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First published online 9 September 2003
doi: 10.1242/jcs.00726

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Shear flow-induced motility of Dictyostelium discoideum cells on solid substrate

¹ Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département Réponse et Dynamique Cellulaires, CEA-Grenoble, DRDC/BBSI, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France
² Structures et Propriétés des Architectures Moléculaires, Département de Recherche Fondamentale sur la Matière Condensée, CEA-Grenoble, DRFMC/SI3M, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France
³ Laboratoire de Thermodynamique et de Physico-Chimie du Métal, ENS d'Electrochimie Electrométallurgie, LTPCM, Domaine Universitaire, 38402 Saint-Martin d'Hères, France

View larger version (20K): [in a new window]   Fig. 1. Experimental setup. (A) A uniform hydrodynamic flow was generated between the lucite top part and the microscope glass plate on which cells adhere. Cell movements were imaged by an inverted microscope under various illumination types and recorded by a digital camera coupled to a computer. (B) The hydrodynamic shear stress applied to the cells is related to the chamber width l and height e and to flow rate D through Eqn 1 (see Materials and Methods). (C) Representation of cell movements. The cell position is represented as successive points in Cartesian coordinates, where the x axis denotes the direction of the flow. The translational cell velocity during a track <jv> is defined by Eqn 3 from the cell positions at the initial time (t0) and the last cell position recorded (tend). The projections of the translational cell velocity over the directions parallel and perpendicular to the flow are denoted <vx> and <vy>. The instant cell velocity jv(ti) is calculated according to Eqn 2 from the cell positions at the previous (ti–1) and following (ti+1) instant times and given in orthoradial coordinates, where i is the angle between the speed and the flow.

View larger version (52K): [in a new window]   Fig. 2. Shear flow-induced cell motility. Cells were submitted to a 1 Pa shear stress and monitored under dark field illumination at a 2.5x magnification for 10 minutes. A set of representative cell tracks is shown superimposed over the first image recorded. Each track starts on the cell identified by the indicated number and ends at the free end of the line. Scale bar, 100 µm. Arrow points into the flow direction. (A) Untreated cells. These data correspond to Movie 1. (B) In the presence of 20 µg ml–1 CIPC. These data correspond to Movie 2. (C) Example of individual cell track analysis. The instant velocity and angle with respect to flow of the cells denoted 1, 2 and 3 in (A) were calculated according to Eqn 2 and represented in orthoradial coordinates. Left: speed modulus as a function of time. The dotted lines indicate the average speed modulus. Right: speed angle as a function of time. The solid lines represent portions of straight-line movements. The dotted lines indicate cell turns.

View larger version (29K): [in a new window]   Fig. 3. Kinematic analysis of shear flow induced cell motility. Cell motions were recorded under application of the indicated shear flow. Individual cell tracks are analysed as a set of instant velocity between successive time frames according to Eqn 2. (A) Probability distribution of speed modulus. The distribution of speed modulus was normalized so that the area under each curve is 1. (B) Angular distribution of cell instant velocities with respect to the flow axis. Directions of cell movement were decomposed into eight 45° classes, centered on the eight directions indicated.

View larger version (45K): [in a new window]   Fig. 4. Flow-induced evolution of cell-substrate contact areas. Individual cell-substrate contact area was imaged during shear flow application (=4 Pa) by RICM at a 40x magnification. The peeling velocity has already been analysed for its relationship with the applied shear stress (Decave et al., 2002a). (A,B) Representative set of cell substrate contact area position over time. The dotted line indicates the position of the rear of the cell at the beginning of the recording. The cell's rear and front edges are indicated by black and white circles, respectively. Arrows indicate the flow direction. Arrowheads indicate rapid increases of cell-substrate contact area (bursts). (A) Untreated cells. The solid line indicates the overall direction of the cell during the considered straight movement. (B) In the presence of 20 µg ml–1 CIPC. (C,D) Positions of cell rear (closed diamonds) and front (open squares) edges over time for untreated (C) and CIPC-treated (D) cells. The data correspond to the cells shown in A and B, respectively. Arrows indicate rapid increases of cell-substrate contact area (burst phases). The sixth extension of the front edge (C, t=215 seconds) is longer than the others (7 µm instead of 2 µm). This corresponds to several bursts occurring simultaneously. Notice that the rear peeling velocities of CIPC-treated and untreated cells are identical in this case, in which both cells are going to detach. (E,F) Distribution of rear (E) and front (F) edge instant velocities. The data correspond to the cell shown in A. The cell rear movement exhibits a single peak at 0.2 µm second–1 velocity. Bursts and immobility phases in the cell front movement correspond to peaks at 0.5 µm second–1 and 0 µm second–1, respectively.

View larger version (25K): [in a new window]   Fig. 5. During SFICM, cell speed is modulated by the frequency of fast increases in contact area (burst). Individual cell-substrate contact areas were imaged under SFICM at the indicated shear stress values by RICM at 40x magnification. The relative positions of cell rear (closed diamonds) and front (open squares) edges are plotted as a function of time. Arrows indicate the rapid increases in cell-substrate contact area (bursts).

View larger version (39K): [in a new window]   Fig. 6. Cells respond to increasing forces by an increased pseudopod frequency. D. discoideum cells adhering to glass were continuously monitored by phase-contrast microscopy. Cell response was triggered by application of a continuous 1.8 Pa shear stress (at t=0 seconds). Pseudopodia were detected by inspection over 50 cells as described under Methods and the pseudopodium emission frequency per cell was calculated at each time point. (A) Time course of cell pseudopodium emission frequency. The solid and dotted lines indicate the spontaneous and adapted pseudopodium emission frequencies, respectively. (B) A cell submitted to shear flow exhibiting nascent (empty triangles) and fully extended (filled triangles) pseudopodia, used in building up the curve shown in (A). The images are centered on the same cell and the indicated time is relative to the application of the flow. Different phases of cell response were selected: resting state (–2 seconds), onset of pseudopodium extension (+2 seconds), full immediate response (+6 seconds) and adapted phase (between +10 seconds and +30 seconds). Notice that most of the pseudopodia emitted after application of the flow protrude in the direction of the flow. Scale bar, 5 µm.

View larger version (63K): [in a new window]   Fig. 7. Orientation of the internal PtdIns(3,4,5)P3 gradient in response to shear flow. CRAC-GFP-expressing cells were observed at high magnification under constant shear flow (=2 Pa). Cells were observed between 2 minutes and 10 minutes upon application of the flow. The figure shows a gallery of cells presenting a unique CRAC-GFP front aligned with the direction of the flow (arrow). Statistics of the CRAC-GFP orientation is provided in Table 4. Top: GFP fluorescence. Middle: fluorescence intensity profile along the line shown in the top panel. Bottom: phase contrast images of the same cells. These images were taken 5 seconds after the fluorescence ones. The triangles point to membrane extensions that had occurred between the two pictures. In the fluorescence image of the cell reported in (A), an endocytic structure can be seen (arrowhead).

View larger version (42K): [in a new window]   Fig. 8. Effect of PI3K inhibition on SFICM. (A) Cells were submitted to a 1.8 Pa shear stress in the presence of 34 µM LY294002 and monitored for 10 minutes under dark field illumination at a 2.5x magnification. A set of representative cell tracks is shown superimposed over the first image recorded. These data correspond to Movie 4. Each track starts on the cell near the corresponding number and ends up at the free end of the line. Scale bar, 100 µm. (B) Cells were submitted to a 1.8 Pa shear stress in the presence of the indicated LY294002 concentration and monitored for 10 minutes as in (A). The orthogonal projections of the average translational cell velocity over 10 minutes (<vx> and (<vy>) were determined using Eqn 2 and plotted as a function of LY294002 concentration. (C) Distributions of instant cell velocity modulus in the presence (gray bars) or the absence (white bars) of 34 µM LY294002. Applied shear stress is 1.8 Pa. Data are from Fig. 2A and Fig. 8A. (D) Angular distributions of cell instant velocities with respect to the flow axis in the presence (gray bars) or the absence (white bars) of 34 µM LY294002. The histogram of the directions of cell movement is represented as in Fig. 3C. Applied shear stress is 1.8 Pa. Data are from Fig. 2A and Fig. 8A.

View larger version (18K): [in a new window]   Fig. 9. Possible mechanisms of SFICM. (A) Shear stress at the cell-substrate contact area facing the flow triggers two signaling pathways. One stimulates pseudopodium emission in every direction (+), whereas the other locally inhibits pseudopodium emission near the stressed zone. This latter local signal is related to PI3K activity, because its inhibition allows pseudopodium emission against the flow. (B) Cell detachment results from the competition between SFICM and passive membrane peeling. The solid and dotted lines depict the average front and rear edge speed as a function of applied shear flow, respectively, based on the values determined in Figs 3, 5. Closed squares: cell instant speed (Fig. 3). Open squares: peeling velocity (Fig. 5). For =6.2 Pa, the peeling velocity is approximated by the translational velocity of rapidly detaching cells and the front velocity by the instant speed of slowly detaching cells. The threshold stress for cell detachment 1/2 corresponds to the speed at which the average front edge velocity vf cannot adapt to the average rear edge velocity vr; vp designates the burst growth rate and vl the limit in front edge velocity leading to the detachment of 50% of the cells. The dotted line corresponds to the following equation: , with v0 =(1.1±0.1)x10–2µm second–1 and 0 =(8±0.5)x10–2 Pa. The rationale for using this relation to describe cell rear velocity as a function of applied or internal forces is given elsewhere (Décavé et al., 2002b). The solid line relating the front speed to the applied shear stress is hand-drawn.