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. 2009 Jun;1793(6):1086-95.
doi: 10.1016/j.bbamcr.2008.10.007. Epub 2008 Oct 29.

Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate

Matthew J Hayes  1 , Dong-Min Shao, Adam Grieve, Tim Levine, Maryse Bailly, Stephen E Moss
Affiliations

Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate

Matthew J Hayes et al. Biochim Biophys Acta. 2009 Jun.

Abstract

Vesicle rocketing has been used as a model system for understanding the dynamics of the membrane-associated F-actin cytoskeleton, but in many experimental systems is induced by persistent, non-physiological stimuli. Localised changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in membranes stimulate the recruitment of actin-remodelling proteins to their sites of action, regulate their activity and favour vesicle rocketing. The calcium and anionic phospholipid-binding protein annexin A2 is necessary for macropinocytic rocketing and has been shown to bind both PI(4,5)P2 and the barbed-ends of F-actin filaments. Here we show that annexin A2 localises to the comet tails which form constitutively in fibroblasts from patients with Lowe Syndrome. These fibroblasts are deficient in OCRL1, a phosphatidylinositol polyphosphate 5-phosphatase with specificity for PI(4,5)P2. We show that upon depletion of annexin A2 from these cells vesicle rocketing is reduced, and that this is also dependent upon PI(4,5)P2 formation. Annexin A2 co-localised with comet-tails induced by pervanadate and hyperosmotic shock in a basophilic cell line, and in an epithelial cell line upon activation of PKC. In vitro annexin A2 promoted comet formation in a bead-rocketing assay and was sufficient to link F-actin filaments to PI(4,5)P2 containing vesicles. These observations are consistent with a role for annexin A2 as an actin nucleator on PI(4,5)P2-enriched membranes.

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Figures

Fig. 1
Fig. 1. HOS and pervanadate induce macropinocytic rocketing in RBL cells.
(A) Sections through the cells, from the bottom to the midline, reveal formation of actin rockets predominantly at the points of cell contact. Blue arrow indicates the cup-shaped region at the tip of a large vesicle. Scale bar=35 μm. (B) When the extracellular matrix was labelled with a fluorescent marker (red) it was seen to be internalised when cells underwent rocketing. Arrows show vesicular structures labelled with TRITC-collagen I (red) at the end of comet tails. Scale bar in A=30 μm, in B top row=30 μm, bottom row=8 μm.
Fig. 2
Fig. 2. Annexin A2 colocalises with F-actin and PI(4,5)P2 in rockets induced by HOS and pervanadate.
RBLs were induced to rocket using hyperosmotic stress (HOS) and sodium pervanadate (200 μM). (A) Annexin A2 was observed to colocalise with actin on the tails of comets, particularly those at the periphery of cells. Scale bar=12 μm. (B) TMR-labelled PI(4,5)P2 (red) localised to the tips (top panels) and the in some cases also the actin tails (green) (lower panels) of comets. Scale bar in A=5 μm). (C) Annexin A2 partially colocalised with actin and PI(4,5)P2 (localised using the PH domain of PLCδ-fused to GFP) in the tails of HOS/pervanadate induced rockets. Scale bar=5 μm.
Fig. 3
Fig. 3. Annexin A2 is associated with rockets in Lowe’s fibroblasts.
Lowe’s fibroblasts were fixed and stained for F-actin. In a small proportion of cells we observed large comet tails (A) scale bar=15 μm. In the majority of cells however we identified a large population of tiny rockets which clustered together in a perinuclear zone (B) Scale bar=5 μm. Co-staining of the cells with Annexin A2 revealed that it associated with these rockets in a discontinuous, punctate fashion. (C) scale bar=25 μm, close-ups scale bar=3 μm.
Fig. 4
Fig. 4. PI(4,5)P2 and calcium are necessary for actin-based rocketing in Lowe’s fibroblasts.
(A) Lowe’s fibroblasts or control skin fibroblasts were treated with nothing, 2-butanol (2-but) or 1-butanol (1-but) for 20 min before fixing and staining for actin. Cells were classified as containing > 100, <100, large rockets or no rockets. Large numbers of rockets were observed in Lowe’s fibroblasts but not in control fibroblasts, and rocketing was inhibited by the addition of the primary alcohol butan-1-ol, but not by the secondary alcohol butan-2-ol. (B) Lowe’s fibroblasts treated for 20 min with BAPTA-AM (50 μM) fail to generate internal rockets, but external ‘filopod-like’ extensions, appear on their apical surface. (a) Section close to the base of the cell showing stress fibres but no internal rockets. Scalebar=50 μm. (b) Apical surface of the cell, showing filopod-like extensions. (c) Shallow view of a 3D reconstruction of the cell showing the long apical processes. (d) Apical surface of a cell showing filopods, blue boxes represent close-ups scale bar=50 μm (e and f) of filopods with club-like actin structures at their tips, which may be associated with vesicles. Scale bare=8 μm. (C) Treatment with BAPTA-AM inhibits rocking in Lowe’s fibroblasts (results represent mean and SEM of 3 independent experiments at least 100 cells were examined in each case.) ** Represent P values <0.01.
Fig. 5
Fig. 5. Annexin A2 is involved in the actin-mediated rocketing observed in Lowe’s fibroblasts.
A) Western blot showing partial depletion of annexin2 from Lowe’s fibroblasts using siRNA. B) Complete knockdown of Annexin A2 was only apparent in a minority of cells. Close examination of the cytoplasm reveal punctate actin in Annexin A2-depleted cells, but actin rockets in the cells in which Annexin A2 is present. (C) Histogram showing inhibition of actin-rocketing in Lowe’s fibroblasts depleted of Annexin A2. Cells were classified as having greater than 100, less than a hundred or no visible rockets. We also examined the cells for the presence of large rockets. At least 100 cells were counted in 3 independent experiments. siRNA treated cells showed a significant reduction in the number of rockets (P<0.001). Scale bars= 50 μm or 6 μm in close-ups.
Fig. 6
Fig. 6. Annexin A2 is present on actin rockets induced by PMA in MDCK cells.
(A) Actin rockets were induced in MDCK cells using 10 μM PMA. The cells were fixed and stained for Annexin A2 and actin. Rocket formation predominantly occurred in cells at the periphery of small colonies. These stubby rockets co-stained strongly for Annexin A2. (B) butan-1-ol but not butan-2-ol completely abrogated rocketing suggesting that PI(4,5)P2 production down-stream of PKA activation was necessary for comet formation. Scale bars=80 μm. (C) Close-ups showing the actin cytoskeleton. Yellow arrows indicate rocket tails. Scale bars=20 μm.
Fig. 7
Fig. 7. Annexin A2 promotes actin tail formation in vitro.
(A) Annexin A2 promotes actin comet-tail formation on scar-VCA coated polystyrene beads. By 30 min in the presence of Annexin A2 (4 μM) all beads have a prominent actin tail, whereas in the absence of Annexin A2 only a minority of beads have succeeded in nucleating tail formation (representative fields of view, 5% rhodamine-labelled Mg-ATP actin). (B) Histogram quantifying the data in (A) (accumulated from 25 fields of view – approximately 300 beads). Representative experiment of 3 repeats. ** data significant to P<0.001. Also: growth of actin filament comet-tails on scar-VCA coated beads as a function of time. Measurements of actin tail-length were calculated using Metamorph (at least 200 tails at each time point). Annexin A2 was added at 4 μM, at lower concentrations there was no significant effect (data not shown). Control beads were incubated with 4 μM BSA in the same buffer. We noted a significant increase in the percentage of beads with tails at all time points. (P<0.001). (C) Annexin A2 localises to the ends of F-actin filaments. Purified recombinant Annexin A2 was added to pre-formed F-actin filaments, and was immuno-labelled using an anti-Annexin A2 antibody and a secondary anti-mouse antibody conjugated to 10 nM gold. The sample was then shadowed and prepared for electron microscopy.
Fig. 8
Fig. 8. Annexin A2 is sufficient to link F-actin filaments to vesicles charged with anionic phospholipids.
(A) Liposomes were formed containing 10% PS and 90% PC. They were added to a preformed F-actin filaments which were stabilized and labelled with FITC-phalloidin in a buffer containing 50 μM calcium. There was no obvious association of filaments with vesicles (top left panel). When Annexin A2 was added (4 μM) however, the F-actin filaments associated with the vesicles, forming large aggregates. (B) In another experiment Annexin A2 was added to vesicles containing 2.5% PI(4,5)P2 and 97.5% PC. Annexin A2 was able to associate the F-actin filaments to the vesicles to induce the formation of actin-vesicle aggregates (left hand panels). In this case the vesicles were labelled internally with rhodamine-dextran, and the vesicles themselves can be seen in the panels on the right. Aggregation occurred in the presence (top) or absence (bottom) of 50 μM calcium. Scale bar=50 μm.
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