Biology subjects

Garcia-Ruano, D.

Publications and source records attributed to Garcia-Ruano, D..

3 recordsLinked to original sources

bioRxiv2023

Engineering heterothallic strains in fission yeast

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. Take away points- Conventional methods for obtaining heterothallic fission yeast strains are inefficient - We implemented a streamlined genetic editing approach to engineer heterothallism - All fission yeast isolates reported in Jeffares et al. 2015 can be engineered - This method enhances the exploration of genetic diversity in wild fission yeast

genetics↗

bioRxiv2023

Long-term evolution of proliferating yeast cells using the eVOLVER platform

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.

evolutionary biology↗

bioRxiv2021

Fluorescence exclusion: a rapid, accurate and powerful method for measuring yeast cell volume

Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, due in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from models such as budding and fission yeast, but even for these morphologically stereotypical cells, assessment of cell volume has 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 difference 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 reveals how coupling FXm with yeast genetics will bring new insights into the complex biology of cell growth. SUMMARY STATEMENTFluorescence exclusion provides a unique method to accurately measure the volume of yeast cells at both the population and single-cell levels.

cell biology↗