Advanced Single Cell Imaging Method Using Microfluidics and FUCCI to Determine Cell Cycle Progression and Cell Fate Decisions

Abstract In the neural progenitors of the developing central nervous system (CNS), cell proliferation is tightly controlled and coordinated with cell fate decisions. Progenitors divide rapidly during early development and their cell cycle lengthens progressively as development advances to eventually give rise to a tissue of the correct size and cellular composition. However, our understanding of the molecules linking cell cycle progression to developmental time is incomplete. Here, we show that the microRNA (miRNA) let-7 accumulates in neural progenitors over time throughout the developing CNS. Intriguingly, we find that the level and activity of let-7 oscillate as neural progenitors progress through the cell cycle by in situ hybridization and fluorescent miRNA sensor analyses. We also show that let-7 mediates cell cycle dynamics: increasing the level of let-7 promotes cell cycle exit and lengthens the S/G2 phase of the cell cycle, while let-7 knock down shortens the cell cycle in neural progenitors. Together, our findings suggest that let-7 may link cell proliferation to developmental time and regulate the progressive cell cycle lengthening that occurs during development.

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Single cell transcriptomics and developmental trajectories of murine cranial neural crest cell fate determination and cell cycle progression

10.1101/2021.05.10.443503 ◽

2021 ◽

Author(s):

Yu Ji ◽

Shuwen Zhang ◽

Kurt Reynolds ◽

Ran Gu ◽

Moira McMahon ◽

...

Keyword(s):

Cell Cycle ◽

Neural Crest ◽

Single Cell ◽

Cell Fate ◽

Cell Cycle Progression ◽

Developmental Trajectories ◽

Cell Fate Determination ◽

Cycle Progression ◽

Fate Determination ◽

Neural Lineage

Cranial neural crest (NC) cells migrate long distances to populate the future craniofacial regions and give rise to various tissues, including facial cartilage, bones, connective tissues, and cranial nerves. However, the mechanism that drives the fate determination of cranial NC cells remains unclear. Using single-cell RNA sequencing combined genetic fate mapping, we reconstructed developmental trajectories of cranial NC cells, and traced their differentiation in mouse embryos. We identified four major cranial NC cell lineages at different status: pre-epithelial-mesenchymal transition, early migration, NC-derived mesenchymal cells, and neural lineage cells from embryonic days 9.5 to 12.5. During migration, the first cell fate determination separates cranial sensory ganglia, the second generates mesenchymal progenitors, and the third separates other neural lineage cells. We then focused on the early facial prominences that appear to be built by undifferentiated, fast-dividing NC cells that possess similar transcriptomic landscapes, which could be the drive for the facial developmental robustness. The post-migratory cranial NC cells exit the cell cycle around embryonic day 11.5 after facial shaping is completed and initiates further fate determination and differentiation processes. Our results demonstrate the transcriptomic landscapes during dynamic cell fate determination and cell cycle progression of cranial NC lineage cells and also suggest that the transcriptomic regulation of the balance between proliferation and differentiation of the post-migratory cranial NC cells can be a key for building up unique facial structures in vertebrates.

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Understanding cell fate control by continuous single-cell quantification

Blood ◽

10.1182/blood-2018-09-835397 ◽

2019 ◽

Vol 133 (13) ◽

pp. 1406-1414 ◽

Cited By ~ 6

Author(s):

Dirk Loeffler ◽

Timm Schroeder

Keyword(s):

Single Cell ◽

Cell Fate ◽

Cell Imaging ◽

Population Based ◽

Molecular Processes ◽

Cell Fate Decisions ◽

Single Cell Imaging

Abstract Cells and the molecular processes underlying their behavior are highly dynamic. Understanding these dynamic biological processes requires noninvasive continuous quantitative single-cell observations, instead of population-based average or single-cell snapshot analysis. Ideally, single-cell dynamics are measured long-term in vivo; however, despite progress in recent years, technical limitations still prevent such studies. On the other hand, in vitro studies have proven to be useful for answering long-standing questions. Although technically still demanding, long-term single-cell imaging and tracking in vitro have become valuable tools to elucidate dynamic molecular processes and mechanisms, especially in rare and heterogeneous populations. Here, we review how continuous quantitative single-cell imaging of hematopoietic cells has been used to solve decades-long controversies. Because aberrant cell fate decisions are at the heart of tissue degeneration and disease, we argue that studying their molecular dynamics using quantitative single-cell imaging will also improve our understanding of these processes and lead to new strategies for therapies.

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The C terminus of talin links integrins to cell cycle progression

The Journal of Cell Biology ◽

10.1083/jcb.201104128 ◽

2011 ◽

Vol 195 (3) ◽

pp. 499-513 ◽

Cited By ~ 76

Author(s):

Pengbo Wang ◽

Christoph Ballestrem ◽

Charles H. Streuli

Keyword(s):

Cell Cycle ◽

Cell Fate ◽

Focal Adhesion ◽

Intracellular Signaling ◽

Cell Cycle Progression ◽

Mammary Epithelial Cells ◽

Dependent Manner ◽

Cycle Progression ◽

Cell Fate Decisions ◽

C Terminus

Integrins are cell adhesion receptors that sense the extracellular matrix (ECM) environment. One of their functions is to regulate cell fate decisions, although the question of how integrins initiate intracellular signaling is not fully resolved. In this paper, we examine the role of talin, an adapter protein at cell–matrix attachment sites, in outside-in signaling. We used lentiviral small hairpin ribonucleic acid to deplete talin in mammary epithelial cells. These cells still attached to the ECM in an integrin-dependent manner and spread. They had a normal actin cytoskeleton, but vinculin, paxillin, focal adhesion kinase (FAK), and integrin-linked kinase were not recruited to adhesion sites. Talin-deficient cells showed proliferation defects, and reexpressing a tail portion of the talin rod, but not its head domain, restored integrin-mediated FAK phosphorylation, suppressed p21 expression, and rescued cell cycle. Thus, talin recruits and activates focal adhesion proteins required for proliferation via the C terminus of its rod domain. Our study reveals a new function for talin, which is to link integrin adhesions with cell cycle progression.

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First person – Nadine Pollak

Journal of Cell Science ◽

10.1242/jcs.259602 ◽

2021 ◽

Vol 134 (24) ◽

Keyword(s):

Cell Cycle ◽

Cell Fate ◽

Cell Biology ◽

Cell Cycle Progression ◽

Early Career ◽

First Person ◽

Apoptosis Resistance ◽

Cycle Progression ◽

Cell Fate Decisions ◽

Extrinsic Apoptosis

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Nadine Pollak is first author on ‘ Cell cycle progression and transmitotic apoptosis resistance promote escape from extrinsic apoptosis’, published in JCS. Nadine conducted the research described in this article while a postdoc at the Institute of Cell Biology and Immunology, University of Stuttgart, Germany, initially under the supervision of Prof. Peter Scheurich and subsequently in the lab of Prof. Markus Rehm, where she is now investigating the mechanisms underlying cell fate decisions in response to death stimuli throughout the cell cycle at the single-cell level.

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Coordination between cell proliferation and apoptosis after DNA damage in Drosophila

Cell Death and Differentiation ◽

10.1038/s41418-021-00898-6 ◽

2021 ◽

Author(s):

Mireya Ruiz-Losada ◽

Raul González ◽

Ana Peropadre ◽

Alejandro Gil-Gálvez ◽

Juan J. Tena ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Fate ◽

Cell Cycle Progression ◽

Genotoxic Stress ◽

Regulatory Elements ◽

Suppressor Gene ◽

Cycle Progression ◽

Cell Fate Decisions ◽

Induced Apoptosis

AbstractExposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.

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Actual time-series of single-cell transcriptomics reveals circadian clocks as key regulators of cell differentiation in Arabidopsis

10.21203/rs.3.rs-40935/v1 ◽

2020 ◽

Author(s):

Kotaro Torii ◽

Keisuke Inoue ◽

Keita Bekki ◽

Kazuya Haraguchi ◽

Minoru Kubo ◽

...

Keyword(s):

Cell Cycle ◽

Cell Differentiation ◽

Circadian Clock ◽

Single Cell ◽

Cell Fate ◽

Cell Cycle Progression ◽

Circadian Clocks ◽

Cell Fate Determination ◽

Cycle Progression ◽

Actual Time

Abstract The basis of toti/pluripotency is elaborate regulation of cell-cycle progression and cell-fate determination. Circadian clocks are involved in this process, but the underlying mechanisms have not been studied due to technical limitations. In particular, there is a lack of research on the universality of cell differentiation mechanisms in multicellular organisms using plants with high cell-fate plasticity. Here, exploiting in vivo single-cell RNA sequencing and a new actual time reconstitution method, PeakMacth, we analyzed actual time-series of cell reprogramming and differentiation processes at single-cell resolution, and found that the circadian clock modulates cell differentiation via BES1-mediated GSK3 signaling, which has a β-catenin-like function in Arabidopsis. In this pathway, the clock gene LUX in meristematic stem cells directly targets the CYCD and RETINOBLASTOMA-RELATED (RBR) genes, which are commonly involved in cell-cycle progression and cell-fate determination in plants and animals. In addition, the rhythmicity of the circadian clock was associated with cell state, and the establishment of the circadian rhythm preceded cell differentiation. Thus, our study not only reveals the involvement of the circadian clock in the differentiation of plant stem cells but also demonstrates functionally analogous features in the regulatory system of cell differentiation across species.

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