Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei

Abstract

The unicellular parasite Trypanosoma brucei harbors one mitochondrial organelle with a singular genome called the kinetoplast DNA (kDNA). The kDNA consists of a network of concatenated minicircles and a few maxicircles that form the kDNA disc. More than 30 proteins involved in kDNA replication have been described. However, several mechanistic questions are only poorly understood. Here, we describe and characterize minicircle replication factor 172 (MiRF172), a novel mitochondrial genome replication factor that is essential for cell growth and kDNA maintenance. By performing super-resolution microscopy, we show that MiRF172 is localized to the kDNA disc, facing the region between the genome and the mitochondrial membranes. We demonstrate that depletion of MiRF172 leads to a loss of minicircles and maxicircles. Detailed analysis suggests that MiRF172 is involved in the reattachment of replicated minicircles to the kDNA disc. Furthermore, we provide evidence that the localization of the replication factor MiRF172 not only depends on the kDNA itself, but also on the mitochondrial genome segregation machinery, suggesting an interaction between the two essential entities.This article has an associated First Person interview with the first author of the paper.

Keywords: Maxicircles; Minicircle replication factor 172; Minicircles; Mitochondrial DNA replication; TAC; Tripartite attachment complex; Trypanosoma brucei.

Fig. 1.

Phylogeny and protein domains of MiRF172. (A) A phylogenic tree showing the conservation of MiRF172 among Kinetoplastids. The position of MiRF172 in T. brucei brucei is highlighted in red. The scale bar indicates the number of amino acid substitutions. (B) Illustration of the MiRF172 ORF. Depicted are, in green, the mitochondrial-targeting sequence, in magenta, the phosphorylation site at position 999, in dark blue, the poly-Q stretch enriched domain, and in light blue, the alanine and lysine-enriched C-terminal domain.

Fig. 2.

Localization of MiRF172 in BSF and PCF T. brucei cells. (A) Immunofluorescence microscopy of MiRF172–PTP-expressing BSF cells. The localization of MiRF172–PTP (red) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of T. brucei BSF cells. MiRF172–PTP was detected by means of the anti-Protein A antibody. The mature basal bodies were detected with the YL1/2 monoclonal antibody (green). The kDNA and the nucleus were stained with DAPI (cyan). (B) Immunofluorescence analysis of MiRF172–PTP during different stages of the cell cycle (1K1N, dK1N, 2K1N, 2K2N) in BSF cells. K, kDNA; N, nucleus; dK, duplicating kDNA. Localization of MiRF172–PTP (red) and DNA (cyan) were performed as described in A. (C) Immunofluorescence analysis of MiRF172–HA during different stages of the cell cycle (1K1N, dK1N, 2K1N, 2K2N) in PCF cells. The localization of MiRF172–HA (red) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of PCF cells. MiRF172–HA was detected with anti-HA antibody. The kDNA and the nucleus were stained as described in A. PH, phase contrast. Scale bars: 5 µm.

Fig. 3.

Analysis of MiRF172 localization during the cell cycle. (A) Quantification of MiRF172–PTP localization at single or duplicated kDNA discs (1K1N, 2K1N, 2K2N) and duplicating kDNA discs (d1K1N) in BSF cells (n_total=240). K, kDNA; dK, duplicating kDNA; N, nucleus. The left side shows representative immunofluorescence microscopy images depicting the localization of MiRF172–PTP (red) relative to the kDNA disc (cyan). The pie charts show the percentage localization of MiRF172 in the respective kDNA replication stage. (B) 3D-STED immunofluorescence analysis of MiRF172–PTP in T. brucei BSF cells. An MiRF172 (red) and kDNA (cyan) 3D projection (surface rendering) from different angles is shown. MiRF172-PTP was detected with the anti-Protein A antibody and images acquired by 3D-STED microscopy. The kDNA was stained with DAPI (cyan) and images acquired by confocal microscopy. Pictures were deconvolved with the Huygens professional software. (C) Model of MiRF172 localization during the cell cycle. Depicted is a model of the different stages of kDNA disc (cyan) replication in T. brucei and the localization of MiRF172 (red) relative to the kDNA. Scale bars: 1 µm.

Fig. 4.

Phenotype upon knockdown of MiRF172 mRNA by RNAi in T. brucei BSF cells. (A) Growth curve of T. brucei BSF cells expressing MiRF172 RNAi. Results are mean±s.d. The inset depicts a northern blot, showing ablation of MiRF172 mRNA at day 3 (d3) post induction. 18S rRNA serves as a loading control. (B) Quantification of the relative occurrence of kDNA discs and nuclei in MiRF172 RNAi induced (+tet day 3) and uninduced cells (−tet) (n≥100 for each condition). K, kDNA; N, nucleus; sK, small kDNA. Results are mean±s.d. *P<0.05, **P≤0.01 (unpaired two-tailed t-test). (C) Representative fluorescence microscopy images of MiRF172 RNAi BSF cells. The nucleus and the kDNA were stained with DAPI. Arrowheads point to small kDNA. PH, phase contrast. Scale bar: 5 µm. (D) Upper panels, representative images of the ultra-structures of the kDNA of MiRF172 RNAi cells revealed by TEM. White arrows point to the kDNA, yellow arrowheads to the mitochondrial membrane, magenta arrowheads to basal body or base of flagellum. Scale bar: 500 nm. Lower panel, length measurements (n≥30 for each condition) of kDNA ultra-structures from uninduced (mean length=544 nm) and induced (3 days; mean length=368 nm) MiRF172 RNAi BSF cells. ***P≤0.001 (unpaired two-tailed t-test). (E) Growth curve of MiRF172 RNAi BSF γL262P T. brucei cells. The inset depicts a western blot showing ablation of MiRF172–PTP protein at day 3 post induction. EF1α serves as a loading control. (F) Quantification of the relative occurrence of kDNA discs and nuclei in MiRF172 RNAi γL262P T. brucei cells (n≥100 for each condition).

Fig. 5.

Effect of MiRF172 RNAi on the kDNA abundance and free minicircle replication intermediates in T. brucei BSF cells. (A) Detection of total minicircles and maxicircles by Southern blotting. Total DNA, digested with HindIII and XbaI, from either uninduced [day (d)0] or RNAi induced (d3, d5, d7) cells was used. This was probed for minicircles (detection of 1.0-kb linearized minicircles), maxicircles (detection of a 1.4-kb fragment), and tubulin, as a loading control (detection of a 3.6-kb fragment). (B) Quantification of minicircle and maxicircle abundance on Southern blots during MiRF172 depletion (n≥6 for each time point). Black, minicircles; gray, maxicircles. The ratio of the abundance of total minicircle or maxicircle relative to the loading control (tubulin), normalized to day 0 of RNAi induction, is shown. **P≤0.01, ***P≤0.001 (gray values are for minicircles, and black for maxicircles). (C) Detection of free minicircle replication intermediates by Southern blotting. Total DNA isolated from either uninduced (d0) or RNAi induced (d3, d5, d7) cells was Southern blotted, and probed for minicircles. N/G and CC minicircles are indicated. Tubulin was used as a loading control. (D) Quantification of CC and N/G minicircles, as determined by Southern blotting, during MiRF172 depletion (n≥6 for each time point). Black circles, CC minicircles, black triangles, N/G minicircles. The ratio of the abundance of minicircles relative to the loading control tubulin and normalized to day 0 of RNAi induction is shown. *P<0.05.

Fig. 6.

MiRF172 and TAC102 after p197 RNAi depletion and recovery after removal of tet in γL262P p197 RNAi BSF T. brucei cells. (A) Colocalization of MiRF172–PTP with TAC102 in γL262P p197 RNAi BSF cells. Localization of MiRF172–PTP (magenta) and TAC102 (green) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of γL262P p197 RNAi BSF T. brucei cells. MiRF172–PTP was detected with anti-Protein A antibody. TAC102 was detected with anti-TAC102 monoclonal mouse antibody. The kDNA and the nucleus were stained with DAPI (cyan). The inset shows a higher magnification view. (B) TAC recovery experiment in γL262P p197 RNAi BSF T. brucei cells. To detect MiRF172–PTP, TAC102 and DNA the same antibodies and reagents as in A were used. The pictures were obtained under the same conditions as in A. The basal bodies (red) were detected with the YL1/2 monoclonal antibody. - tet, uninduced cells; d5 post induction, MiRF172-depleted cells at day 5 of RNAi (RNAi was induced by addition of tet); d4 post recovery, after 5 days of RNAi, tet was removed and cells were grown for 4 additional days. (C) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P p197 RNAi induced and uninduced cells (n≥113 for each time point). K, kDNA; N, nucleus. (D) Quantitative analysis of TAC102 in γL262P p197 RNAi cells without tet (no tet), with tet at day five (d5 p.i.) as well as 2 days after removal of tet (post recovery; d2 p.r.) and at day 4 post recovery (d4 p.r.) (n≥105 for each time point). (E) Quantitative analysis of the MiRF172–PTP signal in in γL262P p197 RNAi cells as in D (n≥105 for each time point). (F) Western blot analysis of γL262P p197 RNAi BSF cells. Total protein isolated from uninduced cells (−tet), cells induced with tet for 5 days (d5 p.i.) and cells released from p197 RNAi at day 2 (d2 p.r.) and day 4 post recovery (d4 p.r.) was used. C-terminally PTP-tagged MiRF172 was detected with the anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. EF1α serves as a loading control. Arrowheads point to the TAC102 and MiRF172 signals. PH, phase contrast. Scale bars: 5 µm.

Fig. 7.

Quantification of TAC102 in MiRF172 RNAi BSF cells. (A) MiRF172 RNAi BSF cells stained for TAC102 (green) and basal bodies (red) from either uninduced (-tet) or RNAi induced [day (d)3] cells. Pictures show maximum intensity projections from immunofluorescence microscopy image stacks of MiRF172 RNAi BSF T. brucei cells. TAC102 was detected with the anti-TAC102 polyclonal rat antibody and the basal bodies with the monoclonal mouse antibody BBA4. The kDNA and the nucleus were stained with DAPI (cyan). (B) γL262P MiF172 RNAi BSF cells stained for MiRF172–PTP (magenta), TAC102 (green), basal bodies (red) and DAPI (cyan). Proteins and DNA were detected with the same antibodies and reagents as in A. MiRF172–PTP was detected with the anti-Protein A antibody. The pictures show maximum intensity projections as in A. (C) Western blot analysis of γL262P MiRF172 RNAi BSF cells. Total protein isolated from uninduced cells (d0) and cells induced with tet for 3 days (d3). C-terminally PTP-tagged MiRF172 was detected with an anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. Tubulin serves as a loading control. (D) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P MiRF172 RNAi induced and uninduced cells (n≥180 for each time point). K, kDNA; N, nucleus. (E) Quantification of TAC102 in γL262P MiRF172 RNAi uninduced (−tet) and cells induced for three days with tet (d3 tet). Black represents the wild-type TAC102 signal and gray stands for a weak TAC102 signal. (F) Quantification of the relative occurrence of the TAC102 signal in γL262P MiRF172 RNAi cells with different kDNA and nucleus DNA content. PH, phase contrast. Scale bars: 5 µm.