Computationally enhanced quantitative phase microscopy reveals autonomous oscillations in mammalian cell growth

The fine balance of growth and division is a fundamental property of the physiology of cells, and one of the least understood. Its study has been thwarted by difficulties in the accurate measurement of cell size and the even greater challenges of measuring growth of a single cell over time. We address these limitations by demonstrating a computationally enhanced methodology for quantitative phase microscopy for adherent cells, using improved image processing algorithms and automated cell-tracking software. Accuracy has been improved more than twofold and this improvement is sufficient to establish the dynamics of cell growth and adherence to simple growth laws. It is also sufficient to reveal unknown features of cell growth, previously unmeasurable. With these methodological and analytical improvements, in several cell lines we document a remarkable oscillation in growth rate, occurring throughout the cell cycle, coupled to cell division or birth yet independent of cell cycle progression. We expect that further exploration with this advanced tool will provide a better understanding of growth rate regulation in mammalian cells.

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Computationally Enhanced Quantitative Phase Microscopy Reveals Autonomous Oscillations in Mammalian Cell Growth

10.1101/631119 ◽

2019 ◽

Cited By ~ 2

Author(s):

Xili Liu ◽

Seungeun Oh ◽

Leonid Peshkin ◽

Marc W. Kirschner

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cell Growth ◽

Single Cell ◽

Cell Lines ◽

Mammalian Cells ◽

Cell Tracking ◽

Quantitative Phase ◽

Phase Microscopy ◽

Quantitative Phase Microscopy

AbstractThe fine balance of growth and division is a fundamental property of the physiology of cells and one of the least understood. Its study has been thwarted by difficulties in the accurate measurement of cell size and the even greater challenges of measuring growth of a single-cell over time. We address these limitations by demonstrating a new computationally enhanced methodology for Quantitative Phase Microscopy (ceQPM) for adherent cells, using improved image processing algorithms and automated cell tracking software. Accuracy has been improved more than two-fold and this improvement is sufficient to establish the dynamics of cell growth and adherence to simple growth laws. It is also sufficient to reveal unknown features of cell growth previously unmeasurable. With these methodological and analytical improvements, we document a remarkable oscillation in growth rate in several different cell lines, occurring throughout the cell cycle, coupled to cell division or birth, and yet independent of cell cycle progression. We expect that further exploration with this improved tool will provide a better understanding of growth rate regulation in mammalian cells.Significance StatementIt has been a long-standing question in cell growth studies that whether the mass of individual cell grows linearly or exponentially. The two models imply fundamentally distinct mechanisms, and the discrimination of the two requires great measurement accuracy. Here, we develop a new method of computationally enhanced Quantitative Phase Microscopy (ceQPM), which greatly improves the accuracy and throughput of single-cell growth measurement in adherent mammalian cells. The measurements of several cell lines indicate that the growth dynamics of individual cells cannot be explained by either of the simple models but rather present an unanticipated and remarkable oscillatory behavior, suggesting more complex regulation and feedbacks.

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Size control in mammalian cells involves modulation of both growth rate and cell cycle duration

10.1101/152728 ◽

2017 ◽

Cited By ~ 4

Author(s):

Clotilde Cadart ◽

Sylvain Monnier ◽

Jacopo Grilli ◽

Rafaele Attia ◽

Emmanuel Terriac ◽

...

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cell Division ◽

Cell Growth ◽

Single Cell ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Size Control ◽

Cycle Duration ◽

Mathematical Framework

SummaryDespite decades of research, it remains unclear how mammalian cell growth varies with cell size and across the cell division cycle to maintain size control. Answers have been limited by the difficulty of directly measuring growth at the single cell level. Here we report direct measurement of single cell volumes over complete cell division cycles. The volume added across the cell cycle was independent of cell birth size, a size homeostasis behavior called “adder”. Single-cell growth curves revealed that the homeostatic behavior relied on adaptation of G1 duration as well as growth rate modulations. We developed a general mathematical framework that characterizes size homeostasis behaviors. Applying it on datasets ranging from bacteria to mammalian cells revealed that a near-adder is the most common type of size control, but only mammalian cells achieve it using modulation of both cell growth rate and cell-cycle progression.

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Conserved Ark1-related kinases control TORC2 signaling

10.1101/870550 ◽

2019 ◽

Author(s):

Maria Alcaide-Gavilán ◽

Selene Banuelos ◽

Rafael Lucena ◽

Douglas R. Kellogg

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Plasma Membrane ◽

Cell Growth ◽

Cell Size ◽

Cell Cycle Progression ◽

Coordinated Control ◽

Cycle Progression ◽

Membrane Growth ◽

Slow Growing

AbstractIn all orders of life, cell cycle progression is dependent upon cell growth, and the extent of growth required for cell cycle progression is proportional to growth rate. Thus, cells growing rapidly in rich nutrients are substantially larger than slow growing cells. In budding yeast, a conserved signaling network surrounding Tor complex 2 (TORC2) controls growth rate and cell size in response to nutrient availability. Here, a search for new components of the TORC2 network identified a pair of redundant kinase paralogs called Ark1 and Prk1. Previous studies found that Ark/Prk play roles in endocytosis. Here, we show that Ark/Prk are embedded in the TORC2 network, where they appear to influence TORC2 signaling independently of their roles in endocytosis. We also show that reduced endocytosis leads to increased cell size, which indicates that cell size homeostasis requires coordinated control of plasma membrane growth and endocytosis. The discovery that Ark/Prk are embedded in the TORC2 network suggests a model in which TORC2-dependent signals control both plasma membrane growth and endocytosis, which would ensure that the rates of each process are matched to each other and to the availability of nutrients so that cells achieve and maintain an appropriate size.

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A conserved Lkb1 signaling axis modulates TORC2 signaling in budding yeast

10.1101/293209 ◽

2018 ◽

Author(s):

Maria Alcaide-Gavilán ◽

Rafael Lucena ◽

Katherine Schubert ◽

Karen Artiles ◽

Jessica Zapata ◽

...

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cell Growth ◽

Carbon Source ◽

Cell Size ◽

Nutrient Availability ◽

Cell Cycle Progression ◽

Budding Yeast ◽

Small Cells ◽

Cycle Progression

ABSTRACTNutrient availability, growth rate and cell size are closely linked. For example, in budding yeast, the rate of cell growth is proportional to nutrient availability, cell size is proportional to growth rate, and growth rate is proportional to cell size. Thus, cells grow slowly in poor nutrients and are nearly half the size of cells growing in rich nutrients. Moreover, large cells grow faster than small cells. A signaling network that surrounds Tor kinase complex 2 (TORC2) plays an important role in enforcing these proportional relationships. Cells that lack components of the TORC2 network fail to modulate their growth rate or size in response to changes in nutrient availability. Here, we show that budding yeast homologs of the Lkb1 tumor suppressor kinase are required for normal modulation of TORC2 signaling and in response to changes in carbon source. Lkb1 kinases activate Snf1/AMPK to initiate transcription of genes required for utilization of poor carbon sources. However, Lkb1 influences TORC2 signaling via a novel pathway that is independent of Snf1/AMPK. Of the three Lkb1 homologs in budding yeast, Elm1 plays the most important role in modulating TORC2. Elm1 activates a pair of related kinases called Gin4 and Hsl1. Previous work found that loss of Gin4 and Hsl1 causes cells to undergo unrestrained growth during a prolonged mitotic arrest, which suggests that play a role in linking cell cycle progression to cell growth. We found that Gin4 and Hsl1 also control the TORC2 network. In addition, Gin4 and Hsl1 are themselves influenced by signals from the TORC2 network, consistent with previous work showing that the TORC2 network constitutes a feedback loop. Together, the data suggest a model in which the TORC2 network sets growth rate in response to carbon source, while also relaying signals via Gin4 and Hsl1 that set the critical amount of growth required for cell cycle progression. This kind of close linkage between control of cell growth and size would suggest a simple mechanistic explanation for the proportional relationship between cell size and growth rate.

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A G1 sizer mechanism coordinates growth and division in the mouse epidermis

10.1101/754424 ◽

2019 ◽

Cited By ~ 1

Author(s):

Shicong Xie ◽

Jan M. Skotheim

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Size ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Single Cells ◽

Mouse Skin ◽

Mouse Epidermis ◽

Cycle Progression

SummaryCell size homeostasis is often achieved by coupling cell cycle progression to cell growth. Studies of cell size homeostasis in single-celled bacteria and yeast have observed several distinct phenomena. Growth can be coupled to division through a range of mechanisms, including a ‘sizer’, wherein cells of varying birth size divide at similar final sizes [1–3], and an ‘adder’, wherein cells increase in size a fixed amount per cell cycle [4–6]. Importantly, intermediate control mechanisms are observed, and even the same organism can exhibit distinct control phenomena depending on growth conditions [2,7,8]. While 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. Sizer, adder, and intermediate mechanisms have been observed in vitro [9–12], but it is unclear how these diverse size homeostasis phenomena relate to mammalian cell proliferation in vivo. To address this gap, we analyzed time-lapse images of the mouse epidermis taken over one 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 mechanism operating largely in the G1 phase. Thus, while the majority of tissue culture studies to-date identified adder mechanisms, our analysis demonstrates that sizer mechanisms are important in vivo and highlights the need to determine their underlying molecular origin.

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Optical imaging of cell mass and growth dynamics

AJP Cell Physiology ◽

10.1152/ajpcell.00121.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. C538-C544 ◽

Cited By ~ 288

Author(s):

Gabriel Popescu ◽

YoungKeun Park ◽

Niyom Lue ◽

Catherine Best-Popescu ◽

Lauren Deflores ◽

...

Keyword(s):

Cell Growth ◽

Cell Model ◽

Cell Mass ◽

Dry Mass ◽

Living Cells ◽

Specific Information ◽

Surface Integral ◽

Quantitative Phase ◽

Phase Microscopy ◽

Quantitative Phase Microscopy

Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or “dry mass” of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells.

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Quantitative phase microscopy for visualization, quantification and analysis oflive cells

The Review of Laser Engineering ◽

10.2184/lsj.36.218 ◽

2008 ◽

Vol 36 (Supplement) ◽

pp. 218-219

Author(s):

Takahiro Ikeda

Keyword(s):

Quantitative Phase ◽

Phase Microscopy ◽

Quantitative Phase Microscopy

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Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)

Pharmaceutics ◽

10.3390/pharmaceutics13050590 ◽

2021 ◽

Vol 13 (5) ◽

pp. 590

Author(s):

Jennifer Cauzzo ◽

Nikhil Jayakumar ◽

Balpreet Singh Ahluwalia ◽

Azeem Ahmad ◽

Nataša Škalko-Basnet

Keyword(s):

Rapid Development ◽

Fluorescent Labeling ◽

Label Free ◽

Batch Mode ◽

Full Field ◽

Quantitative Phase ◽

Phase Microscopy ◽

Quantitative Phase Microscopy ◽

Tracking Ability

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems.

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Oncogenic role of ALX3 in cervical cancer cells through KDM2B-mediated histone demethylation of CDC25A

BMC Cancer ◽

10.1186/s12885-021-08552-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jinhong Qi ◽

Li Zhou ◽

Dongqing Li ◽

Jingyuan Yang ◽

He Wang ◽

...

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Cell Growth ◽

Cancer Cells ◽

Cell Cycle Progression ◽

Cervical Squamous Cell Carcinoma ◽

Cycle Progression ◽

Normal Tissues ◽

Positive Regulator ◽

Cervical Cancer Cells

Abstract Background Cell division cycle 25A (CDC25A) is a well-recognized regulator of cell cycle progression and is involved in cancer development. This work focused on the function of CDC25A in cervical cancer cell growth and the molecules involved. Methods A GEO dataset GSE63514 comprising data of cervical squamous cell carcinoma (CSCC) tissues was used to screen the aberrantly expressed genes in cervical cancer. The CDC25A expression in cancer and normal tissues was predicted in the GEPIA database and that in CSCC and normal cells was determined by RT-qPCR and western blot assays. Downregulation of CDC25A was introduced in CSCC cells to explore its function in cell growth and the cell cycle progression. The potential regulators of CDC25A activity and the possible involved signaling were explored. Results CDC25A was predicted to be overexpressed in CSCC, and high expression of CDC25A was observed in CSCC cells. Downregulation of CDC25A in ME180 and C33A cells reduced cell proliferation and blocked cell cycle progression, and it increased cell apoptosis. ALX3 was a positive regulator of CDC25A through transcription promotion. It recruited a histone demethylase, lysine demethylase 2B (KDM2B), to the CDC25A promoter, which enhanced CDC25A expression through demethylation of H3k4me3. Overexpression of ALX3 in cells blocked the inhibitory effects of CDC25A silencing. CDC25A was found as a positive regulator of the PI3K/Akt signaling pathway. Conclusion This study suggested that the ALX3 increased CDC25A expression through KDM2B-mediated demethylation of H3K4me3, which induced proliferation and cell cycle progression of cervical cancer cells.

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