Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome
Fig 4
Evidence of probasal body movement in T. brucei.
(A) Schematic of protocol used in the study. Trypanosomes were treated with AEE788 (5 μM) or DMSO (control) for 4 hours, released from drug pressure, and allowed to recover for 1.5, 2, or 3 hours. Antibodies against TbRP2 (YL1/2) and TbSAS6 were used to identify basal bodies via immunofluorescence microscopy. ImageJ was used to measure inter-basal body distances by tracking separation between centers of TbSAS6 puncta. (B) Representative images of cells from AEE788-treated group, and cells allowed to recover from drug for 1.5 h. Separation between basal bodies is highlighted in yellow. Scale bar = 5 μm. (C) Plot shows distances between pro-basal bodies (TbSAS6 positive) and mature basal bodies (TbRP2/TbSAS6 positive) in cells with one mature basal body (mBB). Bars on graph indicate median and inter-quartile range. Numbers to the right indicate median inter-basal body distances for each group. Trypanosomes were drawn from a single experiment. Cells analyzed = 41 (DMSO), 131 (AEE788), 99 (1.5 h recovery), 106 (2 h recovery), 62 (3 h recovery). Inter-basal body distances were compared between groups with a Mann-Whitney U test. The difference in distribution of inter-basal body distances between DMSO treated group and AEE788 treated group was statistically significant (p = 2.4*10−2). The difference in inter-basal body distances between the group harvested immediately after AEE788 treatment and the population given 1.5 h to recover was statistically significant (p = 4.8*10−2). The difference between the 1.5 h recovery and 2 h recovery groups was highly statistically significant (p = 9.1*10−12). The difference in inter-basal body distance between the 2 h and 3 h group was statistically significant (p = 4.1*10−5). (D) Distances between pro-basal bodies (pairs of TbSAS6-positive foci) in cells with two mature basal bodies (mBB) are plotted. Bars on graph show median and inter-quartile range. Numbers to the right in black indicate median distances between a mature basal body and a pro-basal body for each group. Numbers in red denote distances (median) between pairs of mature basal bodies for each group. Cells analyzed = 59 (DMSO), 29 (AEE788), 16 (1.5 h recovery), 32 (2 h recovery), 92 (3 h recovery). Inter-basal body distances were compared between groups with a Mann-Whitney U test. The difference in distribution of inter-basal body distances between DMSO treated group and AEE788 treated group was not statistically significant. The difference in distribution of inter-basal body distances between the AEE788 treatment group and the group at 1.5 h recovery was statistically significant (p = 2.1*10−2). The difference in inter-basal body distances in 1.5 h recovery and 2 h recovery groups was not statistically significant. The difference in inter-basal body distance between the 2 h and 3 h group was statistically significant (p = 2.4*10−5). Distances between mature basal bodies (pairs of TbRP2-positive foci) in the same cells are listed to the right in red.