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Article ; Online: Artificial Modulation and Rewiring of Cell Cycle Progression Using Synthetic Circuits in Fission Yeast.

Jain, Akanksha / Wu, Pei-Yun Jenny / Coudreuse, Damien

Methods in molecular biology (Clifton, N.J.)

2024 Volume 2740, Page(s) 89–105

Abstract: Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms ... ...

Abstract	Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms that sustain cell proliferation and the ways in which they interact with other cellular pathways. In this context, the use of a synthetic approach to simplify the cell cycle network in unicellular genetic models such as fission yeast has opened the door to studying the biology of proliferating cells from unique perspectives. Here, we provide a series of methods based on a minimal cell cycle module in the fission yeast Schizosaccharomyces pombe that allows for an unprecedented artificial control of cell cycle events, enabling the rewiring and remodeling of cell cycle progression.
MeSH term(s)	Schizosaccharomyces/metabolism ; Cell Cycle ; Cell Division ; Cell Cycle Proteins/metabolism ; Schizosaccharomyces pombe Proteins/metabolism
Chemical Substances	Cell Cycle Proteins ; Schizosaccharomyces pombe Proteins
Language	English
Publishing date	2024-02-23
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3557-5_5
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Live-cell imaging defines a threshold in CDK activity at the G2/M transition.

Sugiyama, Hironori / Goto, Yuhei / Kondo, Yohei / Coudreuse, Damien / Aoki, Kazuhiro

Developmental cell

2024 Volume 59, Issue 4, Page(s) 545–557.e4

Abstract: Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how ... ...

Abstract	Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.
MeSH term(s)	Animals ; Phosphorylation ; Schizosaccharomyces/genetics ; Schizosaccharomyces/metabolism ; Mitosis ; Cyclin-Dependent Kinases/genetics ; Cyclin-Dependent Kinases/metabolism ; Cell Cycle Proteins/genetics ; Cell Cycle Proteins/metabolism ; Mammals/metabolism ; Protein-Tyrosine Kinases/genetics ; Schizosaccharomyces pombe Proteins/genetics ; Schizosaccharomyces pombe Proteins/metabolism
Chemical Substances	Cyclin-Dependent Kinases (EC 2.7.11.22) ; Cell Cycle Proteins ; wee1 protein, S pombe (EC 2.7.1.-) ; Protein-Tyrosine Kinases (EC 2.7.10.1) ; Schizosaccharomyces pombe Proteins
Language	English
Publishing date	2024-01-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	2054967-2
ISSN	1878-1551 ; 1534-5807
ISSN (online)	1878-1551
ISSN	1534-5807
DOI	10.1016/j.devcel.2023.12.014
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Article ; Online: Insights from synthetic yeasts.

Coudreuse, Damien

Yeast (Chichester, England)

2016 Volume 33, Issue 9, Page(s) 483–492

Abstract: Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building ...

Abstract	Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building artificial genetic circuits and studying their behaviours has rapidly demonstrated its potential in a broad range of fields in the life sciences. From the assembly of synthetic genomes to the generation of novel biological functions, yeast cells have imposed themselves as the most powerful eukaryotic model for this approach. However, we are only beginning to explore the possibilities of synthetic biology, and the perspectives it offers in a genetically amenable system such as yeasts are endless. Copyright © 2016 John Wiley & Sons, Ltd.
Language	English
Publishing date	2016-09
Publishing country	England
Document type	Journal Article
ZDB-ID	632636-5
ISSN	1097-0061 ; 0749-503X
ISSN (online)	1097-0061
ISSN	0749-503X
DOI	10.1002/yea.3169
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Article ; Online: Engineering heterothallic strains in fission yeast.

García-Ruano, Daniel / Hsu, Ian / Leray, Baptiste / Billard, Bénédicte / Liti, Gianni / Coudreuse, Damien

Yeast (Chichester, England)

2023 Volume 41, Issue 3, Page(s) 87–94

Abstract: In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- ... ...

Abstract	In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- and h+ mating types with a high frequency, thereby ensuring the presence of both mating partners in a population of cells. However, alteration of the mating type locus can abolish mating type switching or reduce it to a very low frequency. Such heterothallic strains have been isolated and are common in research laboratories due to the simplicity of their use for Mendelian genetics. In addition to the standard laboratory strains, a large collection of natural S. pombe isolates is now available, representing a powerful resource for investigating the genetic diversity and biology of fission yeast. However, most of these strains are homothallic, and only tedious or mutagenic strategies have been described to obtain heterothallic cells from a homothallic parent. Here, we describe a simple approach to generate heterothallic strains. It takes advantage of an alteration of the mating type locus that was previously identified in a mating type switching-deficient strain and the CRISPR-Cas9 editing tool, allowing for a one-step engineering of heterothallic cells with high efficiency.
MeSH term(s)	Schizosaccharomyces/genetics ; Reproduction/genetics ; Meiosis/genetics ; Genes, Mating Type, Fungal
Language	English
Publishing date	2023-12-15
Publishing country	England
Document type	Journal Article
ZDB-ID	632636-5
ISSN	1097-0061 ; 0749-503X
ISSN (online)	1097-0061
ISSN	0749-503X
DOI	10.1002/yea.3914
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Article: Insights from synthetic yeasts

Coudreuse, Damien

Yeast. 2016 Sept., v. 33, no. 9

2016

Abstract	Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building artificial genetic circuits and studying their behaviours has rapidly demonstrated its potential in a broad range of fields in the life sciences. From the assembly of synthetic genomes to the generation of novel biological functions, yeast cells have imposed themselves as the most powerful eukaryotic model for this approach. However, we are only beginning to explore the possibilities of synthetic biology, and the perspectives it offers in a genetically amenable system such as yeasts are endless. Copyright Â© 2016 John Wiley & Sons, Ltd.
Keywords	biological properties and phenomena ; genome ; models ; prokaryotic cells ; synthetic biology ; yeasts
Language	English
Dates of publication	2016-09
Size	p. 483-492.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	632636-5
ISSN	1097-0061 ; 0749-503X
ISSN (online)	1097-0061
ISSN	0749-503X
DOI	10.1002/yea.3169
Database	NAL-Catalogue (AGRICOLA)

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Article: Long-term evolution of proliferating cells using the eVOLVER platform.

García-Ruano, Daniel / Jain, Akanksha / Heins, Zachary J / Wong, Brandon G / Yimer Wolle, Ezira / Khalil, Ahmad S / Coudreuse, Damien

bioRxiv : the preprint server for biology

2023

Abstract: Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past ... ...

Abstract	Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently-developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new standalone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled setup for long-term experimental evolution.
Language	English
Publishing date	2023-04-19
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.03.28.534552
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Article ; Online: Long-term evolution of proliferating cells using the eVOLVER platform.

García-Ruano, Daniel / Jain, Akanksha / Heins, Zachary J / Wong, Brandon G / Yimer Wolle, Ezira / Khalil, Ahmad S / Coudreuse, Damien

Open biology

2023 Volume 13, Issue 7, Page(s) 230118

Abstract	Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new stand-alone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled set-up for long-term experimental evolution.
MeSH term(s)	Software ; Biological Evolution
Language	English
Publishing date	2023-07-26
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ZDB-ID	2630944-0
ISSN	2046-2441 ; 2046-2441
ISSN (online)	2046-2441
ISSN	2046-2441
DOI	10.1098/rsob.230118
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Article ; Online: Fluorescence exclusion - a rapid, accurate and powerful method for measuring yeast cell volume.

García-Ruano, Daniel / Venkova, Larisa / Jain, Akanksha / Ryan, Joseph C / Radhakrishnan Balasubramaniam, Vasanthakrishnan / Piel, Matthieu / Coudreuse, Damien

Journal of cell science

2022 Volume 135, Issue 13

Abstract: Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, owing in large part to the challenges associated with accurate determination of cell volume. Much of our ... ...

Abstract	Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, owing in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from yeast models, but even for these morphologically stereotypical cells, assessment of cell volume has mostly relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle differences in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work highlights how the coupling of FXm with yeast genetics will bring new insights into the complex biology of cell growth.
MeSH term(s)	Animals ; Cell Cycle ; Cell Size ; Mammals ; Microfluidics ; Saccharomyces cerevisiae/genetics ; Schizosaccharomyces
Language	English
Publishing date	2022-06-30
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2993-2
ISSN	1477-9137 ; 0021-9533
ISSN (online)	1477-9137
ISSN	0021-9533
DOI	10.1242/jcs.259392
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Article: Drug delivery and temperature control in microfluidic chips during live-cell imaging experiments.

Muñoz-Garcia, Javier / Babic, Julien / Coudreuse, Damien

Methods in cell biology

2018 Volume 147, Page(s) 3–28

Abstract: Microfluidic technologies have become a standard tool in cell biological studies, offering unprecedented control of the chemical and physical environment of cells grown in microdevices, the possibility of multiplexing assays, as well as the capacity to ... ...

Abstract	Microfluidic technologies have become a standard tool in cell biological studies, offering unprecedented control of the chemical and physical environment of cells grown in microdevices, the possibility of multiplexing assays, as well as the capacity to monitor the behavior of single cells in real time while dynamically manipulating their growth medium. However, the properties of the materials employed for the fabrication of microchips that are compatible with live-cell imaging has limited the use of these techniques for a broad range of experiments. In particular, the strong absorption of a large panel of small molecules by these materials prevents the accurate delivery of compounds of interest. Here we describe a novel microsystem dedicated to live-cell imaging that (1) uses alternative materials devoid of absorptive properties, and (2) allows for dynamic in-chip control of sample temperature. Based on a proof-of-concept design that we have routinely used with non-adherent fission yeast cells, this chapter details all the steps for the fabrication and utilization of these microdevices.
MeSH term(s)	Alkenes/chemistry ; Animals ; Calibration ; Cell Survival ; Drug Delivery Systems ; Imaging, Three-Dimensional ; Microfluidic Analytical Techniques/methods ; Polymers/chemistry ; Temperature
Chemical Substances	Alkenes ; Polymers
Language	English
Publishing date	2018-07-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	0091-679X
ISSN	0091-679X
DOI	10.1016/bs.mcb.2018.06.004
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Article ; Online: An easy-to-build and re-usable microfluidic system for live-cell imaging.

Babic, Julien / Griscom, Laurent / Cramer, Jeremy / Coudreuse, Damien

BMC cell biology

2018 Volume 19, Issue 1, Page(s) 8

Abstract: Background: Real-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with ... ...

Abstract	Background: Real-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with precision and high time resolution, and the simultaneous alteration of multiple parameters is a major challenge. Recently, microfluidics has provided powerful solutions for such analyses, bringing an unprecedented level of control over the conditions and the medium in which cells under microscopic observation are grown. However, such technologies have remained under-exploited, largely as a result of the complexity associated with microfabrication procedures. Results: In this study, we have developed simple but powerful microfluidic devices dedicated to live-cell imaging. These microsystems take advantage of a robust elastomer that is readily available to researchers and that presents excellent bonding properties, in particular to microscopy-grade glass coverslips. Importantly, the chips are easy-to-build without sophisticated equipment, and they are compatible with the integration of complex, customized fluidic networks as well as with the multiplexing of independent assays on a single device. We show that the chips are re-usable, a significant advantage for the popularization of microfluidics in cell biology. Moreover, we demonstrate that they allow for the dynamic, accurate and simultaneous control of multiple parameters of the cellular environment. Conclusions: While they do not possess all the features of the microdevices that are built using complex and costly procedures, the simplicity and versatility of the chips that we have developed make them an attractive alternative for a range of applications. The emergence of such devices, which can be fabricated and used by any laboratory, will provide the possibility for a larger number of research teams to take full advantage of these new methods for investigating cell biology.
MeSH term(s)	Cell Survival ; Elastomers/chemistry ; Fluorescence ; HeLa Cells ; Humans ; Imaging, Three-Dimensional ; Microfluidics/methods ; Perfusion ; Pressure ; Rheology ; Saccharomyces cerevisiae/metabolism ; Temperature
Chemical Substances	Elastomers
Language	English
Publishing date	2018-06-20
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1471-2121
ISSN (online)	1471-2121
DOI	10.1186/s12860-018-0158-z
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