Temperature control for spindle assembly studies in fission yeast

Temperature controlled experimets

The use of fission yeast temperature-sensitive mutants has contributed to the discovery of new genes involved in yeast biology. They also have been used to gain better understanding in essential genes function. Indeed, essential genes have a critical role and mutations in those genes lead to lethal phenotypes, thus preventing further insights on their actions. (Ben Aroya et al., 2010).

Temperature sensitive mutations are often missense mutations. Mutated proteins retain their functionality at low, permissive, temperature, but at higher, restrictive temperature, they become inactive. Fission yeast biology is intrinsically sensitive to temperature. Furthermore, at low temperatures, microtubules depolymerize. Here, we present the validation of ElveflowTemp use for efficient cytoskeleton studies and cell cycle phenotype analysis

Temperature control of spindle assembly experimental set-up

ElveflowTemp temperature controller device, our PDMS chip, is composed of microchannels allowing the circulation of thermalized fluid. The chip is connected to Peltier devices set at two different hot/cold temperatures making possible to quickly change from low to high temperature. Our chip is mounted on a thin glass coverslip, that comes on top of the biological sample. Spacers can be used depending on sample thickness. In our system, the fluid is never in contact with the sample thus preventing any shear stress.

Cell imaging and microscopy Microtubule depolymerization experiments were performed on cdc25-22expressing GFP-Atb2 tubulin. Mitotic spindle experiments were done using temperature sensitive cut7-24ts expressing GFP-Atb2. Images were acquired using a Yokogawa CSU-10 spinning disc scan head on a Nikon TE2000e inverted microscope. 3D imaging was done using a 100/1.45 NA objective, Metamorph 7.5 software for microscope and Hamamatsu ORCA-AG-CCD camera control.

Temperature control of microtubules and spindle assembly

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Reversible ultra-fast heat-induced microtubules depolymerization

To validate the efficacy of ElveflowTemp, we used GFP-Atb2 tubulin expressing yeast and follow fluorescence in vivo. At 22°C, microtubules exhibit a typical elongated shape (Fig1A). At 16°C, we could already quantify a decrease in microtubule polymer mass (Fig1B), and we observed a complete depolymerization when cells were at 6°C (Fig1A and 1B) with a loss of microtubule-associated fluorescence signal (Fig1A) and an increase in cytoplasmic-associated fluorescence (Fig1C). This suggests that tubulin dimers were released from the microtubules and moved to the pool of free-tubulin dimers. We found that the time to reach full depolymerization was cell-size dependent. This process was reversible, and microtubules repolymerized when we shifted back the temperature from 6°C to 22°C. Using our temperature controller, we could efficiently monitor cold-induced microtubule depolymerization and re-polymerization while at the microscope stage.