The Phosphorylation Status of Drp1-Ser637 by PKA in Mitochondrial Fission Modulates Mitophagy via PINK1/Parkin to Exert Multipolar Spindles Assembly during Mitosis - PubMed (original) (raw)
The Phosphorylation Status of Drp1-Ser637 by PKA in Mitochondrial Fission Modulates Mitophagy via PINK1/Parkin to Exert Multipolar Spindles Assembly during Mitosis
Huey-Jiun Ko et al. Biomolecules. 2021.
Abstract
Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we first re-visited how mitochondrial ETC inhibition disturbed mitosis progression, resulting in multipolar spindles formation in HeLa cells. Inhibitors of ETC complex I (rotenone, ROT) and complex III (antimycin A, AA) decreased the phosphorylation of Plk1 T210 and Aurora A T288 in the mitotic phase (M-phase), especially ROT, affecting the dynamic phosphorylation status of fission protein dynamin-related protein 1 (Drp1) and the Ser637/Ser616 ratio. We then tested whether specific Drp1 inhibitors, Mdivi-1 or Dynasore, affected the dynamic phosphorylation status of Drp1. Similar to the effects of ROT and AA, our results showed that Mdivi-1 but not Dynasore influenced the dynamic phosphorylation status of Ser637 and Ser616 in Drp1, which converged with mitotic kinases (Cdk1, Plk1, Aurora A) and centrosome-associated proteins to significantly accelerate mitotic defects. Moreover, our data also indicated that evoking mito-Drp1-Ser637 by protein kinase A (PKA) rather than Drp1-Ser616 by Cdk1/Cyclin B resulted in mitochondrial fission via the PINK1/Parkin pathway to promote more efficient mitophagy and simultaneously caused multipolar spindles. Collectively, this study is the first to uncover that mito-Drp1-Ser637 by PKA, but not Drp1-Ser616, drives mitophagy to exert multipolar spindles formation during M-phase.
Keywords: Drp1; PKA; centrosomes; mitochondria; mitophagy; multipolar spindles; phosphorylation.
Conflict of interest statement
The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Figures
Figure 1
Inhibition of mitochondrial ETC complexes I and III induces multiple spindles formation. (A) HeLa cells lost almost all respiratory capacity after ROT or AA treatment. The OCR was determined in the presence of ROT (200 nM), AA (10 μM) and control at 24 h using an oxygen electrode (Ouroboros O2k). The PCP protocol (phosphorylation control protocol) was applied to evaluate OCR, routine, leak, ETS (maximum electron transfer system) and ROX (residual oxygen consumption) values in ROT (pink) or AA (blue) treated HeLa cells compared to control (green) HeLa cells using O2k. (B) HeLa cells were synchronized at G2/M by nocodazole. More than 300 cells were calculated. Quantitation pie charts indicate the percentage of bipolar, tripolar, and multipolar mitotic cells. (C) Normalized ATP measurements in HeLa cells. (D) Mitochondrial morphological features in the presence of ROT or AA were evaluated by the changes in mitochondrial dynamics. The mitochondria were stained with MitoTracker Red to evaluate mitochondrial morphology. Blue DAPI represents chromosome. (E) HeLa cells were analyzed for mitochondrial dynamics-associated proteins by treating with ROT and AA for 24 h synchronized with nocodazole and then released into the M phase. GAPDH was used as an internal control. The bar graph represents the mean of triplicates ± SD. * p < 0.05, ** p < 0.01 compared with the Mock group.
Figure 2
Mdivi-1 causes the formation of multiple spindles poles and uneven segregation. (A) HeLa cells were synchronized at G2/M by nocodazole, followed by the incubation for 24 h in drug-free medium in the absence (Mock) or presence of Mdivi-1. More than 300 cells were calculated. Quantitation pie charts indicate the percentage of bipolar, tripolar, and multipolar mitotic cells. (B) Normalized ATP measurements in HeLa cells. (C) HeLa cells were synchronized by either a double nocodazole block, release into fresh medium at the indicated time points, and then analysis by immunoblotting with antibodies against the indicated proteins. (D) Mitochondrial morphological features in the presence of Mdivi-1 were evaluated by the changes in mitochondrial dynamics. GAPDH was used as an internal control. The bar graph represents the mean of triplicates ± SD. * p < 0.05, ** p < 0.01 compared with the Mock group.
Figure 3
Dynasore did not significantly affect the mitotic defects. (A) HeLa cells were synchronized at G2/M by nocodazole, followed by incubation for 24 h in drug-free medium in the absence (control) or presence of Dynasore. More than 300 cells were calculated. Quantitation pie charts indicate the percentage of bipolar, tripolar, and multipolar mitotic cells. (B) Normalized ATP measurements in HeLa cells. (C) Mitochondrial morphological features in the presence of Dynasore were evaluated by the changes in mitochondrial dynamics. (D) HeLa cells were synchronized by double nocodazole block, released into fresh medium at the indicated time points, and then analyzed by immunoblotting with antibodies against the indicated proteins. GAPDH was used as an internal control. The bar graph represents the mean of triplicates ± SD. * p < 0.05 compared with the Mock group.
Figure 4
Increase in multipolar spindles is associated with mitochondrial ETC blockade and cell-cycle progression in HeLa cells. (A) Immunoblotting analysis of OXPHOS mitochondrial complexes in ROT (200 nM), AA (10 μM), Mdivi-1 (20 μM), and Dynasore (40 μM) treated HeLa cells. (B) Normalized ATP measurements in HeLa cells. (C–E) The mitochondrial ROS, cytosolic ROS and mitochondrial membrane potential (ΔΨm) levels were measured using flow cytometry and fluorescence microscopy with DHE Deep Red, CellROX Deep green, and TMRM Deep Red staining, respectively, and indicated by the mean fluorescent intensity (MFI), or percentage of the cells. (F) Mitochondrial ETC delays cell-cycle G2–M to G1 phase transition. FACS analyses were carried out using cells released from nocodazole block in the presence of ROT, AA, Mdivi-1, and Dynasore for 16 and 24 h. Cell cycle distribution was determined by flow cytometric analysis of propidium iodide-stained cells. Cell number (%) in each cell-cycle phase is indicated in the graph. The bar graph represents the mean of triplicates ± SD. * p < 0.05, ** p < 0.01 compared with the control group.
Figure 5
Blockade of mitochondrial ETC and morphological features by ROT, AA, Mdivi-1, and Dynasore along with the presence of FSK or H89 results in phosphorylation of Drp1-Ser637 and Drp1-Ser616 in PKA performance at mitosis. (A) FSK-enhanced Dynasore stimulated Drp1-dependent mitochondrial fission. (B) FSK-enhanced Dynasore induced multiple spindles formation. (C) H-89 suppressed the mito-Drp1-S637 phosphorylation induced by ROT, AA, and Mdivi-1. (D) H-89 inhibited multiple spindles formation induced by ROT, AA, and Mdivi-1. Cells were pretreated with the PKA inhibitor H89 (50 μM) or PKA activator FSK (25 μM) for 2 h, followed by the treatment with ROT, AA Mdivi-1, and Dynasore for 24 h; whole-cell lysates were then prepared and subjected to Western blot. Quantitation pie charts indicate the percentage of bipolar, tripolar, and multipolar mitotic cells. VDAC1 and GAPDH were used as mitochondria and cytosol markers, respectively. GAPDH was checked for subcellular purity.
Figure 6
Blockade of mitochondrial ETC results in phosphorylation of mito-Drp1 Ser-637, which is essential for the PINK1/Parkin pathway to regulate mitophagy. (A) ROT or AA decreased Drp1-dependent mitochondrial fission. (B) Mdivi-1 decreased Drp1-dependent mitochondrial fission. (C) Dynasore is not a specific Drp1 inhibitor. Levels of total Drp1, Drp1-Ser616, and Drp1-Ser637 were checked in cytoplasmic and mitochondrial fractions in M phase with ROT, AA, Mdivi-1, and Dynasore for 24 h. (D) ROT, AA, and Mdivi-1 treatments can upregulate expression of Parkin and promote mitophagy in M-phase arrest. (E) Levels of PINK1/Parkin, P62, and LC3B were checked in cytoplasmic and mitochondrial fractions in M phase with ROT, AA, Mdivi-1, and Dynasore. (F) CCCP decreased Drp1-dependent mitochondrial fission. VDAC1 was assessed as an internal loading control. GAPDH was checked for subcellular purity. The bar graph represents the mean of triplicates ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group and Mock group.
Figure 7
Blockade of mitochondrial ETC results in aberrant performance of the Aurora A/Plk1 cascade in M phase. (A) HeLa cells were either synchronized by a nocodazole block or were treated with ROT, AA, Mdivi-1, and Dynasore for 24 h, followed by Western blot analysis of key cell cycle regulators. (B) HeLa cells were synchronized in G2/M by 16 h nocodazole followed by release. Cells were treated as indicated. The Aurora A/Plk1 cascade was immunoprecipitated and samples were probed with the indicated antibodies. GAPDH was used as an internal control. The bar graph represents the mean of triplicates ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group. Note that all Mock lanes also as normalized M phase.
Figure 8
Model of cross-talk between mitochondrial ETC proteins and centrosomal kinases/proteins for centrosome function and bipolar spindles assembly. After nocodazole treatment, at M phase four inhibitors (two ETC inhibitors, ROT and AA; two Drp1 inhibitors; Mdivi-1 and Dynasore) were added to induce mitochondrial dynamic imbalance. AKAP1 is believed to be a scaffold protein that recruits PKA to modulate mitochondrial function at Drp1-Ser637 (also see supplementary Figure S8). In summary, upon normal conditions, during mitosis mitochondria are fragmented to facilitate segregation of this organelle to daughter cells. Mitochondrial fission is mediated by Cdk1-dependent phosphorylation and activation of Drp1-Ser616 [39]. Proper partitioning of mitochondria is required for inheritance during cell division, and then proceeds to the next cell cycle. However, upon inhibitor insulting conditions, mito-Drp1-Ser637 drives mitophagy via activated PKA during mitotic arrest. Dynamic Drp1-Ser637/Ser616 ratio affects the PINK1/Parkin pathway and promotes more efficient mitophagy while inducing multipolar spindles formation simultaneously. It appears that mito-Drp1-Ser637 induces mitochondrial fission to drive mitophagy and induces multipolar spindles formation to cause cell aneuploidy or cell death.
References
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