Signaling through the phosphatidylinositol 3-kinase regulates mechanotaxis induced by local low magnetic forces in Entamoeba histolytica

Magnetic labeling. (A) A schematic view of a polarized amoeba cell, with a pseudopod at the leading edge and a uropod at the rear. (B) Typical intracellular magnetic labeling (2.8-μm magnetic beads) with no applied force () and (C) under force application (, dotted arrow). (D) Typical examples of magnetic labeling of the uropod (2.8-μm magnetic beads) with no applied force () and (E) under force application (, dotted arrow).

Locomotion and polarization analysis. Image analysis of amoebic cell migration. The movement of individual cells was tracked every second for 500s. The images were then analyzed to compute locomotion parameters (A) and polarization parameters (B). (A) Two cell trajectories are represented. For each, the net distance covered by the cell between the first and last point (L) and total length of the cell trajectory (L_t) were calculated. The CME was calculated directly as the ratio between the net displacement L and the total distance L_t covered by the cell. At each point the angles γ and α were computed. Angle γ is the angle between the direction of the force (gradB) and a vector , A₀ and A_i being the initial position and each subsequent position, respectively. Angle α is the directional angle between the axis parallel to the force and a vector , A_i and A_i+1 being two successive positions of the cell centroid. (B) Cell polarization analysis is illustrated for a given cell movement. For each position of the cell, the extracellular medium was cleared (1), the instantaneous aspect ratio (a_i/b_i)_inst of the ellipse equivalent was measured (2), the cell cross-sectional surface area was normalized to π(14μm)² and was filled with gray levels 256/500 (3). The 500 filled surfaces were then fused (4). The center of the mean cell thus obtained is black (gray level 256) and the shades of gray reveal shape fluctuations. The aspect ratio (a/b)_mean of the equivalent ellipse (white line) was finally calculated (5).

Intracellular magnetic labeling, locomotion analysis. Examples of cell trajectories for intracellular labeling without an applied force ((A) ) and under force application ((B) F_intra) are given. The direction of the magnetic field gradient (gradB) is indicated. The first image of each corresponding cell is superimposed with the trajectory, indicating the starting position. Arrows indicates initial trajectory direction. (C–E) Mean parameters describing the movement of individual cells under intracellular force application (F_intra), with its respective control (). Error bars represent amoebas intra-group standard deviation. (C) Mean value for the coefficient of Movement Efficiency (CME). (D) Mean value for 〈cosγ〉 and (E) Mean value for 〈cosα〉. (F) At each point of cell trajectory and for each analyzed cell, the angles (α,γ) are plotted.

Intracellular magnetic labeling, polarization analysis. (A) Illustrations of instantaneous polarization analysis of cells at time i (stage 2 in Fig. 2). A typical cell example is given for intracellular labeling without an applied force (, left image) and under force application (F_intra, right image). (a_i/b_i)_inst is calculated from the shape of the cell at each position during tracking (stage 2 in Fig. 2). a_i and b_i being the major and minor axes, respectively. (B) Illustrations of mean polarization analysis of cells (a/b)_mean, which was obtained by fusing all the instantaneous shapes. A typical cell example is given for intracellular labeling without an applied force (, left image) and under force application (F_intra, right image). The shape of the equivalent ellipse is superimposed (white line) on the fused mean cell. The corresponding major and minor axes, a and b, give parameter (a/b)_mean. This parameter gives a measure of the persistence of the orientation of cell polarization. (C–D) Average parameters describing the polarization of individual cells under intracellular force application (F_intra), with its respective control (). Error bars represent amoebas intra-group standard deviation. (C) Average instantaneous cell polarization 〈(a/b)_inst〉. (B) Mean aspect ratio (a/b)_mean.

Extracellular magnetic labeling, locomotion analysis. Examples of cell trajectories for uropod labeling without an applied force ((A) ) and under force application ((B) F_rear) are given. The direction of the magnetic field gradient (gradB) is indicated. The first image of each corresponding cell is superimposed with the trajectory, indicating the starting position. Arrows indicates initial trajectory direction. (C–E) Mean parameters describing the movement of individual cells when force is applied at the rear pole of the cell (F_rear), with its respective control (). Error bars represent amoebas intra-group standard deviation. (C) Mean value for the coefficient of Movement Efficiency (CME). (D) Mean value for 〈cosγ〉 and (E) Mean value for 〈cosα〉. (F) At each point of cell trajectory and for each analyzed cell, the angles (α,γ) are plotted.

Extracellular magnetic labeling, polarization analysis. (A) Illustrations of instantaneous polarization analysis of cells at time i (stage 2 in Fig. 2). A typical cell example is given for uropod labeling without an applied force (, left image) and under force application (F_rear, right image). (a_i/b_i)_inst is calculated from the shape of the cell at each position during tracking. a_i and b_i being the major and minor axes, respectively. (B) Illustrations of mean polarization analysis of cells (a/b)_mean, which was obtained by fusing all the instantaneous shapes. A typical cell example is given for intracellular labeling without an applied force (, left image) and under force application (F_rear, right image). The shape of the equivalent ellipse is superimposed (white line) on the fused mean cell. The corresponding major and minor axes, a and b, give parameter (a/b)_mean. This parameter gives a measure of the persistence of the orientation of cell polarization. (C–D) Average parameters describing the polarization of individual cells when the force is applied at the rear pole of the cell (F_rear), with its respective control (). Error bars represent amoebas intra-group standard deviation. (C) Mean instantaneous cell polarization 〈(a/b)_inst〉. (B) Mean aspect ratio (a/b)_mean.

Extracellular magnetic labeling: locomotion and polarization analysis under the effect of the drug Wortmanin. Comparison of cell movement and cell polarization when the force was applied to the uropod without wortmanin (F_rear) and with wortmanin (F_rear+Wm), and the respective controls ( and ). (A, B) Parameters describing directionality (CME (A), and 〈cosα〉. (B), as described in Fig. 5). (C, D) Parameters describing polarization (instantaneous aspect ratio 〈(a/b)_inst〉 (C) and mean aspect ratio (a/b)_mean (D) as described in Fig. 6). Error bars represent amoebas intra-group standard deviation.