Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.
Project description:In a cell, g-tubulin establishes a cellular network of threads named g-string meshwork. However, the functions of this meshwork remain to be determined. We investigated the traits of the meshwork and show that g-strings have the ability to connect the cytoplasm and the mitochondrial DNA together. We also show that g-tubulin has a role in the maintenance of the mitochondrial meshwork and homeostasis as reduced levels of g-tubulin or impairment of its GTPase domain disrupts the mitochondria network and alters both their respiratory capacity and the expression of mitochondria-related genes. By contrast, reduced mitochondria number or increased protein levels of g-tubulin DNA-binding domain favour the association of g-tubulin with mitochondria. Our results demonstrate that g-tubulin is an important mitochondrial structural component that maintains the mitochondria meshwork, providing mitochondria with a cellular infrastructure. We propose that g-tubulin provides a cytoskeletal element that maintains mitochondria homeostasis and gives form to the mitochondria meshwork. Keywords: Genome binding/occupancy profiling by high throughput sequencing