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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01178

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Overexpression of myosin IB in living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis

Sabrina Marion¹, Claire Wilhelm², Heike Voigt^3,*, Jean-Claude Bacri² and Nancy Guillén^1,

¹ Unité de Biologie Cellulaire du Parasitisme, INSERM U389, Institut Pasteur, 28 rue du Dr Roux 75724, Paris CEDEX 15, France
² Laboratoire des Milieux Désordonnés et Hétérogènes, UMR7603, and FR2438 CNRS `Matière et Systèmes Complexes', Université Pierre et Marie Curie, 4 Place Jussieu, 75005 Paris, France
³ Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Dr Roux 75724, Paris CEDEX 15, France

View larger version (29K): [in a new window]   Fig. 1. The rotating magnetic field system to measure the cytoplasm viscosity in living cells. (A) Amoebae that have previously phagocytosed magnetic beads for 3 hours adhered to the bottom of a chamber hermetically sealed containing nutrition medium and maintained at 37°C. The two pairs of magnetic coils are then adjusted within the horizontal plan around the measuring chamber. (B,C) The two types of deformation applied inside living amoebae by the rotation of the magnetic field. (B) 45° step rotation of the phagosome pairs. (C) Permanent rotation at a frequency of f=0.25 Hz. In both cases, defines the position of the phagosome pair relative to the x axis: for the 45° step rotation, varies between 0 (initial position of the pair) and 45° (position of the magnetic field). When t>1 second, the pair is aligned with the applied magnetic field. In the case of the permanent rotation, varies continuously from 0° to 360° and shows the angular delay between the direction of the phagosome pair and the direction of the magnetic field B. The value of this angle directly correlates with the viscosity value of the medium surrounding the pair. (D,E) Image sequences showing the phagosome pair movement inside adherent amoeba, in the case of the 45° step rotation (D) or the permanent rotation (E), in which several magnetic pairs are examined in the same microscope field. (F) Representative creep rotational curve of one pair of phagosome obtained with the 45° step rotation of the magnetic field within the control strain (black) or in cells treated with latrunculin A (grey). The response of the phagosome pair can be described in two steps: an immediate elastic jump, G, followed by a viscous relaxation, . For the curves depicted in this graph, G=2.1 Pa and =0.27 Pa seconds in the control strain and G=2.4 Pa and =0.09 Pa seconds after Lat-A treatment. (G) The complete rotation of a phagosome pair within 4 seconds. Considering that, at t=4 seconds, the magnetic field is positioned along the x axis, corresponds to the delay for the pair to reach this axis. In this case, =18°.

View larger version (29K): [in a new window]   Fig. 2. The cytoplasm viscosity in E. histolytica is modified by affecting the F-actin dynamics. (A) Adherent wild-type amoeba cells were incubated in the absence or the presence of latrunculin A (Lat A) or jasplakinolide (Jas), and the cytoplasm viscosity was measured. The graphs depict the distribution of the viscosity values obtained for N phagosome pair measurements, performed in different cells and for three independent experiments. The mean value calculated () is indicated together with its standard deviation (s.d.). Lat-A treatment of the cells leads to a 50% decrease in cytoplasm viscosity compared with the untreated amoeba, whereas stabilizing actin filaments with Jas leads to a 40% increase. (B) F-actin distribution in drug-treated E. histolytica. Adherent wild-type amoeba incubated in the absence or the presence of Lat A or Jas were stained for F-actin after fixation, using rhodaminephalloidin. In wild-type strain, F-actin is enriched at the plasma membrane and in pseudopodia when the amoeba is polarized. By contrast, after treatment with Lat A, amoebae became round and actin filaments remained as aggregates inside the cell. In the presence of Jas, cells were flattened and exhibited a diffuse increase in the actin staining intensity throughout the cytoplasm. Scale bar, 5 µm. (C) Effect of Jas or Lat A on the amount of Triton-X-100-insoluble extractable actin. Wild-type parasites were incubated in the absence or the presence of Lat A or Jas before performing the fractionation procedure and examining the Triton-X-100-insoluble fraction. The amount of F-actin in untreated amoebae was arbitrarily set to 1. Lat-A incubation resulted in an effective fourfold decrease in the F-actin content, whereas Jas led to fourfold increase

View larger version (22K): [in a new window]   Fig. 3. Cytoplasm viscosity is increased in MyoIB+ and SH3 strains. Cytoplasm viscosity was measured in the strains that overproduce the different myosin IB truncated forms. The graphs show the distribution of the values obtained for N phagosome pairs studied in different cells for each strain. The mean value of the cytoplasmic viscosity () obtained for each strain is indicated±s.d. The SH3 strain exhibits an equivalent increase of the cytoplasm viscosity to the MyoIB+ strain. By contrast, the strain overproducing myosin IB deleted for the head domain (Myo-tail+) or the ATP-insensitive actin-binding site TH2 (TH2) display the same viscosity value as the control cells.

View larger version (21K): [in a new window]   Fig. 4. Overexpression of truncated forms of myosin IB. (A) The E. histolytica myosin IB heavy chain domains and the truncated forms of the protein used in this study. (B) The same amount of total cell lysates from amoebae transfected with the indicated constructs were obtained and the expression level of myosin IB proteins was quantified by immunoblot. The black arrow indicates the endogenous myosin IB (129 kDa) and the asterisks indicate the truncated forms: Myo-tail+, 47 kDa; SH3, 124 kDa; TH2, 125 kDa. The different truncated forms of myoIB were overexpressed at equivalent levels to the full-length protein in MyoIB+ strain and about three times more than in the wild-type cells.

View larger version (39K): [in a new window]   Fig. 5. The two actin-binding sites in myosin IB heavy chain account for the inhibition of phagocytosis without changes in the F-actin content. (A) Overexpression of myosin IB does not change the F-actin/G-actin ratio compared with the treatment of cells with jasplakinolide. Whole-cell lysates from equivalent cell numbers of the indicated amoeba strain were separated into a Triton-X-100-insoluble fraction, containing actin filaments (F-actin), and a Triton-X-100-soluble fraction, containing globular actin (G-actin). The fractions obtained were analysed by immunoblotting with a monoclonal antibody against actin. The effect of Jas in the wild-type strain on the amount of actin in each fraction was also examined. The MyoIB+ and SH3 strains showed a significant 10% increase (P<0.01) in the proportion of F-actin on total actin in the cell compared with the other strains examined in this study (P>0.5). By contrast, after incubation with Jas, the proportion of F-actin in the treated amoebae was drastically increased from 22% to 82%. (B) MyoIB+ and SH3 strains show a defect in phagocytosis. Amoebae were incubated with human erythrocytes for 10 minutes at 37°C, lysed and analysed for their haem concentration at 400 nm. The graph depicts the mean optical densities±s.d. of three independent experiments. Only cells overproducing myosin IB or myosin IB deleted for the SH3 domain present a threefold decrease in their phagocytic activity compared with the control strain or the strains overproducing myosin IB deleted for one of the two actin-binding sites.

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