To investigate the autophagic response of LRRK2 mutation carrying cells using an alternative technique, autophagic vesicle formation was assessed by ICC under normal growing and starvation conditions.
A correlation between the growth rate and the quantity of ribosomes was previously reported in cells growing under starvation conditions [ 41, 42].
Growth conditions and the environment can affect tRNA modifications both quantitatively and qualitatively, e.g. bacteria growing under starvation conditions for certain amino acids or iron leads to under-modification of tRNA [ 10].
Tandem mass spectrometry (MS/MS) identified lysine 19 (K19) and K514 as the acetylation sites of Pck1p and site-directed mutagenesis revealed that acetylation of K514 is critical for its enzymatic activity and promotes extension of life span in yeast growing under starvation conditions.
Conidia of the ugeAΔ strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation.
In contract, we found a significant requirement for the gene when hppy mutant larvae grow under starvation conditions.
48 h after plating, cells were washed and grown in starvation medium (75% DMEM, 25% HAM/F12 and antibiotics).
As a result, imaginal disk growth becomes decoupled from somatic growth, and wing disks continue to grow when starvation, or entry into the wandering stage, stops somatic growth.
Cells were grown in starvation medium (75% DMEM, 25% F12 and antibiotics) or exposed to 20 µM CDDP in the presence or absence of IGF1, growth factors or inhibitors, as indicated in each experiment.
48 h after plating, cells were washed and grown with starvation medium for 12 h, followed by fixation with 3% paraformaldehyde containing 0.5% TritonX100 for 30 sec and with 3% paraformaldehyde for 30 min. Cells were stained with anti-cytochrome C antibody (Santa Cruz).
