Mass measurements of polyploid lymphocytes reveal that growth is not size limited but depends strongly on cell cycle

AbstractCell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e. growth efficiency) than intermediate sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size and cell cycle dependent growth is challenging. To decouple and quantify cell size and cell cycle dependent growth effects we monitor growth efficiency of freely proliferating and cycling polyploid mouse lymphocytes with high resolution. To achieve this, we develop large-channel suspended microchannel resonators that allow us to monitor mass of single cells ranging from 40 pg (small diploid lymphocyte) to over 4000 pg, with a resolution ranging from ~1% to ~0.05%. We find that mass increases exponentially with respect to time in early cell cycle but transitions to linear dependence during late S and G2 stages. This growth behavior repeats with every endomitotic cycle as cells grow in to polyploidy. Overall, growth efficiency changes 29% due to cell cycle. In contrast, growth efficiency did not change due to cell size over a 100-fold increase in cell mass during polyploidization. Consistently, growth efficiency remained constant when cell cycle was arrested in G2. Thus, cell cycle is a primary determinant of growth efficiency and increasing cell size does not impose transport limitations that decrease growth efficiency in cultured mammalian cells.Significance statementCell size is believed to influence cell behavior through limited transport efficiency in larger cells, which could decrease the growth rate of large cells. However, this has not been experimentally investigated due to a lack of non-invasive, high-precision growth quantification methods suitable for measuring large cells. Here, we have engineered large versions of microfluidic mass sensors called suspended microchannel resonators in order to study the growth of single mammalian cells that range 100-fold in mass. This revealed that the absolute size of a cell does not impose strict transport or other limitations that would inhibit growth. In contrast to cell size, however, cell cycle has a relatively large influence on growth and our measurements allow us to decouple and quantify the growth effects caused by cell cycle and cell size.

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Mass measurements during lymphocytic leukemia cell polyploidization decouple cell cycle- and cell size-dependent growth

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922197117 ◽

2020 ◽

Vol 117 (27) ◽

pp. 15659-15665

Author(s):

Luye Mu ◽

Joon Ho Kang ◽

Selim Olcum ◽

Kristofor R. Payer ◽

Nicholas L. Calistri ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Size ◽

Single Cells ◽

Leukemia Cell ◽

Growth Efficiency ◽

Lymphocytic Leukemia ◽

Size Dependent ◽

Large Channel ◽

Dependent Growth

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2. Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.

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Simulation of Cell Cycle Dependent Growth and Metabolism in Animal Cell Fermentation

IFAC Proceedings Volumes ◽

10.1016/s1474-6670(17)45655-2 ◽

1995 ◽

Vol 28 (3) ◽

pp. 326-329

Author(s):

B. Szperalski ◽

F. Geipel ◽

U. Behrendt ◽

J. Wahl

Keyword(s):

Cell Cycle ◽

Animal Cell ◽

Dependent Growth ◽

Cycle Dependent

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Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

The Journal of Cell Biology ◽

10.1083/jcb.200104104 ◽

2001 ◽

Vol 154 (2) ◽

pp. 331-344 ◽

Cited By ~ 80

Author(s):

Daniel Kornitzer ◽

Rakefet Sharf ◽

Tamar Kleinberger

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Mammalian Cells ◽

Growth Arrest ◽

Anaphase Promoting Complex ◽

Reading Frame ◽

Cycle Growth ◽

M Phase ◽

Dependent Growth ◽

Synthetically Lethal

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)–dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4–PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

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Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.532 ◽

1987 ◽

Vol 7 (1) ◽

pp. 532-534 ◽

Cited By ~ 22

Author(s):

J M Leeds ◽

C K Mathews

Keyword(s):

Cell Cycle ◽

Mammalian Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

G1 Phase ◽

Ovary Cells ◽

Dna Labeling ◽

Metabolic Pool ◽

Specific Activities ◽

Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

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E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7268 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7268-7282 ◽

Cited By ~ 142

Author(s):

R Verona ◽

K Moberg ◽

S Estes ◽

M Starz ◽

J P Vernon ◽

...

Keyword(s):

Cell Cycle ◽

Subcellular Localization ◽

Nuclear Localization ◽

Mammalian Cells ◽

Critical Role ◽

Biological Properties ◽

Dependent Manner ◽

Restriction Point ◽

Cycle Dependent ◽

Almost All

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

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Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

10.1101/470013 ◽

2018 ◽

Cited By ~ 5

Author(s):

Evgeny Zatulovskiy ◽

Daniel F. Berenson ◽

Benjamin R. Topacio ◽

Jan M. Skotheim

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Growth ◽

Cell Size ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Inverse Correlation ◽

Cell Types ◽

Similar Amount ◽

Cell Cycle Inhibitor

Cell size is fundamental to function in different cell types across the human body because it sets the scale of organelle structures, biosynthesis, and surface transport1,2. Tiny erythrocytes squeeze through capillaries to transport oxygen, while the million-fold larger oocyte divides without growth to form the ~100 cell pre-implantation embryo. Despite the vast size range across cell types, cells of a given type are typically uniform in size likely because cells are able to accurately couple cell growth to division3–6. While some genes whose disruption in mammalian cells affects cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive7–12. Here, we show that cell growth acts to dilute the cell cycle inhibitor Rb to drive cell cycle progression from G1 to S phase in human cells. In contrast, other G1/S regulators remained at nearly constant concentration. Rb is a stable protein that is synthesized during S and G2 phases in an amount that is independent of cell size. Equal partitioning to daughter cells of chromatin bound Rb then ensures that all cells at birth inherit a similar amount of Rb protein. RB overexpression increased cell size in tissue culture and a mouse cancer model, while RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of G1 phase. Thus, Rb-dilution by cell growth in G1 provides a long-sought cell autonomous molecular mechanism for cell size homeostasis.

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Cell cycle-dependent localization of Ca2+/calmodulin-dependent protein kinase II in mammalian cells

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)55086-7 ◽

1990 ◽

Vol 52 ◽

pp. 86

Author(s):

Yasutaka Ohta ◽

Takashi Ohba ◽

Eishichi Miyamoto

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Mammalian Cells ◽

Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein ◽

Cycle Dependent

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Cell Cycle-Dependent Flagellar Disassembly in a Firebug Trypanosomatid Leptomonas pyrrhocoris

mBio ◽

10.1128/mbio.02424-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 4

Author(s):

Cynthia Y. He ◽

Adarsh Singh ◽

Vyacheslav Yurchenko

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Mammalian Cells ◽

Current Understanding ◽

Model Organisms ◽

Regulation Mechanism ◽

Length Variation ◽

Content Type ◽

Length Regulation ◽

Cycle Dependent

ABSTRACT Current understanding of flagellum/cilium length regulation focuses on a few model organisms with flagella of uniform length. Leptomonas pyrrhocoris is a monoxenous trypanosomatid parasite of firebugs. When cultivated in vitro, L. pyrrhocoris duplicates every 4.2 ± 0.2 h, representing the shortest doubling time reported for trypanosomatids so far. Each L. pyrrhocoris cell starts its cell cycle with a single flagellum. A new flagellum is assembled de novo, while the old flagellum persists throughout the cell cycle. The flagella in an asynchronous L. pyrrhocoris population exhibited a vast length variation of ∼3 to 24 μm, casting doubt on the presence of a length regulation mechanism based on a single balance point between the assembly and disassembly rate in these cells. Through imaging of live L. pyrrhocoris cells, a rapid, partial disassembly of the existing, old flagellum is observed upon, if not prior to, the initial assembly of a new flagellum. Mathematical modeling demonstrated an inverse correlation between the flagellar growth rate and flagellar length and inferred the presence of distinct, cell cycle-dependent disassembly mechanisms with different rates. On the basis of these observations, we proposed a min-max model that could account for the vast flagellar length range observed for asynchronous L. pyrrhocoris. This model may also apply to other flagellated organisms with flagellar length variation. IMPORTANCE Current understanding of flagellum biogenesis during the cell cycle in trypanosomatids is limited to a few pathogenic species, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The most notable characteristics of trypanosomatid flagella studied so far are the extreme stability and lack of ciliary disassembly/absorption during the cell cycle. This is different from cilia in Chlamydomonas and mammalian cells, which undergo complete absorption prior to cell cycle initiation. In this study, we examined flagellum duplication during the cell cycle of Leptomonas pyrrhocoris. With the shortest duplication time documented for all Trypanosomatidae and its amenability to culture on agarose gel with limited mobility, we were able to image these cells through the cell cycle. Rapid, cell cycle-specific flagellum disassembly different from turnover was observed for the first time in trypanosomatids. Given the observed length-dependent growth rate and the presence of different disassembly mechanisms, we proposed a min-max model that can account for the flagellar length variation observed in L. pyrrhocoris.

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