Biology subjects

Lopez, J.

Publications and source records attributed to Lopez, J..

4 recordsLinked to original sources

bioRxiv2018

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV; an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (Light Oxygen Voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution, and a broad dynamic range of over 1300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin encoding gene FLO1, by the FUN-LOV switch, yielded Flocculation in Light (FIL), whereas the light-controlled expression of the co-repressor TUP1 provided Flocculation in Darkness (FID). Overall, the results revealed the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV s ability to accurately manipulate gene expression, with a high-temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.\n\nImportanceOptogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Herein, we report a novel optogenetic switch (FUN-LOV) based on the LOV-domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes: heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

microbiology↗

bioRxiv2018

Activated BAX/BAK enable mitochondrial inner membrane permeabilisation and mtDNA release during cell death

During apoptosis, pro-apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase-independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS-STING signaling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS-STING signaling pathway. Strikingly, using super-resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK-mediated mitochondrial outer membrane pores gradually widen over time. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation can occur during cell death in a BAX/BAK-dependent manner. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase-independent cell death.

cell biology↗

bioRxiv2018

Quantifying the performance of MEG source reconstruction using resting state data

Resting state networks measured with magnetoencephalography (MEG) form transiently stable spatio-temporal patterns in the subsecond range, and therefore fluctuate more rapidly than previously appreciated. These states populate and interact across the whole brain, are simple to record, and possess the same dynamic structure of task related changes. They therefore provide a generic, heterogeneous, and plentiful functional substrate against which to test different MEG recording and reconstruction approaches. Here we validate a non-invasive method for quantifying the resolution of different inversion assumptions under different recording regimes (with and without head-casts) based on resting state MEG. Spatio-temporally partitioning of data into self-similar periods confirmed a rich and rapidly dynamic temporal structure with a small number of regularly reoccurring states. To test the anatomical precision that could be resolved through these transient states we then inverted these data onto libraries of systematically distorted, subject specific, cortical meshes and compared the quality of the fit using Cross Validation and a Free Energy metric. This revealed which inversion scheme was able to best support the least distorted (most accurate) anatomical models. Both datasets showed an increase in model fit as anatomical models moved towards the true cortical surface. In the head-cast MEG data, the Empirical Bayesian Beamformer (EBB) algorithm showed the best mean anatomical discrimination (3.7 mm) compared with Minimum Norm / LORETA (6.0 mm) and Multiple Sparse priors (9.4 mm). This pattern was replicated in the second (conventional dataset) although with a marginally poorer prediction of the missing (cross-validated) data. Our findings suggest that the abundant resting state data now commonly available could be used to refine and validate MEG source reconstruction methods or recording paradigms.

neuroscience↗

bioRxiv2017

Leaf rolling in maize crops: from leaf scoring to canopy level measurements for phenotyping

Leaf rolling in maize crops is one of the main plant reactions to water stress that may be visually scored in the field. However, the leaf scoring did not reach the high-throughput desired by breeders for efficient phenotyping. This study investigates the relationship between leaf rolling score and the induced canopy structure changes that may be accessed by high-throughput remote sensing techniques.\n\nResults gathered over a field phenotyping platform run in 2015 and 2016 show that leaf starts to roll for the water stressed conditions around 9:00 and reaches its maximum around 15:00. Conversely, genotypes conducted under well watered conditions do not show any significant rolling during the same day. Leaf level rolling was very strongly correlated to canopy structure changes as described by the fraction of intercepted radiation fIPARWS derived from digital hemispherical photography. The changes in fIPARWS were stronly correlated (R2=0.86, n=50) to the leaf level rolling visual score. Further, a very good consistency of the genotype ranking of the fIPARWS changes during the day was found ({rho}=0.62). This study demonstrating the strong coordination between leaf level rolling and its impact on canopy structure changes poses the basis for new high-throughput remote sensing methods to quantify this water stress trait.\n\nHighlighThe diurnal dynamics of leaf rolling scored visually is strongly related to canopy structure changes that can be documented using Digital hemispherical photography. Consequences for high-throughput field phenotyping are discussed

plant biology↗