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First published online 29 June 2004
doi: 10.1242/jcs.01208

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Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes

Ursula Rescher^*, Daniela Ruhe^*, Carsten Ludwig^*, Nicole Zobiack^* and Volker Gerke

Institute for Medical Biochemistry, Center for Molecular Biology of Inflammation, von Esmarch-Str. 56, Münster, 48149, Germany

View larger version (75K): [in a new window]   Fig. 1. EPEC-infection induces annexin 2-p11 accumulation at PtdIns(4,5)P2-enriched sites of actin pedestal formation. HeLa cells, transiently expressing the different tagged proteins indicated, were infected with EPEC. Three hours after infection, cells were fixed, permeabilized and analyzed for the distribution of the respective, ectopically expressed protein. Staining with Texas Red-phalloidin (Tx-phalloidin) revealed the characteristic reorganization of the actin cytoskeleton beneath micro-colonies of adhering bacteria. Annexin 2 (anx2) -GFP and YFP-p11 accumulate at sites of EPEC attachment. PHD-YFP localizes to the same actin-rich structures, indicating elevated PtdIns(4,5)P2 levels at sites of pedestal formation. Since PtdIns4P 5-kinase is also enriched, the rise in PtdIns(4,5)P2 is possibly caused by its enzymatic activity. Bars 10 µm.

View larger version (103K): [in a new window]   Fig. 2. Annexin 2 is recruited to PtdIns(4,5)P2-rich structures elicited by active Arf6. (A) HeLa cells, transiently expressing wild-type Arf6 or co-expressing Arf6 and PHD-YFP, were treated for 30 minutes with AlF (30 mM NaFl and 50 µM AlCl3) and processed for annexin 2 immunostaining or plasma membrane labeling with CM-DiI, respectively. Both annexin 2 (anx2) and PHD-YFP localize to plasma membrane protrusions induced by AlF-mediated Arf6 activation (arrowheads). To ascertain that increased signals at sites of membrane ruffling are not owing to a general increase in membrane thickness, mean fluorescence intensity ratios for signals in ruffled (arrowheads) to non-ruffled (arrows) regions were determined for PHD-YFP and DiI, respectively. Results are presented as percent-ratio of the mean fluorescence intensity ± s.e.m. *P<0.05, bars 10 µm. (B) HeLa cells were co-transfected with constitutively active Arf6 Q67L and PHD-YFP, GFP-actin, annexin 2 (anx2)-GFP, or YFP-p11. Annexin 2-GFP and YFP-p11 both clearly localize to the GFP-actin-positive, PtdIns(4,5)P2-rich vacuoles that were induced by Arf6 Q67L expression. Bars 10 µm.

View larger version (25K): [in a new window]   Fig. 3. Annexin 2 binds directly to PtdIns(4,5)P2. (A) Co-sedimentation assays, employing the annexin 2-p11 heterotetramer and phospholipid liposomes. Annexin 2-p11 complex was mixed with brain-extract liposomes containing no (–) or 0.5% (w/w) of the indicated phosphoinositides in the presence of 1 mM Ca2+ (+) or in the presence of 1 mM EGTA (–). Liposomes were collected by ultracentrifugation and bound annexin 2-p11 complex was detected by immunoblotting with an antibody specific for the annexin 2 subunit. Experiments were carried out several times and a representative blot is shown. Signal intensities of blots of three independent experiments were quantified by densitometric scanning. Relative intensities are presented as fold-binding (mean value±s.e.m.) over control, i.e. brain-extract liposomes without added phosphoinositides, in the diagramm below the blot. Note the increase in co-pelleted annexin 2 in the case of liposomes containing PtdIns(4,5)P2 which is seen in the absence of Ca2+. (B) Lipid-plate binding-assays. Microtiter wells were coated with the indicated lipids and blocked with BSA. Purified annexin 2-p11 complex was added in the presence of Ca2+ or EGTA as indicated; (–) denotes reactions carried out in buffer alone. The amount of bound complex was determined by a colorimetric reaction using annexin 2-specific antibodies and peroxidase-coupled secondary antibodies. In the absence of phosphoinositides (control), a weak background signal was detected. Binding assays were performed at least three times and the bar graphs give the mean value ± s.e.m. calculated from triplicate samples of a representative individual experiment. (C) The annexin 2-p11 complex was dissociated and the binding of the individual subunits to both, immobilized PtdIns(4,5)P2 and PtdIns(3,4,5)P3 was investigated with the experimental setup described in (B), using either annexin 2- or p11-specific antibodies. (D) The binding of monomeric annexin 2 to immobilized PtdIns(4,5)P2 was compared with that of the recombinantly expressed PH domain of human PLC-1. Experiments were carried out using increasing molar ratios of PHD-PLC to annexin 2 and bound annexin 2 was determined as described in (B).