QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02461

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fache, S. Articles by Bruckert, F. Search for Related Content PubMed PubMed Citation Articles by Fache, S. Articles by Bruckert, F. Social Bookmarking                         What's this?

Calcium mobilization stimulates Dictyostelium discoideum shear-flow-induced cell motility

¹ Structures et Propriétés des Architectures Moléculaires (UMR 5919 CNRS), Département de Recherche Fondamentale sur la Matière Condensée, CEA-Grenoble, DRFMC/SI3M, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France
² Image Analysis & Computer Graphics, Informatics and Mathematical Modelling, Technical University of Denmark, Richard Petersens Plads, Building 321, DK-2800 Kgs. Lyngby, Denmark
³ Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 5092 CNRS), Département Réponse et Dynamique Cellulaires, CEA-Grenoble, DRDC/BBSI, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France
⁴ Johns Hopkins University, School of Medicine, 725 N. Wolfe St., 114 WBSB, Baltimore, MD 21205, USA
⁵ Laboratoire des Matériaux et Génie des Procédés, ENS de Physique de Grenoble, Domaine Universitaire, 38402 Saint-Martin d'Hères, France

View larger version (31K): [in a new window]   Fig. 1. Calcium entry is required for shear-flow-induced cell motility. (A,B) Ax2 cells were allowed to adhere to glass at a low (5-10 µM) calcium concentration, then the calcium concentration was raised to the indicated value by changing the bathing solution at low shear stress. The flow was then stopped and video recording started (t=0 minutes). After 2 minutes, a constant shear flow (2.4 Pa) was applied (arrow). The instant cell speed (A) and directionality (B) are plotted as a function of time. , and : 10 µM, 30 µM and 1 mM CaCl2, respectively, added to MES-Na buffer. Standard deviations in (A) and (B) are 3 µm minute-1 and 0.1, respectively (data not shown). (C,D) Cells were allowed to adhere to glass at a low (5 µM) calcium concentration (MES-Na buffer), then a constant shear flow (2.4 Pa) was applied and video recording started (t=0 minutes). (C) At the indicated times, the flowing solution was exchanged, first for MES-Na buffer supplemented with 100 µM EGTA, then for MES-Na buffer supplemented with 1 mM CaCl2. (D) The same procedure was applied, except that MES-Na buffer was first supplemented with 1 mM CaCl2, then with 1 mM CaCl2 + 100 µM GdCl3. The average instant cell speed <vi> is plotted as a function of time.

View larger version (20K): [in a new window]   Fig. 2. Calcium requirements for cell motility and cell adhesion to glass. (A) The average cell speed is plotted as a function of the free calcium concentration in the bathing fluid, for cells submitted to shear stress (2.4 Pa, ) or not (). In the first case, the average cell speed is determined in the steady-state portion of the motility response (from 8 to 15 minutes after onset of the flow, see Fig. 1), except when EGTA is added (1 µM), where it is determined 30 seconds to 3 minutes after EGTA addition (see Fig. 1C). In the second case, the average cell speed is determined from the entire recording. The solid and the dashed lines correspond to a fit with an apparent calcium affinity of 22 µM and a maximum speed of 25 and 10 µm minute-1, respectively. Standard deviation is 2 µm minute-1 (data not shown). (B) The critical shear stress for cell detachment is plotted as a function of the calcium concentration in the flowing fluid. Ax2 cell adhesion was probed with the radial flow detachment assay. The solid line corresponds to a fit with an apparent calcium affinity of 2.5 µM and a maximum detachment shear stress of 7.5 Pa. Error bars, 0.5 Pa (data not shown). Free calcium concentrations above or below 5 µM were obtained by adding CaCl2 or EGTA, respectively to MES-Na buffer (A) or SB (B).

View larger version (32K): [in a new window]   Fig. 3. Force sensitivity is modulated by the external calcium concentration. (A,B) Ax2 cells were allowed to adhere to glass at a low (5-10 µM) calcium concentration in MES-Na buffer, after which the calcium concentration was raised to 1 mM at low shear stress. The flow was then stopped and video recording started (t=0 minutes). After 2 minutes, a constant shear stress was applied (arrow):, =0.1 Pa; , =0.5 Pa; circles, =1 Pa. The instant cell velocity modulus (A) and directionality (B) are plotted as a function of time. (C,D) Average cell speed (C) and directionality (D) are plotted as a function of the applied shear stress at a 1 mM () or 5 µM () calcium concentration. The average cell speed and directionality are determined from the steady-state portion of the motility response to shear stress (from 7 to 15 minutes in Fig. 3A,B). Data are collected from several independent experiments performed in Sörensen or MES-Na buffer.

View larger version (35K): [in a new window]   Fig. 4. Cell speed increases and cell morphology changes upon a rise of extracellular calcium concentration under applied stress. (A,B) Ax2 cells were allowed to adhere to glass at a low (5-10 µM) calcium concentration in MES-Na buffer, then a constant shear stress (2.4 Pa) was applied and video recording started (t=0 minutes). After 2 minutes, the calcium concentration was raised to 1 mM () or 100 µM (open circles) at the same shear stress. The instant cell velocity modulus (A) and directionality (B) are plotted as a function of time. (C,D) High-resolution imaging of cells undergoing shear-flow-induced motility (=2.4 Pa) in the presence of 1 mM CaCl2. Cells were imaged during either the immediate (C) or the adapted (D) phases, defined as on Fig. 4A. In C are shown examples of the large protrusions frequently observed when calcium is raised under shear stress (white arrowheads). In D are shown small protrusions observed either at low calcium concentrations or when cells are adapted to the extracellular calcium concentrations (black arrowheads).

View larger version (38K): [in a new window]   Fig. 5. Dynamics of cell-substrate contact areas during shear-flow-induced motility. Ax2 cells undergoing shear-flow-induced motility (=2.4 Pa) in the presence of the indicated calcium concentration were imaged at high resolution under RICM and morphology of cell-substrate contact areas was analyzed as described in Materials and Methods. (A) Growth rates of gained (left) and lost (right) contact areas are represented as a function of time, for individual cells recorded at the indicated calcium concentration. Average peak height and frequency are determined from such recordings. (B,C) Average peak height and frequency are plotted as a function of cell speed, for ten individual cells. , protrusions; , retractions. In B, the dotted line is a linear least square fit of all data points (slope 7.9 µm; ordinate at origin 1.7 µm2 second-1). Standard deviations for protrusion and retraction peak heights are 1.9 and 1.3 µm2 second-1, respectively. In C, the dotted lines represent the mean values of the retraction and protrusion frequencies.

View larger version (31K): [in a new window]   Fig. 6. Cells devoid of the ß subunit of the heterotrimeric G protein family are insensitive to an increase of the external calcium concentration. (A) Average speed of LW6 Gß-null cells (Gß, ) or LW20 Gß-rescued cells (Gß-rescued Gß, ) is plotted as a function of the free calcium concentration in the flowing fluid (=2.4 Pa). The solid line corresponds to a fit with an apparent calcium affinity of 150 µM and a minimum and maximum speed of 3.5 and 13 µm minute-1, respectively. (B) LW20 (Gß-rescued Gß, ) and LW6 (Gß, ) cells were allowed to adhere to glass at a low (5 µM) calcium concentration, then a constant shear stress (2.4 Pa) was applied and video recording started (t=0 minutes). After 2 minutes, the calcium concentration was raised to 1 mM at the same shear stress. Instant cell speed is plotted as a function of time. Note that at low calcium concentrations, LW6 cell speed is slightly, but significantly higher than that of LW20 cells. (C,D) Average cell speed (C) and directionality (D) are plotted as a function of the applied shear stress at a 1 mM calcium concentration either for LW20 (Gß-rescued Gß, triangles) or LW6 (Gß, ) cells. Average cell speed and directionality are determined from the steady-state portion of the motility response to shear stress.

View larger version (25K): [in a new window]   Fig. 7. Protrusions and retractions are less active during shear-flow-induced cell motility in cells devoid of the beta subunit of the heterotrimeric G protein family. LW6 or LW20 cells undergoing shear-flow-induced motility (=2.4 Pa) in the presence of the indicated calcium concentration were imaged at high resolution under RICM and morphology of cell-substrate contact areas was analyzed as described in Materials and Methods. (A) Growth rates of gained (left) and lost (right) contact areas are represented as a function of time, for a single Gß-null cell at a 1 mM calcium concentration. (B,C) Mean peak height and frequency are plotted as a function of individual cell speed. , protrusions; , retractions. Filled symbols, Gß-rescued cells (LW20); open symbols, Gß-null cells (LW6). In B, the dotted line is a linear least square fit of the LW20 data points (slope 4.9 µm; ordinate at origin 0.7 µm2 second-1). Standard deviations for protrusion and retraction peak height are 0.9 and 0.7 µm2 second-1 for Gß-null cells and 0.7 and 0.5 µm2 second-1 for Gß-rescued cells, respectively. In C, the dotted lines represent the mean values of the retraction and protrusion frequencies.

View larger version (27K): [in a new window]   Fig. 8. Shear-flow-induced motility of IP3-receptor null Dictyostelium cells. (A,B) Average speed (A) and directionality (B) of IP3-receptor null cells (IP3-Receptor , circles) or parental RK-Ax2 cells (wild type, triangles) are plotted as a function of the free calcium concentration in the flowing fluid (=2.4 Pa). The solid line corresponds to a fit with an apparent calcium affinity of 120 µM and a minimum and maximum speed of 5 and 32 µm minute-1, respectively. The dotted line is a guide for the eye. (C,D) IP3-receptor null cells (IP3-Receptor , ) or parental Ax2-RK cells (wild type, ) were allowed to adhere to glass at a low (5 µM) calcium concentration, then a constant shear-flow (2.4 Pa) was applied and video recording started (t=0 minutes). After 3 minutes, the calcium concentration was raised to 1 mM under the same shear stress. (C) Cell speed is plotted as a function of time. The lines are hand-drawn. (D) Cell directionality is measured during the 1-3 minutes (white bars) and 8-12 minutes (black bars) portions of the recordings, corresponding to a 5 µM and 1 mM extracellular concentration, respectively.

View larger version (27K): [in a new window]   Fig. 9. (A) Model of Dictyostelium signaling pathways involved in shear stress-induced motility. Experimental evidence supporting a role for PI3K in the directionality response is presented elsewhere (Décavé et al., 2003). A link between mechanical stress and heterotrimeric G protein activation is shown in the present work since Gß-invalidation reduces cell speed in response to shear stress (Fig. 6C). In both cases, molecular details are unknown. Heterotrimeric G protein mediated Ca2+ entry (arrows 1 and 2) is supported by the effect of Gd3+ ions (Fig. 1D) and Gß-invalidation (Fig. 6B). Ca2+-induced PLC activation (arrow 3) is shown in (Cubitt and Firtel, 1992). Direct biochemical evidence for IP3-mediated calcium release (arrows 4 and 5) is given elsewhere (Schaloske et al., 2000). Our work suggests Dictyostelium IP3-receptor-like protein as a possible mediator (Fig. 8). The link between intracellular calcium and cell speed is shown in our work and (Van Duijn and Van Haastert, 1992). Note that calcium pumping activities, which are essential to restore low cytosolic calcium concentrations, are omitted. (B,C) Protrusive and retractile activities of cell-substrate contact area as a function of cell speed. Data are gathered from Fig. 5 (Ax2 cells), Fig. 7 (LW6 and LW20 cells) and from data obtained with IP3-receptor null cells (not shown). In B, the solid line is a linear least square fit of all data points (slope 9.2 µm; ordinate at origin 0.84 µm2 second-1). In C, solid and dotted lines are linear least square fits of protrusion and retraction frequencies (slope -0.03 and -0.02 µm-1, respectively; ordinate at origin 0.13 and 0.09 Hz, respectively).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter