Publications

Live-cell fluorescence spectral imaging is an evolving modality of microscopy that uses specific properties of fluorophores, such as excitation or emission spectra, to detect multiple molecules and structures in intact cells. The main challenge of analyzing live-cell fluorescence spectral imaging data is the precise quantification of fluorescent molecules despite the weak signals and high noise found when imaging living cells under non-phototoxic conditions. Beyond the optimization of fluorophores and microscopy setups, quantifying multiple fluorophores requires algorithms that separate or unmix the contributions of the numerous fluorescent signals recorded at the single pixel level. This review aims to provide both the experimental scientist and the data analyst with a straightforward description of the evolution of spectral unmixing algorithms for fluorescence live-cell imaging. We show how the initial systems of linear equations used to determine the concentration of fluorophores in a pixel progressively evolved into matrix factorization, clustering, and deep learning approaches. We outline potential future trends on combining fluorescence spectral imaging with label-free detection methods, fluorescence lifetime imaging, and deep learning image analysis.

The role of meiotic proteasome-mediated degradation has been extensively studied. At the same time, macroautophagy/autophagy only emerged recently as an essential regulator for meiosis progression. Our recent publication showed that autophagy in meiotic cells exhibits a temporal pattern distinct from that in quiescent cells or mitotic cells under prolonged starvation. Importantly, autophagic activity oscillates during meiotic cell divisions, i.e., meiosis I and meiosis II, which can accelerate meiotic progression and increase sporulation efficiency. Our in vitro and in vivo assays revealed that the conserved phosphatase Cdc14 stimulates autophagy initiation during meiotic divisions, specifically in anaphase I and II, when a subpopulation of active Cdc14 relocates to the cytosol and interacts with phagophore assembly sites (PAS) triggering the dephosphorylation of Atg13 to stimulate Atg1 kinase activity and autophagy. Together, our findings reveal a mechanism for the coordination of autophagy activity in the context of meiosis progression.

Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.

Cellular quiescence is a nonproliferative state required for cell survival under stress and during development. In most quiescent cells, proliferation is stopped in a reversible state of low Cdk1 kinase activity; in many organisms, however, quiescent states with high-Cdk1 activity can also be established through still uncharacterized stress or developmental mechanisms. Here, we used a microfluidics approach coupled to phenotypic classification by machine learning to identify stress pathways associated with starvation-triggered high-Cdk1 quiescent states in Saccharomyces cerevisiae. We found that low- and high-Cdk1 quiescent states shared a core of stress-associated processes, such as autophagy, protein aggregation, and mitochondrial up-regulation, but differed in the nuclear accumulation of the stress transcription factors Xbp1, Gln3, and Sfp1. The decision between low- or high-Cdk1 quiescence was controlled by cell cycle-independent accumulation of Xbp1, which acted as a time-delayed integrator of the duration of stress stimuli. Our results show how cell cycle-independent stress-activated factors promote cellular quiescence outside G1/G0.

Plant breeding is directly linked to the development of crops that can effectively adapt to challenging conditions such as soil nutrient depletion, water pollution, drought, and anthropogenic climate change. These conditions are extremely relevant in developing countries already burdened with population growth and unchecked urban expansion, especially in the tropical global southern hemisphere. Engineering new crops thus has potential to enhance food security, prevent hunger and spur sustainable agricultural growth. A major tool for the improvement of plant varieties in this context could be the manipulation of homologous recombination and genome haploidization during meiosis. The isolation or the design of mutations in key meiotic genes may facilitate DNA recombination and transmission of important genes quickly and efficiently. Genome haploidization through centromeric histone mutants could be an option to create new crosses rapidly. This review covers technical approaches to engineer key meiotic genes in tropical crops as a blueprint for future work and examples of tropical crops in which such strategies could be applied are given.