The anti-viral dynamin family member MxB participates in mitochondrial integrity

The membrane deforming dynamin family members MxA and MxB are large GTPases that convey resistance to a variety of infectious viruses. During viral infection, Mx proteins are known to show markedly increased expression via an interferon-responsive promoter to associate with nuclear pores. In this study we report that MxB is an inner mitochondrial membrane GTPase that plays an important role in the morphology and function of this organelle. Expression of mutant MxB or siRNA knockdown of MxB leads to fragmented mitochondria with disrupted inner membranes that are unable to maintain a proton gradient, while expelling their nucleoid-based genome into the cytoplasm. These findings implicate a dynamin family member in mitochondrial-based changes frequently observed during an interferon-based, anti-viral response.

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Fluorescence co-localization data was analyzed using ImageJ software (version 1.52a, National Institutes of Health, USA) and the JACOP (Just Another Co-localization Plug-in) plug-in. Quantitative real-time PCR data was analyzed using Roche analysis software (LCS480 version 1.5.0.39, Roche life science, Indianapolis, IN).
The data sets generated and analyzed during this study are available from the corresponding author upon reasonable request. Figures 1-5  Samples were excluded from analysis if Western blotting showed poor knockdown in siRNA or shRNA experiments.
Reproducibility of data was addressed by keeping culturing of cells the same as well as keeping the timing of experiments consistant.
No randomization was performed. Samples were allocated to different groups based upon over-expression or knockdown of specific proteins.
For computer based quantitation of Rhodamine 123 uptake, images of random fields were acquired, and all samples were analyzed using the same software parameters. Morphological analysis of mitochondria was performed by a qualitative grouping of organelle shapes into 4 broad categories. For mtDNA image quantitation, samples were manually thresholded then mtDNA localization was determined by non bias analysis software.
Commercial antibodies were used per manufacturers recommendations and additionally validated by Western blotting of overexpression or knockdown samples. Rabbit polyclonal antibodies made by McNiven lab were purified on a peptide column and validated by Western blot against pre-immune serum, and over-expression or knockdown samples.