Annexin A1 and A2: roles in retrograde trafficking of Shiga toxin

Abstract

Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA(2)), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA(2) activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA(2).

Figure 1. ShigaB transport to the Golgi is regulated by annexin A1 and A2.

HeLa cells were transfected with control, annexin A1 or A2 siRNA for 72 h. For sulfation measurements, cells were starved in the presence of radioactive sulfate for 3 h. ShigaB-sulf2 or Ricin-sulf1 was then added and the incubation proceeded for an additional one or two hours, respectively. Cells were lyzed, ShigaB or ricin immunoprecipitated, separated by electrophoresis and analyzed by autoradiography. The protein knockdown level was investigated in total cell lysates by immunoblotting. (A) Cell lysates were analyzed by western blotting (upper panel) with the indicated antibodies demonstrating protein knockdown of annexin A1 and A2 by the indicated siRNA oligos. Hsp90 represents loading control. Autoradiography (lower panel) showing results from the corresponding sulfation experiment. (B) and (C) Quantative data from protein sulfation for ShigaB and ricin respectively, plotted as percentages of control values. Quantifications of sulfation are the average of 3–8 independent experiments, each performed in parallel, error bars indicating standard error of the mean; *p<0.05, **p<0.005 indicate statistically significant change.

Figure 2. Rescue of phenotype by over-expression of wild type annexins.

HeLa cells where transfected with indicated siRNA and subjected for 24 h either to Fugene treatment as control, A1-GFP, A1-Y21F-GFP or A2-CFP transfection as indicated. (A) Western blots of cells lysates for the conditions stated under each lane, stained with the indicated antibody and showing expression of constructs and/or endogenous annexins for one representative experiment (duplicates are not shown here for more clarity). (B) Quantative data from ShigaB sulfation (black bars) and total protein sulfation (white bars) are plotted as an average percentage of control values for at least 3 independent experiments, each performed in parallel, error bars indicating standard error of the mean; *p<0.05 indicates statistically significant change.

Figure 3. Time course of ShigaB sulfation in annexin A1 or A2 depleted cells.

(A) HeLa cells transfected with control or annexin A1 siRNA for 72 h were analyzed for the amount of ShigaB being sulfated at different time points after addition of StxB. Sulfation data are plotted as percentages of the amount of sulphated ShigaB after 30 min incubation with control siRNA treated cells. Black and white bars represent sulfation of ShigaB in control and annexin A1 siRNA treated cells, respectively. Data presented are the average of 3–4 independent experiments, each performed in parallel, error bars indicating standard error of the mean, *p<0.05; **p<0.005 indicates statistically significant change. (B) After the same treatment as in (A), cells were fixed and stained with antibodies against TGN46 and ShigaB. Top panel shows representative confocal pictures for 30 min incubation with StxB, scale bars 20 μm. Left graphic shows quantification of amount of ShigaB colocalized with TGN46. In the right graphic, mean intensity of ShigaB in the Golgi area for the same representative experiment is plotted as percentage of mean intensity in the whole cell. Data presented are the average of at least 35 cells per condition. Quantifications where obtained with ImageJ software, error bars indicating standard error of the mean.

Figure 4. Stx transport in annexin A1 depleted cells is regulated by PKCδ and PLA2.

(A) Golgi transport of Shiga toxin was evaluated as described in materials and methods by quantification of sulfated ShigaB in HeLa cells transfected with siRNA against annexin A1 or non targeting siRNA, pretreated with the indicated inhibitors. Data from Stx sulfation are plotted as percentages of the value obtained for HeLa cells transfected with control siRNA and treated with DMSO. The white and black bars represent ShigaB-sulf2 sulfation for control and annexin A1 knockdown cells respectively. Data presented are the average of 3 independent experiments, each performed in parallel, error bars indicating standard error of the mean. *p<0.05 indicates statistically significant change between annexin A1 knockdown cells and the corresponding control siRNA treated cells. (B) After treatment with either 5 µM ONO-RS-082 for 30 min or 30 µM MAFP for 1 h, HeLa cells were incubated for 30 min with ShigaB before fixation and staining as indicated in the materials and methods section with antibodies against TGN46 and ShigaB. Panel shows representative confocal pictures, scale bars 20 μm. Graphic shows quantification of amount of ShigaB colocalized with TGN46 in one representative experiment plotted as percentage of control condition. Data presented for one representative experiment (n = 3) are the average of at least 30 cells per condition. Quantifications were obtained with Zen 2009 software from Zeiss, error bars indicating standard error of the mean.

Figure 5. Close proximity of Annexin A1 and cPLA2α in HeLa cells.

(A) Close proximity of annexin A1 with cPLA₂α was evaluated using proximity ligation assay from Duolink. Fixed and permeabilized cells were incubated with the indicated primary antibodies by pair or alone as negative control. Scale bar is 20 μm. Dots per cell were automatically counted using ImageJ software and the data presented in the diagram is the average of at least 40 cells per condition in one representative experiment (n = 3), error bars indicating standard deviation. Values for negative controls were 10.3±2.7 and 0.3±0.1 dots per cell for cPLA₂ α and annexin A1 antibodies respectively. (B) Before proximity ligation assay, cells were washed and incubated for 30 min in Hepes buffered medium. They were subsequently incubated for 10 min with ShigaB before staining with annexin A1 and cPLA₂α antibodies in combination. Scale bar is 20 μm. Interaction events where evaluated as in A. Data presented are the average of at least 40 cells quantified per condition for one representative experiment (n = 3), error bars indicating standard deviation.