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First published online 14 November 2002
doi: 10.1242/jcs.00190

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EhPAK, a member of the p21-activated kinase family, is involved in the control of Entamoeba histolytica migration and phagocytosis

Elisabeth Labruyère^1,*, Christophe Zimmer², Vincent Galy^1,, Jean-Christophe Olivo-Marin² and Nancy Guillén¹

¹ Unité de Biologie Cellulaire du Parasitisme, INSERM U389, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
² Unité d'Analyse d'Images Quantitative, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
Present address: Gene expression program, EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

View larger version (35K): [in a new window]   Fig. 1. Molecular description of EhPAK. (A) The EhPAK region containing the few differences observed between the EhPAK (1) sequence (this paper) and the EhPAK (2) sequence (Gangopadhyay et al., 1997). (B) Amino-acid sequence comparison of EhPAK to the human PAK2 (HsPAK2). The N-terminal sequence of EhPAK shows a polybasic sequence (bold and italic) and a proline-rich sequence (italic) and the C-terminal sequence contains a predicted ATP-binding site (bold and underlined) and catalytic site (bold). The regulatory N-terminal sequence of human PAK2 contains the Nck-binding motif (bold), the polybasic region (italic), the CRIB domain (italic, bold and underlined) and the PIX interacting sequence (grey). The catalytic domain of HsPAK2 showing the ATP binding site (bold and underlined) and the catalytic site (bold). Accession numbers: EhPAK (1) EMBL X98048 and HsPAK2 (u24153.gb_pr). Sequence comparisons were performed with BESTFIT function in the GCG program. (C) Schematic representation of EhPAK and HsPAK2 showing their N-terminal region (oblique lines) and their catalytic domain (grey). The proline-rich sequence (open circle), CRIB motif (hatched box), ATP-binding site (black box) and catalytic site (white box) are indicated. (D) Shematic representation of the GST-hybrid fusion proteins produced in E. coli and purified.

View larger version (26K): [in a new window]   Fig. 2. Biochemical characteristics of EhPAK. (A) Interaction of truncated EhPAK with small GTPases Rac1 or Cdc42 GST-hybrid proteins carrying —C-PAK (5 µg), —N-PAK (5 µg), —Np-PAK (5 µg) and —WASP (0.5 µg) coupled to glutathione Sepharose beads were incubated with purified recombinant GTPases (1.5 µg), human Rac1 or human Cdc42, activated (+, GTPS loaded) or not (—, loaded with GDP). The potential complex (hybrid protein)/GTPase was separated by electrophoresis, transferred to nitrocellulose, and the presence of GTPases was revealed by immunoblotting with mAbRac1 or mAbCdc42 antibodies. (B) Kinase activity of the C-terminal domain of EhPAK. The hybrid protein GST-C-PAK (1 µg) immobilised on glutathione Sepharose beads and thrombin-cleaved derivative soluble protein C-PAK (500 ng) and uncoated glutathione Sepharose beads were subjected to an in vitro kinase assay using myelin basic protein (MBP) as the substrate in the presence of [-32P]ATP. Samples were run on a gel and analysed with a phosphoimager, and the phosphorylation was quantified in arbitrary units with the IQ Mac V12 molecular imaging system. An autophosphorylation background of MBP is shown in the control line and a large increase in MBP phosphorylated was obtained when the carboxylic domain of EhPAK was added.

View larger version (37K): [in a new window]   Fig. 5. Effect of EhPAK on F-actin distribution. (A) Micrographs represent confocal analysis of the different labelling obtained in optical planes through the middle of the cells. Moving amoebae (control and C-PAK+) were fixed on cover slips, filamentous actin was decorated with phallacidine (green) and PAK was stained with specific anti-EhPAK polyclonal antibodies (red). As observed by DIC, the control strain was elongated, polarised and presented a unique pseudopod, and the strain overexpressing C-PAK is a rounded cell presenting multiple membrane extension. F-actin was diffusely distributed in the cytoplasm of the C-PAK+ strain. For the control strain, F-actin was enriched at one pole of polarised parasites. EhPAK concentrated in the membranous protrusion for both C-PAK+ and the control amoeba. Bars, 10 µm. (B) Cell fractionation in the presence of Triton X100 was performed on C-PAK+ and control strains. The fractions were electrophoresed and analysed by western blot with anti-PAK or anti-actin antibodies. The revealed protein was analysed with a phosphoimager, bands were quantified in arbitrary units with the IQ Mac V12 molecular imaging system. An equivalent amount of PAK was found in both TritonX100-soluble (s) and TritonX100-insoluble (i) fractions. The C-PAK peptide, carried by the C-PAK+ strain, was present in the TritonX100-insoluble fraction. The total amount of PAK remained unchanged in each strain. In the control cells, the total actin partitioned corresponded to 70% TritonX100-soluble and 30% TritonX100-insoluble fractions, whereas in the C-PAK+ strain actin was at 10% in the TritonX100-insoluble and 90% in the TritonX100-soluble fractions.

View larger version (103K): [in a new window]   Fig. 3. Cellular distribution of EhPAK and F-actin in a motile E. histolytica. Amoeba moving from left to right display a prominent pseudopod that guides its locomotion. After fixation and permeabilisation, filamentous actin was decorated with FITC-phallacidine (green), and EhPAK was stained with purified polyclonal antibodies against EhPAK (red). A confocal micrograph of an optical plane in the middle of the parasite showed that F-actin was moderately concentrated in the pseudopod, distributed in the cytoplasm and enriched at the posterior end of the parasite. EhPAK was dispersed throughout the cytoplasm and was concentrated in pseudopodia extensions. Superimposition of the two stainings indicates that F-actin and EhPAK were concentrated at specific opposite sites of the parasite. The differential interference contrast (DIC) image shows E. histolytica with a pseudopod at the front of migration. Bar, 5 µm.

View larger version (72K): [in a new window]   Fig. 4. Effect of EhPAK on E.histolytica morphology. The histograms represent the number of pseudopod counted for each strain. Fixed amoebae were examined by microscopy, and pseudopodia (indicated by the arrow) and/or pseudopodia-like structures (indicated by the stars) were counted as described in Materials and Methods. Note that the majority of control cells (90%) showed one to three pseudopodia. By contrast, 90% of the C-PAK+ strain simultaneously displayed three to six protrusions.

View larger version (17K): [in a new window]   Fig. 7. Accumulation of C-PAK enhances phagocytosis of human RBCs by E. histolytica. The histogram shows the erythrophagocytosis rates of E. histolytica strains. Wild-type (WT) (white) and transfectants C-PAK (black) or MyoIB+ (grey) were used. To quantify engulfed RBCs the concentration of haemoglobin inside parasites was measured as described previously (Voigt et al., 1999). Bars indicate standard deviations of the means after three independent experiments.

View larger version (26K): [in a new window]   Fig. 6. Overproduction of C-PAK affects E. histolytica motility. The mean speed (µm/s) and the trajectory diameter (µm), representing the largest distance between two arbitrary points of the total trajectory of each amoeba (control and C-PAK+ •), were plotted. When the trajectory diameter was greater than the size of an amoeba (25 µm), we concluded that the parasite migrated, and when it was below 25 µm, the parasite was considered to be unable to migrate. Note that 80% of the analysed C-PAK+ population did not migrate. Velocities of amoeba from the control strain reached 0.9 µm/s. Each plot is a compilation of three distinct experiments.