jBillou edited Prophase onset coincide with circadian slowdown.tex  about 9 years ago

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\subsection{Prophase onset coincide with circadian slowdown.}  \label{sec:Prophase_onset_coincide_with_circadian_slowdown}  Next, we hypothesized that the observed slow down of the circadian clock just before division, as the \revalphaYFP signal is in it's rising phase, was due to transcriptional shutdown during M phase. In order to temporally resolve the different mitotic phases we generated a NIH-3T3 cell line stably expressing a \histone fusion protein that allow to follow the chromosome condensation that marks the onset of prophase (TODO:ref), \cite{Sumner1991},  and coincide with the observed shutdown of transcription at division (TODO:ref). We then manually annotated prophase, metaphase and citokinesis on individual cell traces (Figure \ref{fig:pro_met_cyt}A). We marked the onset of prophase as the first frame where the texture of the \histone reporter became granular, the metaphase as the first frame where the \histone reporter is aligned in the nucleus and the citokinesis as the first frame where two separate objects are clearly visible (which is easier to see in the \revalphaYFP channel). We then inferred the circadian and cell cycle phases (Figure \ref{fig:pro_met_cyt}B) and plotted the phase of the different mitotic phases on top of the phase portrait. As shown in Figure \ref{fig:pro_met_cyt}C the onset of prophase coincide with the deceleration of the circadian phase, although the region extend past the measured times of prophase.  To test whether this wasn't due to a resolution limit of our recordings we imaged a few cells with a $60$x magnification and five minutes time resolution, but the results remained similar.