The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes

Abstract

The Ca2+- and lipid-binding protein annexin 2, which resides in a tight heterotetrameric complex with the S100 protein S100A10 (p11), has been implicated in the structural organization and dynamics of endosomal membranes. To elucidate the function of annexin 2 and S100A10 in endosome organization and trafficking, we used RNA-mediated interference to specifically suppress annexin 2 and S100A10 expression. Down-regulation of both proteins perturbed the distribution of transferrin receptor- and rab11-positive recycling endosomes but did not affect uptake into sorting endosomes. The phenotype was highly specific and could be rescued by reexpression of the N-terminal annexin 2 domain or S100A10 in annexin 2- or S100A10-depleted cells, respectively. Whole-mount immunoelectron microscopy of the aberrantly localized recycling endosomes in annexin 2/S100A10 down-regulated cells revealed extensively bent tubules and an increased number of endosome-associated clathrin-positive buds. Despite these morphological alterations, the kinetics of transferrin uptake and recycling was not affected to a significant extent, indicating that the proper positioning of recycling endosomes is not a rate-limiting step in transferrin recycling. The phenotype generated by this transient loss-of-protein approach shows for the first time that the annexin 2/S100A10 complex functions in the intracellular positioning of recycling endosomes and that both subunits are required for this activity.

Figure 1.

Protein levels and intracellular localization of annexin 2 and its cellular ligand S00A10 in HeLa cells transfected with annexin 2-specific siRNAi. (A) Immunoblot showing the protein amounts of annexin 1, annexin 2, vimentin, and S100A10 in HeLa cells lysates prepared from cells transfected with annexin 2-specific siRNA (anx2siRNA) or mock transfected control cells (ct). Note that annexin 1 expression was not altered, indicating the specificity of annexin 2 silencing. Vimentin served as an internal control for equal loading. A quantification of the annexin 2 and S100A10 signals determined by densitometry and calculated from at least six independent transfections is given below the blots. *p < 0.05. (B) Immunofluorescence staining of cells treated with annexin 2-specific siRNA using a monoclonal antibody (mAb) recognizing only free annexin 2 (a), polyclonal antibodies (pAb) recognizing both free and complexed annexin 2 (b and c), or an mAb recognizing the annexin 2 ligand S100A10 (d) revealed a strong decrease in annexin 2 and S100A10 expression, with only the cortical heterotetrameric annexin 2/S100A10 complex still detectable (arrow-heads). Dotted lines indicate position of cells with reduced annexin 2 levels. Bar, 10 μm.

Figure 2.

Annexin 2 gene-silencing specifically alters the distribution of transferrin-positive recycling endosomes. HeLa cells either mock-transfected (A and B) or transfected with annexin 2-specific siRNA (C and D) were incubated with TxTf at 37°C for 5 min followed by a 20-min chase to label the recycling endosomes. The annexin 2 distribution visualized by staining with the monoclonal HH7 antibody is given in A and D, whereas B and E show the distribution of internalized TxTf. Phase contrast images of the same cells are shown in C and F to visualize the localization of the nuclei. Note the marked perinuclear clustering transferrin label in the siRNA-treated cells. Bars, 10 μm.

Figure 3.

Down-regulation of annexin 2/S100A10 does not affect the internalization into sorting endosomes or transport along the late endocytic pathway. (A, top) HeLa cells were transfected with the annexin 2 siRNA (panels labeled anx2siRNA) or mock transfected (panels labeled control). To preferentially label recycling endosomes, cells were then allowed to internalize TxTf at 37°C for 5 min followed by 20-min chase. Distribution of rab11 (green label) was visualized using polyclonal anti-rab11 antibodies and merged with the localization of the labeled transferrin (red label). In cells with reduced annexin 2 levels, internalized TxTf accumulates in perinuclear rab11-positive structures. (A, bottom) Mock-transfected (control) or annexin 2 siRNA-transfected cells (anx2siRNA) were subjected to a 10-min pulse, 15-min chase procedure by using DiI-LDL (red label). Cells were then stained for LAMP-1 (green label) to confirm that late endosomes were labeled by the internalized DiI-LDL. No obvious differences in the distribution of DiI-LDL were observed in annexin 2 down-regulated compared with control cells. (B, top) TxTf was internalized into mock-transfected (control) or annexin 2-depleted cells (anx2siRNA) at 16°C for 30 min to label only sorting endosomes. These sorting endosomes did not accumulate in the perinuclear region of the annexin 2-silenced cells but showed a dispersed pattern identical to the distribution observed in control cells. A significant fraction of the TxTf-labeled sorting endosomes (red label) is positive for EEA1 as revealed by colabeling with monoclonal anti-EEA1 antibodies (green label). (B, bottom) Mock transfected (control) or siRNA-treated cells (anx2siRNA) were allowed to internalize TxTf at 16°C for 30 min and the distribution of rab11 (green label) was visualized in comparison with that of internalized TxTf (red label). Note some perinuclear accumulation of rab11 in the annexin 2-depleted cells. Bars, 10 μm. The enlarged insets show a twofold magnification of the boxed regions.

Figure 3.

Down-regulation of annexin 2/S100A10 does not affect the internalization into sorting endosomes or transport along the late endocytic pathway. (A, top) HeLa cells were transfected with the annexin 2 siRNA (panels labeled anx2siRNA) or mock transfected (panels labeled control). To preferentially label recycling endosomes, cells were then allowed to internalize TxTf at 37°C for 5 min followed by 20-min chase. Distribution of rab11 (green label) was visualized using polyclonal anti-rab11 antibodies and merged with the localization of the labeled transferrin (red label). In cells with reduced annexin 2 levels, internalized TxTf accumulates in perinuclear rab11-positive structures. (A, bottom) Mock-transfected (control) or annexin 2 siRNA-transfected cells (anx2siRNA) were subjected to a 10-min pulse, 15-min chase procedure by using DiI-LDL (red label). Cells were then stained for LAMP-1 (green label) to confirm that late endosomes were labeled by the internalized DiI-LDL. No obvious differences in the distribution of DiI-LDL were observed in annexin 2 down-regulated compared with control cells. (B, top) TxTf was internalized into mock-transfected (control) or annexin 2-depleted cells (anx2siRNA) at 16°C for 30 min to label only sorting endosomes. These sorting endosomes did not accumulate in the perinuclear region of the annexin 2-silenced cells but showed a dispersed pattern identical to the distribution observed in control cells. A significant fraction of the TxTf-labeled sorting endosomes (red label) is positive for EEA1 as revealed by colabeling with monoclonal anti-EEA1 antibodies (green label). (B, bottom) Mock transfected (control) or siRNA-treated cells (anx2siRNA) were allowed to internalize TxTf at 16°C for 30 min and the distribution of rab11 (green label) was visualized in comparison with that of internalized TxTf (red label). Note some perinuclear accumulation of rab11 in the annexin 2-depleted cells. Bars, 10 μm. The enlarged insets show a twofold magnification of the boxed regions.

Figure 4.

Analysis of transferrin recycling and endosomal membrane fractionation in annexin 2/S100A10 down-regulated cells. (A) Kinetics of transferrin recycling. Cells transfected with the annexin 2-specific siRNA (▾) or mock-transfected control cells (•) were incubated with HRP-Tf for 30 min at 16°C. After removing surface bound Tf by acid wash, cells were incubated in the presence (open symbols) or absence (closed symbols) of 100 nM wortmannin (WN), shifted to 37°C, and the release of recycled Tf was monitored at the indicated times by measuring released and remaining intracellular HRP enzymatic activity. Recycled Tf was calculated as percentage of released to total HRP activity. Each time point represents the mean of triplicate samples. The experiment was repeated three times with similar results, and a typical set of data is given. (B) Endosomal membranes and distribution of internalized transferrin. HeLa cells transfected with the annexin 2-specific siRNA or mock-transfected cells (control) were fed with biotinylated transferrin to preferentially label recycling endosomes and homogenized. Membrane fractions of the postnuclear supernatant (PNS) were separated in a discontinuous sucrose flotation gradient and fractions enriched in early endosomes (EE), late endosomes (LE), and heavy membranes (HM), respectively, were analyzed by SDS-PAGE and subsequent Western blotting. As shown in the top panel, internalized bTf is mainly recovered in the EEA1-positive fraction containing sorting and recycling endosomes. The bottom panels show the presence of internalized bTf, EEA1, and rab11 in fractions enriched in early, i.e., sorting and recycling, endosomes. No differences in these fractions from control compared with annexin 2/S100A10-depleted cells could be detected. LE and HM fractions were also analyzed in these experiments but not shown because no aberrant accumulation of EEA1, bTf, or rab11 was seen in these fractions. The subcellular fractionations indicate that the density and composition of recycling endosomes and the delivery of internalized transferrin to these structures is unaffected by annexin 2/S100A10 depletion.

Figure 5.

siRNA-mediated silencing of S100A10 specifically alters the positioning of transferrin receptor positive recycling compartment. (A) S100A10 and annexin 2 protein amounts were determined by immunoblotting of lysates prepared from S100A10siRNA-transfected cells. Whereas S100A10 expression is strongly decreased, annexin 2 protein level remains unchanged. Vimentin served as an internal control for equal loading. The corresponding bar graphs were calculated from the densitometric data of at least six independent transfections. *p < 0.05. (B) Immunofluorescence staining with anti-S100A10 antibodies confirmed the siRNA-mediated gene silencing of S100A10. Dotted lines indicate the position of cells with reduced S100A10 level (a). Pulse-chase labeling with TxTf revealed an accumulation of transferrin-positive perinuclear endosomes in cells with down-regulated S100A10 (b). Bar, 10 μm. (C) Kinetics of transferrin recycling. Cells transfected with the S100A10-specific siRNA (▾) or control cells (•) were incubated with HRP-Tf for 30 min at 16°C. After removing surface bound Tf by acid wash, cells were incubated in the presence (open symbols) or absence (closed symbols) of 100 nM wortmannin (WN), shifted to 37°C, and the release of recycled Tf was monitored at the indicated times by measuring released and remaining intracellular HRP enzymatic activity. Recycled Tf was calculated as percentage of released to total HRP activity. Each time point represents the mean of triplicate samples. The experiment was repeated three times with similar results, and a typical set of data is given.

Figure 6.

Ultrastructural analysis of endosomes in annexin 2 siRNA-treated cells. HeLa cells were either mock-transfected (control, A, C, and E) or transfected with annexin 2 siRNA (anx2siRNA, B, D, and F). Cells were then labeled by using a pulse-chase protocol to accumulate HRP-Tf in recycling endosomes and subsequent incubation with DAB that polymerizes in HRP-containing compartments. After permeabilization and immunogold labeling, the cells were inspected by transmission electron microscopy. Due to the HRP-Tf labeling/HRP pulse/DAB polymerization protocol only early, i.e., HRP-positive sorting and recycling endosomes look like dark structures in the electron micrographs. Low magnifications showing a part of the cell, excluding the nucleus, are given in A and B. Here, the labeled endosomes looked like tubular structures throughout the control cells (A), whereas they were more condensed and clustered in the perinuclear region in annexin 2/S100A10 down-regulated cells (B). Bars, 1 μm. Higher magnification of cells labeled with antibodies against TfR (C–F, 5-nm gold), annexin 2 (C and D, 10-nm gold), and clathrin (E and F, 10-nm gold). In control cells (C and E), annexin 2 (C, 10-nm gold) was found on tubular structures enriched in transferrin receptors (C, 5-nm gold). Clathrin (E, 10-nm gold) was associated with individual transferrin receptor (E, 5-nm gold)-positive buds along tubular membranes. In cells transfected with annexin 2-specific siRNA (D and F), no label of endosome-associated annexin 2 was detectable (D) with the few 10-nm gold particles visible representing background of the secondary antibody. The transferrin receptor-positive structures (5-nm gold) often formed highly bent tubules of almost circular shape (D), not seen in control cells (C). They also contained an increased number of endosomal buds heavily decorated with clathrin antibodies (F). Counting of clathrin and TfR immunogold label in nine representative areas selected from three individual micrographs revealed a ratio (clathrin:TfR) of 1:7 in control cells and 1:2 in the depleted cells. Bars, 0.2 μm. The enlarged insets show a twofold magnification of the boxed regions.

Figure 7.

Expression of S100A10 or the N-terminal domain of annexin 2 rescues the S100A10 or annexin 2 siRNA phenotype, respectively. HeLa cells silenced and additionally transfected with expression plasmids indicated were pulsed for 5 min with TxTf followed by a 20-min chase. The intrinsic YFP and GFP fluorescence identifying YFP-S100A10 – and PMNanx2-GFP–expressing cells is given in A, C, E, and G, respectively, whereas B, D, F, and H show the distribution of internalized TxTf. Although the characteristic clustering of the internalized transferrin was seen in S100A10-silenced cells (A and B), expression of YFP-S100A10 restored the phenotype with the label now occurring in vesicles more widely spread throughout the cell (A and B). In annexin 2 siRNA-treated cells depleted of the annexin 2/S100A10 complex, the perinuclear accumulation of TxTf in clustered recycling endosomes could not be rescued by YFP-S100A10 expression (C and D; note the clustered appearance of recycling endosomes in both YFP-S100A10–expressing and –nonexpressing cells). In contrast, recycling endosome clustering induced by annexin 2/S100A10 down-regulation was reversed by PMNanx2-GFP, which encodes amino acids 1–27 of human annexin 2 fused to GFP (E and F; note the nonclustered appearance of internalized TxTf in the two cells expressing PM-Nanx2-GFP). Expression of the PMNanx2-GFP construct in S100A10 siRNA-treated cells did not result in a significant reversion of the recycling ensdosome clustering (G and H). Dotted lines indicate position of depleted cells, arrowheads point toward the respective transferrin-positive recycling compartments. Bars, 10 μm.