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.
Introduction
Mitochondria are dynamic organelles that undergo fission and fusion events in response to changes in metabolic needs and important cellular processes such as cell division and apoptosis1,2,3. Central to these alterations in mitochondrial form are the dynamin family of large GTPases, which are known to bind and remodel membranes4,5. Distinct from the conventional dynamins (Dyn1, 2, 3) that are known to support membrane scission at distinct cellular sites such as the plasma membrane6, the Golgi apparatus7, as well as endosomes8 and autolysosomes9,10,11, the dynamin-related proteins have been implicated in the dynamics of both mitochondria12 and peroxisomes13. Although highly conserved within the N-terminal GTPase domains, these dynamin-related proteins are only modestly conserved in the middle domain and generally lack pleckstrin binding and proline-rich domains found in the conventional family members. The mitofusins (MFN1/MFN2) are believed to support the outer mitochondrial membrane fusion and optic atrophy 1 (OPA1) has been implicated in the fusion of the inner membrane14,15,16,17. Reciprocally, dynamin-related protein 1 (DRP1) has been shown to constrict the outer mitochondrial membrane4 with the final scission event perhaps driven by conventional Dyn218.
In addition to the mitochondria-associated dynamins are the myxovirus resistance GTPases (MX1/2 human and MxA/B mouse), which are <40% similar to the conventional dynamins but over 60% identical in sequence to each other. These related proteins are expressed in cells upon exposure to type 1 interferon (IFN) and convey a cellular innate immune response to a variety of different pathogenic viruses19,20,21,22,23,24 including influenza, hepatitis B (MxA), and HIV-1 (MxB). We and others have found that MxA can assemble into polymers that act to deform membranes25,26 and appears to reside on components of the smooth endoplasmic reticulum (ER)25,27. MxB also assembles into polymeric structures28 and was originally found to associate with nuclear pores where it was predicted to restrict the access of a viral genome to the host transcriptional machinery29. MxB has been described to bind to the HIV-1 genome, while impairing its chromosomal integration24,30,31, and recently has been identified as a key factor behind IFN-mediated suppression of hepatitis C virus infection32.
In this study we report that although markedly upregulated in cells treated with IFN, MxB is expressed constitutively in a variety of cell types, particularly in primary hepatocytes and hepatoma cell lines. In addition to reported localization at nuclear pores, we find that MxB is intimately associated with the inner mitochondria, where it plays an essential role in the maintenance of mitochondrial cristae and DNA stability of this organelle.
Results
MxB associates with mitochondria in cultured cells
As the related family member MxA has been shown to convey a partial resistance to the hepatocyte-centric HBV33, while associating with membranous structures in hepatocytes, our goal is to compare the distribution and expression of the highly related family member MxB in hepatocyte cell lines and in the whole liver. Using several different antibodies and tagged constructs to MxB, we observe the punctate nuclear staining previously observed by others34,35,36. In addition to this localization, we observe a fusiform and linear staining pattern in the hepatoma cell line Hep3B and HeLa cells, using a previously published MxB antibody and exogenously expressed green fluorescent protein (GFP)/Myc-tagged constructs (Fig. 1a–d and Supplementary Fig. 1a–c). Co-staining these cells with a variety of different organelle marker antibodies reveals that the MxB labeling represents mitochondria as confirmed by antibodies to cytochrome c oxidase 4 (CoxIV) (Fig. 1a–d and Supplementary Fig. 1a–c). This localization is consistent when staining with three distinct MxB antibodies or expressing either GFP or Myc-tagged MxB, whereas the characteristics of labeling using these different probes varies from fine to large puncta coating the mitochondria.
Western blot (WB) analysis of various human tissues for endogenous MxB shows that MxB appears enriched in the liver, lymph node, testis, tonsil, and pig liver. Of note is the fact that human lymph node and tonsil tissues possess two bands, whereas the liver, testis, and pig liver indicate a single band (Fig. 1e). Accordingly, appreciable levels of endogenous MxB are observed in several human hepatoma cell lines (Hep3B, HepG2, Huh7) and primary hepatocytes isolated from human and pig livers (Fig. 1f). HeLa cells appear to have modest MxB levels that become more apparent upon longer exposures (Supplementary Fig. 4). As reported previously34, MxB expression is markedly increased in isolated cells treated with 1000 IU ml−1 of IFNα (Fig. 1g).
MxB has been shown to be expressed in two forms, one of which contains an N-terminal sequence required for nuclear targeting. The distribution of the long and short MxB forms have been examined in significant detail by others33,34. In our hands we find that the short form has modest affinity for the nucleus or mitochondria regardless of the tag used in comparison with the long form that associates with both organelles (Supplementary Fig. 1d-j).
As our initial morphological observations (Fig. 1 and Supplementary Fig. 1) show MxB associating with mitochondria as either small or large puncta, or more diffuse in nature, we think it is important to analyze live cells expressing MxB-mCherry to test for changes in MxB localization over time. One example of this dynamic distribution is depicted in Fig. 2a, showing a single Hep3B cell expressing MxB-mCherry viewed over a 16 h time period. Dramatic and unexpected changes in MxB localization are observed, as the tagged protein appears to cycle between diffuse and punctate forms during the extended viewing period. Subsequently, cells co-transfected with Mito-GFP37 as a mitochondrial marker test whether the coalescing MxB puncta might associate with this organelle. Indeed, as shown in the images from movies of two distinct live cells (Fig. 2b, c), MxB-mCherry appears diffuse and barely detectable at early viewing times but subsequently coalesces into numerous large puncta that associate with the tips of undulating mitochondria (inserts). As observed in Fig. 2a, these puncta appear to dissolve over time, leaving the MxB in a diffuse state.
MxB manipulations disrupt mitochondrial structure and function
Based on the morphological observations described above, we next test whether manipulation of MxB levels might alter mitochondrial morphology. Hep3B cells transfected with either GFP-tagged or untagged wild-type (WT) or GTPase-defective mutant K131A MxB for 24 h are followed by staining with the CoxIV marker antibody. Representative images displayed in Fig. 3a, b show that mutant MxB-transfected cells possess fragmented mitochondria and significantly reduced CoxIV staining compared with adjacent untransfected cells. Importantly, in many transfected cells, mitochondrial distribution, size, and shape appear abnormal. These altered phenotypes include fragmented, twisted, or clustered morphologies (Fig. 3c and Supplementary Fig. 3a). It is particularly surprising that 25–30% of the cells examined have markedly attenuated CoxIV staining (Fig. 3d). To define these changes at a higher resolution, Hep3B cells transfected to express WT or K131A mutant MxB for 2 days are then fixed and embedded for electron microscopy (EM). Although untransfected cells display normal mitochondrial morphologies, many Hep3B cells expressing either of the constructs possess mitochondria with a greatly reduced number of cristae and/or numerous vacuoles or blisters (Fig. 3e–g). Similar results are seen in transfected HeLa cells, which lose almost all of their mitochondrial cristae, leaving hollow, elongated, organelle shells (Supplementary Fig. 2a–c). To better understand the effects of the mutant K131A form, we compare the distribution of this mutant with the WT form in Hep3B cells that are transfected with GFP-tagged constructs then fixed and viewed by fluorescence microscopy. Quantification of mitochondrial and nuclear fluorescence using ImageJ software shows a near-equal preference of the WT form for both organelles, while the K131A mutant exhibits increased association with mitochondria (Supplementary Fig. 2d). As a biochemical comparison, Hep3B cells expressing either the WT or K131A form subjected to subcellular fractionation to obtain nuclear, mitochondrial enriched, and cytosol fractions, are blotted for corresponding organelle markers and MxB (Supplementary Fig. 2e). By this approach, a near-equal distribution of both forms is observed.
To further define the role of MxB in maintaining mitochondrial morphology, Hep3B cells are subjected to MxB knockdown using both small interfering RNA (siRNA) and short hairpin RNA (shRNA) approaches. Following 5 days of treatment, cells are fixed and stained for CoxIV (Fig. 3h–k) or prepared for EM (Fig. 3m–o). As observed in cells expressing exogenous WT or mutant (K131A) MxB protein, these knockdown cells display altered mitochondrial morphologies including fragmented, twisted, or clustered shapes. A comprehensive table showing the effects of MxB knockdown, or overexpression, on mitochondrial shape is provided in Supplementary Fig. 3a. In contrast to the cells overexpressing tagged MxB proteins, the knockdown cells did not exhibit reduced staining for CoxIV or Tom20. EM of siRNA-treated Hep3B cells, however, did reveal highly altered mitochondria with deformed shapes, large vacuoles, and vesiculated cristae (Fig. 3m–o and Supplementary Fig. 2f–h). Based on our findings from the expression of either WT or mutant MxB proteins, and two distinct knockdown strategies, we conclude that this dynamin family member plays an important role in the maintenance of mitochondrial shape and integrity.
A central property of the Mx proteins is a substantially increased expression induced by IFN. Although MxB, but not MxA, is constitutively expressed in some cell and tissue types examined (Fig. 1e–g), the expression levels of both Mx proteins is markedly increased in cells treated with IFN (Fig. 1g, Supplementary Fig. 3j). We found that Hep3B cells treated with IFN for 48 h possess fragmented mitochondria in comparison with control cells (Supplementary Fig. 3b–d, h) and slightly increased levels of MxB on the nuclear envelope vs. mitochondria by immunostaining (Supplementary Fig. 3e-g,i). As a biochemical comparison, Hep3B cells treated as above with IFN for 48 h prior to lysis and differential centrifugation provides a rudimentary separation of nuclei from the mitochondria and cytosol. These fractions are run on SDS-polyacrylamide gel electrophoresis (PAGE) and probed by WB with antibodies to MxB and MxA (Supplementary Fig. 3j). This blotting is consistent with the cell staining and suggests that although there is a large increase in MxB associated with both organelles, a slight preference is observed with the nuclear fraction. As a control comparison the IFN -induced MxA protein is observed exclusively in the cytosolic fraction.
