First published online 14 November 2002
doi: 10.1242/jcs.00190
EhPAK, a member of the p21-activated kinase family, is involved in the control of Entamoeba histolytica migration and phagocytosis
Elisabeth Labruyère1,*,
Christophe Zimmer2,
Vincent Galy1,
,
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
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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.
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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.
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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.
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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.
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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.
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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.
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© The Company of Biologists Ltd 2003
S 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 [