An investigation into the physiological role of annexin A11

The calcium-dependent phospholipid-binding protein annexin A11 was discovered over 25 years ago, however little is known about its function. This thesis supports a role for annexin A11 in the cell cycle and suggests an association with the microtubule network. Recent studies have implicated annexin A11 in the autoimmune disease sarcoidosis, which is further investigated in this study. A single nucleotide polymorphism within annexin A11 (R230C) was identified as a highly associated susceptibility locus for sarcoidosis. Over-expression of annexin A11WT and annexin A11R230C showed no difference in their distribution. Stimulation with ionomycin, which induces a rise in intracellular calcium, resulted in the translocation of both variants to the plasma membrane and nuclear envelope with approximately the same time course. The calcium-dependent translocation of annexin A11 is therefore unaffected by the R230C mutation. In this one aspect there appears to be no difference between the two variants, however much still remains to be investigated such as the potential extracellular roles of these variants and their function in immune cells. Much of the work in this thesis has focused on elucidating the function of wild type annexin A11, particularly during the cell cycle. During mitosis the distribution of annexin A11 is highly dynamic and localises to the mitotic spindle at metaphase and anaphase, as well as the midbody at cytokinesis. The use of methanol fixation highlighted the co-localisation of annexin A11 with the microtubule network throughout mitosis, as well as at interphase. Furthermore annexin A11 was shown to concentrate at the centrosome, again during both mitosis and interphase. Several centrosomal proteins also localise to the midbody and are key regulators of mitotic progression, supporting a similar role for annexin A11. Furthermore the centrosomal localisation of annexin A11 may serve as an ideal docking site from which it can be easily targeted throughout the cell. This study highlights the novel localisation of annexin A11 to the centrosome and the microtubule network. Furthermore these findings contribute to a growing picture for the role of annexin A11 in the cell cycle, particularly with regards to its use of the microtubule network – a feature which may also have a role in interphase cells

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

An investigation into the physiological role of annexin A11.

The calcium-dependent phospholipid-binding protein annexin A11 was discovered over 25 years ago, however little is known about its function. This thesis supports a role for annexin A11 in the cell cycle and suggests an association with the microtubule network. Recent studies have implicated annexin A11 in the autoimmune disease sarcoidosis, which is further investigated in this study. A single nucleotide polymorphism within annexin A11 (R230C) was identified as a highly associated susceptibility locus for sarcoidosis. Over-expression of annexin A11WT and annexin A11R230C showed no difference in their distribution. Stimulation with ionomycin, which induces a rise in intracellular calcium, resulted in the translocation of both variants to the plasma membrane and nuclear envelope with approximately the same time course. The calcium-dependent translocation of annexin A11 is therefore unaffected by the R230C mutation. In this one aspect there appears to be no difference between the two variants, however much still remains to be investigated such as the potential extracellular roles of these variants and their function in immune cells. Much of the work in this thesis has focused on elucidating the function of wild type annexin A11, particularly during the cell cycle. During mitosis the distribution of annexin A11 is highly dynamic and localises to the mitotic spindle at metaphase and anaphase, as well as the midbody at cytokinesis. The use of methanol fixation highlighted the co-localisation of annexin A11 with the microtubule network throughout mitosis, as well as at interphase. Furthermore annexin A11 was shown to concentrate at the centrosome, again during both mitosis and interphase. Several centrosomal proteins also localise to the midbody and are key regulators of mitotic progression, supporting a similar role for annexin A11. Furthermore the centrosomal localisation of annexin A11 may serve as an ideal docking site from which it can be easily targeted throughout the cell. This study highlights the novel localisation of annexin A11 to the centrosome and the microtubule network. Furthermore these findings contribute to a growing picture for the role of annexin A11 in the cell cycle, particularly with regards to its use of the microtubule network – a feature which may also have a role in interphase cells.

Annexins as disease modifiers

The annexins are a family of calciumdependent phospholipid binding proteins which are present in all eukaryotes. There are currently 12 identified human annexins all of which contain unique calcium binding sites, encoded in the highly conserved annexin repeat motifs within the C terminal core. In addition to the C terminal core the annexins contain a significantly more variable N terminal head. It is this domain which endows each annexin with unique functions in a diverse range of cellular processes including; endo- and exocytosis, cytoskeletal regulation and membrane conductance and organisation. Given their involvement in such a variety of processes it is not surprising that the annexins have also been implicated in a range of disease pathologies. Although there is no singular disease state directly attributed to a dysregulation in annexin function, several pathological conditions are suggested to be modified by the annexins. In this review we shall focus on the growing evidence for the role of the annexins in the progression of cancer, diabetes and the autoimmune disorder anti-phospholipid syndrome

Annexin A11 gene polymorphism (R230C variant) and sarcoidosis in a Portuguese population

Helena Alves - investigadora do Departamento de Promoção da Saúde e Prevenção de Doenças Não Transmissíveis do INSA (Porto)A recent genome-wide association study detected a protective effect for the annexin A11 rs1049550*T allele (R230Cvariant) in susceptibility to sarcoidosis. We evaluated the association between rs1049550 C/T and sarcoidosis susceptibility, distinct disease phenotypes and evolution in a Portuguese population. We performed a case-control study of 208 patients and 197 healthy controls. Samples were genotyped for rs1049550 C/T using real-time polymerase chain reaction. The frequency of the annexin A11 rs1049550*T allele was significantly lower in patients than in controls (33.2 vs 44.9%, P < 0.001). Odds ratio of 0.52 and 0.44 were obtained, respectively for carriers of one (CT) and two (TT) copies normalized to the CC wild-type genotype (P < 0.001). There were no significant differences in patients with and without Löfgren syndrome. A significant increase in the frequency of the T allele was observed in patients with bronchoalveolar lavage (BAL) fluid neutrophilia (P = 0.04). No significant associations were seen for lung function pattern, radiological stages or different forms of disease evolution. Our study confirms that rs1049550*T allele exerts a significant protective effect on sarcoidosis susceptibility. Given the role of annexin A11 in cell division, apoptosis and neutrophil function, this polymorphism may affect key elements of granulomatous and interstitial inflammation in sarcoidosis

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

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

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