Polybead microspheres

To estimate the protein degradation rate of CHE-1::GFP, we used Fluorescence Recovery After Photobleaching (FRAP). Animals were immobilized using Polybead microspheres (Polyscience) in M9 buffer on agarose pads covered with a cover glass. To prevent dehydration of animals, the coverslip was sealed with VALAP (vaseline, lanolin and paraffin, ratio 1:1:1). Animals were monitored at several time points during the experiment if they were still alive by checking very subtle movement and/or pumping behaviour. The GFP signal in the ASE neurons of the animals was bleached until approximately 20% of the initial signal was left. After bleaching, the GFP signal was measured every 20 or 30 min until the signal had recovered. Images were taken with the same microscope as in the CHE-1 protein quantification section. We measured for each time point the average GFP intensity in the ASE neurons and subtracted the background intensity measured nearby the ASE neurons. The degradation rate was calculated from the initial slope of the growth curve using the following exponential model: ${\displaystyle R\left(t\right)=\left(\frac{f}{b}\right)+\left(x_0-\left(\frac{f}{b}\right)\right)e^{-bt}}$ , where $x_0$ is the initial fluorescent intensity at the start of the recovery curve right after bleaching. $b$ and $f$ represent the CHE-1 protein degradation and production rate, which are fitted on the individual measured recovery curves, to obtain the average CHE-1 protein degradation rate.