Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas
Figure 1
Enrichment of CK1 in flagella and the influence of CK1 inhibition on the phosphorylation status of flagellum proteins and swimming velocity of C. reinhardtii cells. (a) Cells were grown in TAP in a 12 h light-12 h dark cycle and then released to dim light (LL) according to Section 2. Cells were harvested at LL29 and flagella were isolated and a whole cell crude extract (CE), a flagellar extract (FL), and an extract from cell bodies lacking flagella (CB) were prepared. 25 μg proteins per fraction were separated by SDS-PAGE and analyzed by immunoblotting with anti-CK1 antibodies according to Section 2. (b) For quantifications, the amount of CK1 detected in the whole cell crude extract was set to 100%. Quantifications were done with three biological replicates using the ImageMaster 2D Elite Vs.4.01 software (GE Healthcare). (c) Changes in the phosphorylation pattern of flagellum proteins in cells treated with and without CKI-7. Cells were grown as described above (a) in the presence or absence of CKI-7 and harvested at LL29 before isolation of flagella. Proteins from the MMA fraction of the flagellum (25 μg each lane) were separated by 9% SDS-PAGE along with a molecular mass standard and immunoblotted with specific antibodies against phosphoSer according to Section 2. Changes in the phosphorylation status of proteins after CKI-7 treatment are indicated by “+” and “−” signs, respectively. (d) Swimming velocity of 137c cells in the absence (−CKI-7) or presence of CK1 inhibitor (+CKI-7). Cells were grown at 23°C in a LD cycle. Measurements of swimming velocity were done with a hemocytometer and a differential interference contrast microscope with a total magnification of 400 including a personal computer with a video recording system to measure displacement versus time (). Error bars represent the SEM.
