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Microtubule depolymerisation by cold during C. elegans embryo first division

This application note shows microtubule depolymerisation by cold during C. elegans embryo first division thanks to CherryTemp precise temperature shifts .

Experimental conditions

Strain was cultured at 22°C using standard methods (Brenner, 1974). The strain used was TH65 (YFP::α-tubulin; Schlaitz et al., 2007).

For live imaging, embryos were cut from gravid hermaphrodites in egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 25 mM Hepes, pH 7.5) and mounted on 2% noble agar pads.

Divisions were recorded using a microscope (DM6000; Leica) equipped with epifluorescence and DIC optics and a camera (DFC 360 FX; Leica). Images were collected every 15 sec using a 63×/1.4 NA objective and LAS AF software (Leica). Embryos were imaged at 22°C and the temperature was shifted to 5°C at 0sec. After 195 sec at 5°C,  the temperature was shifted back to 22°C. Temperature control and tempertaure shifts were performed using Cherry Temp.

Images representative of timelapse images of the 1st division of C. elegans embryo movie

Observation

At 5°C = depolymerization of the microtubules (in green), less fluorescence observed.


As soon as the temperature goes back to 22°C, the microtubule polymerize again and the fluorescence intensity increased


Rapid event (we were able to observe the changes within few seconds)


Université-de-Genève

Courtesy of  Dr. Gotta and Dr. Meraldi (Gotta and Meraldi labs)

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