of cellular form is size, which sets the scale of all cell biological processes. Growth, form, and function are all necessarily linked in cell biology, but we often do not understand the underlying molecular mechanisms nor their specific functions. Here, we review progress toward determining the molecular mechanisms that regulate cell size in yeast, animals, and plants, as well as progress toward understanding the function of cell size regulation.

The cargo adapter protein CLINT1 is phosphorylated by the Numb-associated kinase BIKE and mediates dengue virus infection.

The signaling pathways and cellular functions regulated by the four Numb-associated kinases are largely unknown. We reported that AAK1 and GAK control intracellular trafficking of RNA viruses and revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE have not yet been identified.

RB depletion is required for the continuous growth of tumors initiated by loss of RB.

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo.

Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size.

Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes.

G cyclin-Cdk promotes cell cycle entry through localized phosphorylation of RNA polymerase II.

Cell division is thought to be initiated by cyclin-dependent kinases (Cdks) inactivating key transcriptional inhibitors. In budding yeast, the G cyclin Cln3-Cdk1 complex is thought to directly phosphorylate the Whi5 protein, thereby releasing the transcription factor SBF and committing cells to division. We report that Whi5 is a poor substrate of Cln3-Cdk1, which instead phosphorylates the RNA polymerase II subunit Rpb1’s C-terminal domain on S of its heptapeptide repeats.

Cell-size control: Chromatin-based titration primes inhibitor dilution.

Cell growth can drive progression into the cell cycle by diluting a diverse set of cell-cycle inhibitors in yeast, animal, and plant cells. Inhibitor dilution mechanisms implement cell-size control when large and small cells inherit a similar number of inhibitor molecules, and new work shows that these mechanisms in plant cells include specific degradation and chromatin-partitioning components.