Regulation of mitochondrial morphogenesis by annexin a6.

Mitochondrial homeostasis is critical in meeting cellular energy demands, shaping calcium signals and determining susceptibility to apoptosis. Here we report a role for anxA6 in the regulation of mitochondrial morphogenesis, and show that in cells lacking anxA6 mitochondria are fragmented, respiration is impaired and mitochondrial membrane potential is reduced. In fibroblasts from AnxA6(-/-) mice, mitochondrial Ca(2+) uptake is reduced and cytosolic Ca(2+) transients are elevated. These observations led us to investigate possible interactions between anxA6 and proteins with roles in mitochondrial fusion and fission. We found that anxA6 associates with Drp1 and that mitochondrial fragmentation in AnxA6(-/-) fibroblasts was prevented by the Drp1 inhibitor mdivi-1. In normal cells elevation of intracellular Ca(2+) disrupted the interaction between anxA6 and Drp1, displacing anxA6 to the plasma membrane and promoting mitochondrial fission. Our results suggest that anxA6 inhibits Drp1 activity, and that Ca(2+)-binding to anxA6 relieves this inhibition to permit Drp1-mediated mitochondrial fission

Partial co-localisation of AnxA6 to mitochondria.

<p>(A) Mitochondria (M) and cytosol (C) were purified from normal mouse liver and western blotted for AnxA6 and AnxA2. The mitochondrial samples were immunoblotted following repeated washes (lanes w1-w4 show protein in each wash) in buffer containing 1 mM EGTA, as were whole liver cell extracts from control (WT) and <i>AnxA6</i>^−/− (KO) mice. (B) Normal primary mouse fibroblasts were co-immunostained for AnxA6 and cytochrome C and analysed by confocal microscopy. Insets show partial co-localisation of AnxA6 and Cyto-c on mitochondria. (C) Primary mouse fibroblasts were isolated from the ears of <i>AnxA6</i>^−/− mice and fixed and immunostained for AnxA6 and Cyto-c The images show faint non-specific staining with the AnxA6 antibody, and the characteristic pattern of fragmented mitochondria (see inset). Scale bar = 4 µm.</p

Mitochondrial structural abnormalities in <i>AnxA6</i>^−/− mice.

<p>Sections of skin, liver and retina were prepared from control and <i>AnxA6</i>^−/− mice and examined by electron microscopy. Mitochondria (m) were enlarged and rounded in all tissues, and in retinal pigment epithelial cells (RPE) appeared less electron-dense. Pigment granules in the RPE are also indicated (pg). Scale bar = 500 nm.</p

Inhibition of Drp1 reverses mitochondrial fragmentation in AnxA6 null fibroblasts.

<p>Primary mouse fibroblasts were isolated from the ears of <i>AnxA6</i>^−/− mice, loaded with Mitotracker, and exposed to DMSO (control) or 50 µM Mdivi-1 in DMSO for up to 15 min. Images were captured on an inverted confocal microscope at 0, 2.5 and 5 min. The zoomed regions in the top panels show a marked increase in the number and length of mitochondrial extensions (yellow arrows), in contrast to the DMSO treated cells in which the mitochondria remained mostly fragmented. Scale bar = 5 µm.</p

Loss of mitochondria-ER contact sites in <i>AnxA6</i>^−/− cells.

<p>(A) Opa-1 and Mfn-2 co-localization in <i>AnxA6^+/+ and AnxA6^−/−</i> fibroblasts mark the overlap between ER and mitochondria in the two cohorts of cells. Scale bar = 4 µm. (B) Primary mouse fibroblasts fixed and immunostained for Tim23 and Cyto-c The images show co-localisation of Tim23 and Cyto-C in fibroblasts from both mutant and wild type mice, but with the characteristic mitochondrial fragmentation in AnxA6 null cells. Scale bar = 5 µm.</p

Mitochondrial morphology is abnormal in anxA6 null cells.

<p>(A) Images of primary fibroblasts from <i>AnxA6</i>^−/− mice immunostained for cytochrome c and analysed by confocal microscopy to visualise mitochondria. (B) Mitochondrial morphology was graded as tubular, intermediate or fragmented for <i>AnxA6</i>^−/− and control mice. Data represent the mean ± s.d. of 3 independent experiments, with >1000 cells counted for each strain. (C) A431 cells were either labelled in vivo with mitochondrial-targeted GFP (mtGFP), or labelled following fixation with antibodies to Cyto-c. Cells were counter-stained using DAPI and visualised by confocal microscopy. Representative images show that mitochondrial staining is fragmented in control A431 cells, but tubulated in A431 cells stably expressing AnxA6. Scale bar = 2 µm. The western blot shows expression of the larger isoform of anxA6 only in the stable transfected cells.</p

AnxA6 regulates mitochondrial morphology via interaction with Drp1.

<p>(A, B) Whole cell lysates were prepared from control mouse ear fibroblasts or liver) and immunoprecipitated (IP) with antibodies to Drp1, Mfn2 and OPA1 as indicated. For each blot, w1 corresponds to input, and w2, w3 and w7 correspond to wash number. IP is the immunoprecipitate, and the position of AnxA6 is indicated. Blots were probed with antisera to Drp1 (A) and AnxA6 (B), the position of which is indicated, and visualised using enhanced chemiluminescence. The band running beneath AnxA6 in the Mfn2 and OPA-1 IP lanes is IgG heavy chain. (C) Purified mitochondria and whole cell lysates of liver and primary fibroblasts from control (Wt) and <i>AnxA6</i>^−/− (KO) mice were immunoblotted with antisera against Drp1 and the mitochondrial marker Tim23 as a control for loading. (D) Fibroblasts from control and <i>AnxA6</i>^−/− mice were immunostained with antisera to Drp1 (red) and the mitochondrial marker cytochrome C (green), and analysed by confocal microscopy. The insets show reticular staining of Cyto C in control fibroblasts, with little co-localisation with Drp1 (visualised in orange), in contrast to increased co-localisation of the two proteins in the AnxA6 null cells. Scale bar = 5 µm. (E) The proportion of Drp1 immunofluorescence coincident with Cyto-c was calculated using Metamorph, and is presented as mean ± s.d., n = 3000 cells in 3 separate experiments, *p<0.05. (F) A431 cells stably expressing AnxA6 were triple stained for AnxA6 (green), Drp1 (red) and Cytochrome c (magenta). Regions that appear white in the lower right zoomed panel indicate coincidence of the three antigens. Scale bar = 2 µm. (G) A431 cells stably expressing AnxA6 were simulated with 1 µm ionomycin for 5 min, then fixed and stained for AnxA6, Drp1 and Cytochrome c as in (F). Note that AnxA6 relocates from the cytosol to plasma membrane in cells exposed to ionomycin (lower right ‘merge’ panel), with loss of regions of coincident staining of the three proteins (seen as white in the top right ‘merge’ panel). Scale bar = 5 µm.</p