Microtubules Are Essential for Mitochondrial Dynamics-Fission, Fusion, and Motility-in Dictyostelium discoideum

Abstract

Mitochondrial function is dependent upon mitochondrial structure which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Here we examined the relationship between mitochondrial dynamics and the cytoskeleton in Dictyostelium discoideum. Using time-lapse analysis, we quantified mitochondrial fission, fusion, and motility in the presence of cytoskeleton disrupting pharmaceuticals and the absence of the potential mitochondria-cytoskeleton linker protein, CluA. Our results indicate that microtubules are essential for mitochondrial movement, as well as fission and fusion; actin plays a less significant role, perhaps selecting the mitochondria for transport. We also suggest that CluA is not a linker protein but plays an unidentified role in mitochondrial fission and fusion. The significance of our work is to gain further insight into the role the cytoskeleton plays in mitochondrial dynamics and function. By better understanding these processes we can better appreciate the underlying mitochondrial contributions to many neurological disorders characterized by altered mitochondrial dynamics, structure, and/or function.

Keywords: CluA; Dictyostelium discoideum; cytoskeleton; fission; fusion; latrunculin; mitochondria; nocodazole.

Figure 1

Analysis of mitochondria distribution in AX4 and cluA⁻ cytoskeleton disrupted cells. (A) AX4 mitochondrial morphology remained dispersed and was unaffected by all treatments. The cluA⁻ clustered mitochondria phenotype was significantly decreased with more loose clusters and dispersed mitochondrial when treated with latrunculin-B (p < 0.0001) or nocodazole (p < 0.0001). (B,C) Examples of mitochondrial distribution. (B) AX4 cell with dispersed mitochondria, (C) cluA⁻ cells where top cell (with arrow) shows loosely clustered mitochondria, bottom cell (with arrowhead) shows a tight cluster. ^*Indicates significant differences.

Figure 2

Average mitochondrial speed compared across strains and treatments. (A) There was no significant difference between treatments in AX4 (p = 1.0) or cluA⁻ (p = 1.0) when treated with latrunculin-B. Comparing the treatments across strains also failed to prove statistical significance in ethanol (p = 1.0) and latrunculin-B (p = 1.0) treatments. (B) AX4 and cluA⁻ mitochondria treated with nocodazole moved significantly slower than in vehicle treated cells (AX4, p < 0.0001; cluA⁻, p < 0.0001). AX4 DMSO mitochondrial speed was not significantly slower than the cluA⁻ DMSO treatment (p = 0.57) or nocodazole treatment (p = 0.99). (C) Cells were treated with nocodazole and latrunculin-B simultaneously to determine if there was a synergistic effect between actin and microtubules. In AX4 only latrunculin-B (p < 0.0001) but not nocodazole (p = 0.29) single treatments had significantly higher mitochondrial rates compared to the double treatment. In cluA⁻ both single treatments (Lat-B: p < 0.0001; Noc: p = 0.0031) had significantly higher motility rates compared to the double drug treated cluA⁻ cells. CluA plays no direct role in mitochondrial motility, while microtubules determine the speed of mitochondrial movement; though in the absence of CluA, actin does play a significant role also. ^*Indicates significant differences.

Figure 3

Percent of mitochondria moving in cytoskeleton disrupted AX4 and cluA⁻ strains. In AX4 cells, all drug treatments significantly lowered the percent of mitochondria moving in comparison to their appropriate controls (Lat-B: p < 0.0001; Noc: p = 0.0005). This also occurred in the cluA⁻ strain (Lat-B: p < 0.0001; Noc: p < 0.0013). Inhibiting the cytoskeleton significantly decreased the percent of mitochondria moving in both wild-type and cluA⁻ strains. ^*Indicates significant differences.