Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton - PubMed (original) (raw)
Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton
Mireille Lambert et al. J Cell Biol. 2002.
Abstract
Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.
Figures
Figure 1.
Analysis by single-particle tracking of the movement of Ncad-Fc beads bound to the lamellipodia of C2 cells. (A) Coated beads in suspension in the culture medium were trapped by the laser tweezers and held on the cell lamellipodia for 5 s to initiate bead–cell attachment. Beads that did not drift out of focus upon release of the trap were considered bound. The bead movement was followed over a period of 50–200 s and the trajectories extracted by single-particle tracking. (B) Representative trajectory of a Ncad-Fc bead superimposed on differential interference contrast image taken at the end of the recording. Bar, 5 μm. (C) Representative trajectories of Ncad-Fc, anti–N-cadherin, and anti-NCAM antibodies coated beads (X-Y plots, top) and corresponding plots of the two-dimensional diffusion coefficient as a function of time (bottom). Note the directed movement and low diffusion coefficient of the Ncad-Fc and anti– N-cadherin–coated beads. In contrast, anti–N-CAM beads remained diffusive.
Figure 2.
Lower density Ncad-Fc beads show an initial freely diffusive phase. Representative trajectories (X-Y plots, top) and two-dimensional diffusion coefficient versus time plots (bottom) of high, medium, and low Ncad-Fc beads. Note the biphasic behavior of the medium and low Ncad-Fc beads characterized by an initial diffusive phase (green line), followed by a sharp decrease in the diffusion coefficient and the initiation of directed movement (red line).
Figure 3.
Displacement analysis of biphasic low density Ncad-Fc–coated beads. Diffusion coefficient versus time (top) and MSD of a representative biphasic medium Ncad-Fc bead were calculated as described in Materials and methods. (Bottom left) MSD was calculated within a segment of the initial phase (a), plotted as a function of time interval (plain line), and compared with theoretical MSD-Δt plots for simple Brownian diffusion (broken line). MSD increases linearly with time interval, characteristic of a simple Brownian diffusion. (Bottom right) MSD was calculated within a segment in the second phase (b), and plotted as a function of time interval (plain line). The MSD-Δt follows a parabolic curve characteristic of a unidirectional diffusion mode (broken line).
Figure 4.
Lowering the loading density of Ncad-Fc beads increases the latency to achieve anchoring. The scattergram presents the latency of the beads to achieve anchoring as a function of the charge of the beads. Ncad-Fc beads were applied for 5 s on the cell surface as described in Fig. 1. Cell surface–bound beads were considered as anchored at the time they both display a sharp decrease in their diffusion coefficient and undergo a directed rearward movement.
Figure 5.
Lovastatin and N17 Rac 1 inhibit Ncad-Fc bead anchoring without affecting initial binding. (A) Cumulative histograms present the percentage of high Ncad-Fc beads anchored (black bars), bound but not anchored (gray bars), or unbound (white bars) in the 20 s after their application on lamellipodia of untreated, lovastatin-, or vanadate-treated cells. Lovastatin did not alter the bead binding capability but reduced their anchoring. Vanadate reduced Ncad-Fc bead binding to the level of binding of control Fc beads (Fc). (B) Percentage of Ncad-Fc beads anchored (black bars), bound but not anchored (gray bars), or unbound (white bars) to control, N17 Rac1–, or N17 Cdc42–expressing C2 cells. N17 Rac1 prevented bead anchoring without affecting their binding. n = number of beads analyzed.
Figure 6.
Ncad-Fc beads bound at the surface of lovastatin-treated and N17 Rac1–expressing C2 cells remain highly diffusive. Representative movement (X–Y plots, top) and diffusion coefficient versus time (bottom) of high Ncad-Fc beads bound at the surface of untreated, lovastatin-treated, and N17 Rac1–expressing C2 cells. The displacement of Ncad-Fc beads attached on lovastatin-treated or N17 Rac 1–expressing cells remained highly diffusive and nonoriented.
Figure 7.
Neither N17 Rac1 nor V12 Rac1 prevent the accumulation of catenins at the cell–cell contacts. Transfected N17 Rac1-GFP (A–A′, C–C′, and D–D′) or V12 Rac1-GFP (B–B′) C2 cells were processed for immunofluorescent staining with polyclonal anti–β-catenin (A–A′ and B–B′), anti–α-catenin (C–C′) or anti-p120 (D–D′) antibodies and analyzed by confocal laser scanning microscopy. Panels show for each preparation matched three-dimensional projections of 0.5-μm confocal stacks in the red (catenins) and merge images with GFP. α-Catenin, β-catenin, and p120 were similarly accumulated at contact sites between GFP-positive cells expressing Rac1 mutants (open arrowheads) and between untransfected cells (arrowheads). Bar, 20 μm.
Figure 8.
N17 Rac1 does not affect the recruitment and stability of the cadherin–catenin complex. (A) Transfected N17 Rac1-GFP C2 cells were incubated with Ncad-Fc beads for 45 min and processed for immunofluorescent staining with anti–α-catenin antibodies. The panels show matched series of three stacked 1-μm thick confocal optical sections at the level of bead–cell contact. Similar accumulations of α-catenin were detected at contact sites between Ncad-Fc beads and N17 Rac1-GFP–expressing cells (open arrowheads) and between Ncad-Fc beads and untransfected cells (arrowheads). Bar, 20 μm. (B) Puromycin-selected N17 Rac1-GFP–, V12 Rac1-GFP–, or mGFP-expressing cells were allowed to form cell–cell contacts for 5 h before proteins were extracted and analyzed by direct Western blotting with anti–β-catenin, anti– α-catenin, and anti-IQGAP1 antibodies (WB). (IP) Cadherin–catenin complexes were immunoprecipitated with anti–β-catenin antibodies and analyzed by immunoblotting with anti–β-catenin, anti– α-catenin, and anti-IQGAP1 antibodies. α-catenin coimmunoprecipitated with β-catenin at similar levels in N17 Rac1–, V12 Rac1–, and mGFP-expressing cells extracts. In contrast, IQGAP1 was barely detected in the immune complex. Molecular marker size in kD are indicated to the left.
Figure 9.
N17 Rac1 interferes with actin recruitment at the Ncad-Fc bead cell contact. V12 Rac1-GFP– (A) or N17 Rac1-GFP– (B) expressing C2 cells were incubated for 35 min in the presence of Ncad-Fc beads, and then permeabilized and incubated in the presence of rhodamine-conjugated actin for 10 min. Strong incorporation of rhodamine-labeled actin was observed at the contact sites between Ncad-Fc beads (asterisks) and V12 Rac1 expressing cells (A). These results contrast with the absence of actin incorporation at the contact sites between Ncad-Fc beads and N17 Rac1 expressing cells (B). Bar, 5 μm.
References
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