Abstract
The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.
Highlights
The membrane-bound organelles of eukaryotic cells are highly dynamic structures, constantly changing their organization and morphology in response to cellular needs
White arrows show cells with a condensed mitochondrial network, and green arrows show cells considered normal. This effect was quantified visually in 65 to 98 cells depending on the treatment, and a significant difference was found between control and drug-treated cells (control (CT): 11 ± 4% of cells, brefeldin A (BFA): 38 ± 7% and golgicide A (GCA): 26 ± 3%, p ≤ 0.05 versus CT)
To assess if the retrograde transport of the mitochondrial network upon BFA/GCA treatment in RPE1 cells is dependent on microtubule minus end directed dynein transport, we studied the effect of BFA and GCA in cells pretreated with ciliobrevin D, a cytoplasmic dynein inhibitor
Summary
The membrane-bound organelles of eukaryotic cells are highly dynamic structures, constantly changing their organization and morphology in response to cellular needs. Our results show that upon inhibition of either GBF1 or Arf[1] function, mitochondria are relocated to a juxta-nuclear region, proximal to the centrosome, where they form a complex network. This condensation of mitochondria towards the microtubule organizing center (MTOC) is dependent on Miro and dynein. Our data support the conclusion that GBF1 activation of Arf[1] blocks Miro-dependent retrograde mitochondrial transport along microtubules towards the MTOC These results suggest that GBF1 and Arf[1] are involved in the regulation of morphology and positioning of mitochondria within cells
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