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Mitosis control in fission yeast using thermosensitive mutants

In this application note, we show the comparision between blocking fission yeast cdc25-22ts mutants in a thermalized waterbath vs. blocking them on a microscope slide using CherryTemp.

Experimental conditions

Application example: Block and release of cdc25-22 S. pombe ts mutants.

CDC25 ts S. pombe reversible control of mitosis arrest by CherryTemp

Figure 1, cdc25 ts S. pombe reversible control of mitosis arrest by CherryTemp

For the flask experiment, fission yeast cdc25-22cells were blocked at the G2/M transition by placing a flask for 4 hours in a waterbathat restrictive temperature (36.5 °C). Synchronous release into the cell cycle was achieved by a quick immersion of the flask on ice-cold water until 25°C was reached (temperature measured by inserting a thermometer in the flask). Cells were then placed on a microscope slide and were imaged for septation index scoring.

For theCherryTempexperiment, asynchronously growing fission yeast cdc25-22 cells were placed on a CherryTemp mounted system. Cells were blocked at the G2/M transition for 4 hours at restrictive temperature (36.5 °C). Synchronous release into the cell cycle was achieved by a quick shift (10 seconds) into permissive temperature (25°C).

Septation index is an indicator of the progression of fission yeast cells into the cell cycle.Septation index in both experiments shows a high synchrony in the release. Synchrony and time of releasewith both methods are very similar.

High synchrony was obtained as well when blocking nda3-KM311 mutants at 16°C with CherryTemp and measuring septation index upon release to 32°C.

Figure 2, CDC25 ts S. pombe reversible control of mitosis arrest by CherryTemp

Figure 2, CDC25 ts S. pombe reversible control of mitosis arrest by CherryTemp, We thank Damien Coudreuse’s lab (Julien Babic, PhD st. – IGDR, Rennes) for credits.

IGDR

Courtesy of  Damien Coudreuse’s lab
(Julien Babic, PhD st. – IGDR, Rennes)

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