Dataset Information

Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast.

ABSTRACT: The use of luciferase reporters has become a precise, noninvasive, high-throughput method for real-time monitoring of promoter activity in living cells, especially for rhythmic biological processes such as circadian rhythms. We developed a destabilized firefly luciferase as a reporter for rhythmic promoter activity in both the cell division and respiratory cycles of the budding yeast Saccharomyces cerevisiae in which real-time luminescence reporters have not been previously applied. The continuous output of light from luciferase reporters allowed us to explore the relationship between the cell division cycle and the yeast respiratory oscillation, including the observation of responses to chemicals that cause phase shifting of the respiratory oscillations. Destabilized firefly luciferase is a good reporter of cell cycle position in synchronized or partially synchronized yeast cultures, in both batch and continuous cultures. In addition, the oxygen dependence of luciferase can be used under certain conditions as a genetically encodable oxygen monitor. Finally, we use this reporter to show that there is a direct correlation between premature induction of cell division and phase resetting of the respiratory oscillation under the continuous culture conditions tested.

SUBMITTER: Robertson JB

PROVIDER: S-EPMC2584751 | biostudies-other | 2008 Nov

REPOSITORIES: biostudies-other

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Publications

Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast.

Robertson J Brian JB Stowers Chris C CC Boczko Erik E Johnson Carl Hirschie CH

Proceedings of the National Academy of Sciences of the United States of America 20081112 46

The use of luciferase reporters has become a precise, noninvasive, high-throughput method for real-time monitoring of promoter activity in living cells, especially for rhythmic biological processes such as circadian rhythms. We developed a destabilized firefly luciferase as a reporter for rhythmic promoter activity in both the cell division and respiratory cycles of the budding yeast Saccharomyces cerevisiae in which real-time luminescence reporters have not been previously applied. The continuo ...[more]

PMID: 19004762

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Project description:Real-time tracking of vigilance states related to both sleep or anaesthesia has been a goal for over a century. However, sleep scoring cannot currently be performed with brain signals alone, despite the deep neuromodulatory transformations that accompany sleep state changes. Therefore, at heart, the operational distinction between sleep and wake is that of immobility and movement, despite numerous situations in which this one-to-one mapping fails. Here we demonstrate, using local field potential (LFP) recordings in freely moving mice, that gamma (50-70 Hz) power in the olfactory bulb (OB) allows for clear classification of sleep and wake, thus providing a brain-based criterion to distinguish these two vigilance states without relying on motor activity. Coupled with hippocampal theta activity, it allows the elaboration of a sleep scoring algorithm that relies on brain activity alone. This method reaches over 90% homology with classical methods based on muscular activity (electromyography [EMG]) and video tracking. Moreover, contrary to EMG, OB gamma power allows correct discrimination between sleep and immobility in ambiguous situations such as fear-related freezing. We use the instantaneous power of hippocampal theta oscillation and OB gamma oscillation to construct a 2D phase space that is highly robust throughout time, across individual mice and mouse strains, and under classical drug treatment. Dynamic analysis of trajectories within this space yields a novel characterisation of sleep/wake transitions: whereas waking up is a fast and direct transition that can be modelled by a ballistic trajectory, falling asleep is best described as a stochastic and gradual state change. Finally, we demonstrate that OB oscillations also allow us to track other vigilance states. Non-REM (NREM) and rapid eye movement (REM) sleep can be distinguished with high accuracy based on beta (10-15 Hz) power. More importantly, we show that depth of anaesthesia can be tracked in real time using OB gamma power. Indeed, the gamma power predicts and anticipates the motor response to stimulation both in the steady state under constant anaesthetic and dynamically during the recovery period. Altogether, this methodology opens the avenue for multi-timescale characterisation of brain states and provides an unprecedented window onto levels of vigilance.

Dataset Information

Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast.

Publications

Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast.

Similar Datasets

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