Transcriptional timing and noise of yeast cell cycle regulators-a single cell and single molecule approach.

Gene expression is a stochastic process and its appropriate regulation is critical for cell cycle progression. Cellular stress response necessitates expression reprogramming and cell cycle arrest. While previous studies are mostly based on bulk experiments influenced by synchronization effects or lack temporal distribution, time-resolved methods on single cells are needed to understand eukaryotic cell cycle in context of noisy gene expression and external perturbations. Using smFISH, microscopy and morphological markers, we monitored mRNA abundances over cell cycle phases and calculated transcriptional noise for SIC1, CLN2, and CLB5, the main G1/S transition regulators in budding yeast. We employed mathematical modeling for in silico synchronization and for derivation of time-courses from single cell data. This approach disclosed detailed quantitative insights into transcriptional regulation with and without stress, not available from bulk experiments before. First, besides the main peak in G1 we found an upshift of CLN2 and CLB5 expression in late mitosis. Second, all three genes showed basal expression throughout cell cycle enlightening that transcription is not divided in on and off but rather in high and low phases. Finally, exposing cells to osmotic stress revealed different periods of transcriptional inhibition for CLN2 and CLB5 and the impact of stress on cell cycle phase duration. Combining experimental and computational approaches allowed us to precisely assess cell cycle progression timing, as well as gene expression dynamics.

Quantitative analysis of circadian single cell oscillations in response to temperature.

Body temperature rhythms synchronize circadian oscillations in different tissues, depending on the degree of cellular coupling: the responsiveness to temperature is higher when single circadian oscillators are uncoupled. So far, the role of coupling in temperature responsiveness has only been studied in organotypic tissue slices of the central circadian pacemaker, because it has been assumed that peripheral target organs behave like uncoupled multicellular oscillators. Since recent studies indicate that some peripheral tissues may exhibit cellular coupling as well, we asked whether peripheral network dynamics also influence temperature responsiveness. Using a novel technique for long-term, high-resolution bioluminescence imaging of primary cultured cells, exposed to repeated temperature cycles, we were able to quantitatively measure period, phase, and amplitude of central (suprachiasmatic nuclei neuron dispersals) and peripheral (mouse ear fibroblasts) single cell oscillations in response to temperature. Employing temperature cycles of different lengths, and different cell densities, we found that some circadian characteristics appear cell-autonomous, e.g. period responses, while others seem to depend on the quality/degree of cellular communication, e.g. phase relationships, robustness of the oscillation, and amplitude. Overall, our findings indicate a strong dependence on the cell's ability for intercellular communication, which is not only true for neuronal pacemakers, but, importantly, also for cells in peripheral tissues. Hence, they stress the importance of comparative studies that evaluate the degree of coupling in a given tissue, before it may be used effectively as a target for meaningful circadian manipulation.