Biology subjects

Cuny, A. P.

Publications and source records attributed to Cuny, A. P..

3 recordsLinked to original sources

bioRxiv2022

Phenotypic Heterogeneity in the DNA Replication Stress Response Revealed by Quantitative Protein Dynamics Measurements

Cells respond to environmental stressors by activating programs that result in protein abundance and localization changes. The DNA damage and DNA replication stress responses have been heavily studied and provide exemplars of the roles of protein localization and abundance regulation in proper cellular stress response. While vast amounts of data have been collected to describe the dynamics of yeast proteins in response to numerous external stresses, few have assessed and compared both protein localization kinetics and phenotypic heterogeneity in the same context, particularly during DNA replication stress. We developed a robust yet simple quantification scheme to identify and measure protein localization change events (re-localization) and applied it to the 314 yeast proteins whose subcellular distribution changes following DNA replication stress. We captured different kinetics of protein re-localization, identified proteins with localization changes that were not detected in previous analyses, and defined the extent of heterogeneity in stress-induced protein re-localization. Our imaging platforms and analysis pipeline enables efficient measurements of protein localization phenotypes for single cells over time and will guide future work in elucidating the biological parameters that govern cellular heterogeneity.

systems biology↗

bioRxiv2021

Cell region fingerprints enable highly precise single-cell tracking and lineage reconstruction

Experimental studies of cell growth, inheritance, and their associated processes by microscopy require accurate single-cell observations of sufficient duration to reconstruct the genealogy. However, cell tracking--assigning identical cells on consecutive images to a track--is often challenging due to imperfect segmentation, moving cells, or focus drift, resulting in laborious manual verification. Here, we propose fingerprints to identify problematic assignments rapidly. A fingerprint distance measures the similarity between cells in two consecutive images by comparing the structural information contained in the low frequencies of a Fourier transform. We show that it is broadly applicable across cell types and image modalities, provided the image has sufficient structural information. Our tracker (TracX) uses the concept to reject unlikely assignments, thereby substantially increasing tracking performance on published and newly generated long-term data sets from various species. For S. cerevisiae, we propose a comprehensive model for cell size control at the single-cell and population level centered on the Whi5 regulator. It demonstrates how highly precise tracking can help uncover previously undescribed single-cell biology.

systems biology↗

bioRxiv2017

Preventing photomorbidity in long-term multi-color fluorescence imaging of S. cerevisiae and S. pombe

Time-lapse imaging using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL, no-observed-effect level). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide a framework for how to approach photomorbidity in other organisms.

cell biology↗