Fig 1.
Knockdown of TbCK1.2 blocks division of kinetoplasts: Time-course of the effect of RNAi against TbCK1.2 on kinetoplasts and nuclei.
(A) Effects on kinetoplast duplication were assessed by enumeration of the number of kinetoplasts (K) and nuclei (N) per trypanosome in cells cultured in the absence or presence of tetracycline (1 μg/mL, 24 h) (“Other” indicates cells with >2 or <1 K/N). Error bars represent standard deviation of four independent biological experiments (n = 110-268/experimental sample). A χ2 test was used to determine whether the difference in distribution of kinetoplasts and nuclei was statistically significant after knockdown of TbCK1.2 (p = 3.96 x 10−7). Inset: SR-SIM example image of a 1K2N trypanosome following 24 h of RNAi against TbCK1.2. Cell membranes were labeled with mCLING and DNA was detected with DAPI. (B) Effect of knockdown of TbCK1.2 on kinetoplast DNA (kDNA) content. ImageJ was used to measure the fluorescence intensity of individual DAPI-stained kDNA in trypanosomes with one or two kinetoplasts in control (- Tet) or one kinetoplast in TbCK1.2 RNAi (+ Tet) cells. Scatter dot plot relates kDNA fluorescence intensities measured in different trypanosome cell types. The Mann-Whitney U test was used to compare the distribution of fluorescence intensity of DAPI-stained kDNA between -Tet 1K1N and +Tet 1K1N or 1K2N trypanosomes (p = 5.6 x 10−5, and p = < 10−15, respectively). The 95th percentile of the -Tet 1K1N kDNA content is indicated by the horizontal dotted line. Descriptive statistics corresponding to each sample are aligned beneath the graphs. The effect of TbCK1.2 RNAi on kinetoplast duplication was assessed by enumeration of the number of kinetoplasts (K) and nuclei (N) per trypanosome in cells cultured in the absence or presence of tetracycline (1 μg/mL) for 12 h (C) or 18 h (D). (“Other” indicates cells with >2 or <1 K/N). Error bars represent standard deviation of three independent biological experiments (n = >100/experiment). A χ2 test was used to determine whether the difference in distribution of kinetoplasts and nuclei was statistically significant after knockdown of TbCK1.2 for 12 h (p = 0.647) or 18 h (p = 7.94 x 10−5). (E) ImageJ was used to measure DAPI intensity of kDNA fluorescence in trypanosomes with one kinetoplast following 12 h RNAi in control (-Tet) or TbCK1.2 RNAi (+Tet) cells. Violin plot shows the distribution of kDNA fluorescence intensities. A Mann-Whitney U test was used to compare the median fluorescence intensity of DAPI-stained kinetoplasts between -Tet 1K1N (n = 183) and +Tet 1K1N (n = 198) trypanosomes (p = 0.0023). (F) Cartoon explaining the likely origin of 1K2N trypanosomes from 1K1N cells. Kinetoplast DNA is synthesized in S-phase, forming a cell with an undivided kinetoplast and one nucleus (1KU1N) at 12 h. At 18 h of TbCK1.2 knockdown, cells containing two nuclei and one undivided kinetoplast (1KU2N) are detected in the population.
Fig 2.
TbCK1.2 knockdown prevents scission of kDNA.
Knockdown of TbCK1.2 was induced with tetracycline (1 μg/mL for 24 h). (A) Scatter dot plot of kinetoplast length in uninduced (-Tet) and induced (+Tet) RNAi TbCK1.2 population. TEM images show kinetoplasts of different lengths (x ≈ 400 nm, y ≈ 800 and z ≈ 1000 nm) in uninduced and induced TbCK1.2 RNAi cells. The 95th percentile of kinetoplast length in control cells (uninduced) is indicated by horizontal dotted line (n = 216 (108, -Tet; 108, +Tet)). K, Kinetoplast; MtM, Mitochondrial membrane; TEM, Transmission Electron Microscopy. P-value was calculated using the Mann-Whitney U test. (B) Percentage of kinetoplasts longer than 630 nm in uninduced and induced populations of TbCK1.2 RNAi cells. Data was analyzed from three replicates, n = 632 (200, first replicate; 216, second and third replicates), P-value was calculated using an Unpaired Student’s t-test.
Fig 3.
Basal bodies are duplicated after knockdown of TbCK1.2.
Knockdown of TbCK1.2 was induced with tetracycline (1 μg/mL for 24 h) and the effect on basal body duplication was determined using antibodies against TbRP2 (YL1/2, mature basal bodies) or TbSAS6 (detects mature and probasal bodies). DAPI was used to stain DNA. (A) Images represent 1K1N trypanosomes from control cells (-Tet) or 1K2N cells from TbCK1.2-depleted cells (+Tet) with two (2BB) or more (>2BB) basal bodies. Boxed regions are enlarged in the final panel. The average percentage of cell types with the indicated number of mature basal bodies (mBB, YL1/2+) is shown for 1K1N (B) or 1K2N trypanosomes (C) from three independent experiments (n = 108-128/experiment). Blue and red symbols indicate control and RNAi treated samples, respectively. Error bars denote standard deviation. The distribution of mBBs in 1K1N trypanosomes after knockdown of TbCK1.2 was compared to control cells (-Tet) using a χ2 test (1K1N p = 9.1 x 10−26). (A χ2 test could not performed for the 1K2N population because those cells were undetectable (< 1%) in the uninduced population).
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.
Fig 5.
Basal body separation after knockdown of TbCK1.2.
Knockdown of TbCK1.2 was induced with tetracycline (1 μg/mL for 24 h), and the distance between mature basal body pairs was measured. Antibody against TbRP2 (YL1/2) was used to identify basal bodies. ImageJ was used to measure inter-basal body distances (distance between two YL1/2+ mBBs) in TbCK1.2 RNAi cells cultured in the absence (-Tet) or presence of tetracycline (+Tet). A Mann-Whitney U test was used to determine whether differences in inter-basal body distances in control and TbCK1.2 RNAi cells were statistically significant; p values are noted on the graph. Data presented is an aggregate from six biological replicates (n = 302 (-Tet 1K1N), 183 (+Tet 1K1N), 96 (+Tet 1K2N), 111 (-Tet 2K1N), 61 (+Tet 2K1N), 88 (-Tet 2K2N), 51 (+Tet 2K2N)). Descriptive statistics are provided beneath the corresponding data on each graph.
Fig 6.
Intracellular location of TbCK1.2: Identification of candidate TbCK1.2-pathway proteins.
(A) Trypanosomes expressing TbCK1.2 tagged at its N-terminus with V5 epitope were fixed with methanol and probed with anti-V5 antibody. Anti-centrin antibody 20H5 was used to stain basal bodies and bilobes, and DAPI was used to stain DNA. Control images show a V5-TbCK1.2 tagged line without addition of primary antibodies. Arrowheads indicate flagellum (F), basal bodies (BB), bilobes (BL), nuclei, (N), and kinetoplasts (K). Scale bar = 5 μm. (B) Following knockdown (24 h) of TbCK1.2, SILAC phosphoproteomics was used to identify candidate TbCK1.2-pathway proteins. The abundance ratio (H/L) of identified phosphopeptides is plotted as a function of their posterior error probability (PEP) value. Only peptides with a PEP score of 5 x 10−2 (5% chance of error), or lower, are presented. Grey area represents phosphopeptides with a 2-fold or greater increase in abundance. Blue shading indicates phosphopeptides that decreased in abundance 2-fold or greater.
Fig 7.
Kinetoplast division factor hypothesis.
In G1, trypanosomes have one basal body and a single kinetoplast. During S-phase, kDNA synthesis occurs and the probasal body migrates away from the mature basal body (Step 1 and 2). After separating by a distance >895 nm, the probasal body completes maturation (step 3) producing 1K1N trypanosomes with 2 mature basal bodies that are found near opposite ends of a kinetoplast containing uncleaved double-length kDNA [23]. We propose that presence of two basal bodies each at a pole of kinetoplast “licences” kDNA division. Subsequently, kinetoplast division factors (KDFs) are recruited close to, or into, the mitochondrion (Step 4). KDFs may recruit or activate a “kDNA cleavage/scission complex” to divide the kinetoplast (Step 5). In G2, separation of kinetoplasts is visible (Step 6), providing microscopic evidence of kDNA scission. Sorting of kinetoplasts into daughter trypanosomes occurs at cytokinesis (Step 7). Depiction of a “kDNA cleavage/scission complex” is hypothetical. Knockdown of TbCK1.2, or other KDFs, prevents scission of kDNA (see Fig 2). Molecular functions of other KDFs remain to be discovered. TAC-protein sub-complexes are projected to might mediate scission site selection and/or sorting of kinetoplasts (reviewed in [11]).