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First published online 17 July 2006
doi: 10.1242/jcs.03057

Journal of Cell Science 119, 3206-3218 (2006)
Published by The Company of Biologists 2006
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{alpha}1ß1-integrin engagement to distinct laminin-1 domains orchestrates spreading, migration and survival of neural crest cells through independent signaling pathways

Nathalie Desban1,*, Jean-Claude Lissitzky2, Patricia Rousselle3 and Jean-Loup Duband1,{ddagger}

1 Laboratoire de Biologie du Développement, CNRS et Université Pierre et Marie Curie, 9 quai Saint-Bernard, 75252 Paris Cedex 05, France
2 Unité Mixte de Recherche 6032, CNRS et Université de la Méditerranée, Facultés de Médecine et de Pharmacie, Marseille, France
3 IFR 128 BiosSciences Lyon-Gerland, Institut de Biologie et Chimie des Protéines, CNRS et Université Lyon-1, Lyon, France


Figure 1
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  		Fig. 1. Adhesion, spreading and migration responses of crest cells to LN-1 and E8 and E1' proteolytic fragments. (A) Diagram showing the structure of LN-1 and E8 and E1' proteolytic fragments. Arrowheads indicate the major integrin-binding sites situated at both ends of the {alpha}1 chain. (B,C,G) Adhesion, spreading and migration on LN-1 and fragments at coating concentrations of 1-100 µg/ml. (D-F) Cellular morphologies after 1 hour of spreading on LN-1, E8 and E1' at 10 µg/ml. Bar, 50 µm. (H-M) Morphology and migration on (H-J) native or (K-M) heat-denatured LN-1, E8, and E1' at 10 µg/ml. Bar, 100 µm; nt, neural tube. (N) Migration tracks and morphologies of cells on LN-1 and E8 and E1' fragments. Neural tubes were explanted on the dishes and, once crest cells were seen to be separated from the neural tube (defined as t0), their migration was recorded. Migration tracks of several cells as well as their positions and shapes were plotted every hour and the total distance of migration was measured. A typical track and typical values of cell velocity (v) and of persistence of movement (p), defined as the ratio between the linear distance and the total distance covered by the cells, are indicated.

 

Figure 2
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  		Fig. 2. Survival response of crest cells to LN-1 and E8 and E1' proteolytic fragments. (A) Survival after 4, 24 and 48 hours of culture on LN-1 and E8 and E1' fragments at 1-100 µg/ml. (B) Survival on native (white symbols) and heat-denatured (black symbols) LN-1 (squares), E1' (circles) and E8 (triangles) at 10 µg/ml. (C) Survival on LN-1 (squares), E1' (circles) and E8 (triangles) in the presence (black symbols) or absence (white symbols) of 5% serum. (D,E) Proportion of apoptotic and proliferating cells on LN-1 (squares), E1' (circles) and E8 (triangles).

 

Figure 3
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  		Fig. 3. (A-E) Morphologies over time of crest cells cultured on (A) LN-1, (B) E8, (C) E1', (D) LN-5, and (E) LN-10/11. Cells were cultured over substrates at 10 µg/ml and were photographed periodically to evaluate changes in cell shapes and densities. Numerous neurons with long neurite processes (arrowheads) are detected after 48 hours on LN-1 (LN1) only but not on E8 and E1'. On LN-10/11, neurons develop precociously after 24 hours but cannot survive up to 48 hours. Bar, 50 µm.

 

Figure 4
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  		Fig. 4. Cellular response of crest cells to LN-5. (A) Diagram showing the schematic structure of LN-1, LN-5 and LN-10/11. The {alpha}3 subunit of LN-5 in its mature form is much shorter than its {alpha}1 counterpart in LN-1, and only one integrin-binding site situated towards its C-terminal part has been mapped. Likewise, although the {alpha}5 subunit of LN-10/11 resembles the {alpha}1 chain except for its length, it apparently does not contain integrin-binding domains in its N-terminus. (B-D,G) Adhesion, spreading, motility and survival on LN-1, LN-5 and LN-10/11 at 1, 10 and 50 µg/ml. (E,F) Morphology and migration on LN-5 at 50 µg/ml and LN-10/11 at 20 µg/ml. Bar, 100 µm; nt, neural tube.

 

Figure 5
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  		Fig. 5. Spreading and migration of crest cells to LN-1 are mediated primarily by {alpha}1ß1 integrin. (A,B) Effect of function-perturbing polyclonal antibodies against {alpha}1ß1 added to the culture medium on cell spreading (A) and migration (B) on LN-1, E8, and E1' at 10 µg/ml and on LN-10/11 at 20 µg/ml. (C-E) Morphology and migration of cells cultured on E8, E1' and LN-10/11 (C, D and E, respectively) in the presence of antibodies against {alpha}1ß1 added to the culture medium at 100 µg/ml at the onset of migration. The arrow in (D) points at clusters of crest cells that failed to disperse and remained attached to the neural tube. Bar, 100 µm; nt, neural tube.

 

Figure 6
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  		Fig. 6. Crest cell response to LN-1 is mimicked by antibodies against {alpha}1ß1 integrin adsorbed to the substrate. (A-C) Adhesion, spreading and migration on LN-1 and antibodies against {alpha}1ß1 or {alpha}6ß1 integrin. (D,E) Morphology and migration of crest cells on antibodies against {alpha}1ß1 and {alpha}6ß1 at 50 µg/ml. Bar, 100 µm; nt, neural tube. (F) Cell survival on LN-1 at 25 µg/ml (squares) and on antibodies against {alpha}1ß1 (triangles) and {alpha}6ß1 (circles) at 1, 10 and 50 µg/ml. (G,H) Morphologies of crest cells cultured for up to 48 hours on antibodies against {alpha}1ß1 or {alpha}6ß1 at 50 µg/ml. Note the presence of neurons on the antibodies against {alpha}1ß1.

 

Figure 7
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  		Fig. 7. Organization of matrix-adhesion sites and activation of FAK in crest cells cultured on LN-1 and E8 and E1' fragments. (A-F) Immunofluorescence staining for the ß1-integrin subunit (A-C) and paxillin (D-F) in cells cultured for 24 hours on LN-1 (A,D), E8 (B,E) and E1' (C,F). Arrowheads point at focal contacts, arrows at the periphery of the lamellipodium of the cell. (G) Immunoblotting for phosphorylated FAK on Y397 (upper panel) and total FAK (lower panel) on lysates of cells cultured on LN-1, heat-denatured LN-1 (D-LN1), E8 and E1'. (H) Immunoblotting for phosphorylated FAK on Y397 on lysates of cells cultured on LN-1 and antibodies against {alpha}1ß1. Equivalent amounts of material was loaded in G and H.

 

Figure 8
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  		Fig. 8. Activation and functions of the MAPK signaling pathway in crest cells cultured on LN-1 and E8 and E1' fragments. (A) Immunoblotting for phosphorylated Erk (upper panel) and for pan-ErK (lower panel) on lysates of crest cells cultured on LN-1, heat-denatured LN-1 (D-LN1), E8 and E1'. (B) Immunoblotting for phosphorylated Erk on lysates of cells cultured on LN-1 and on antibodies against {alpha}1ß1. Note that, as described previously for chicken embryo fibroblasts (Fincham et al., 2000Go), only a single species of Erk immunoreactivity is detected in crest cells. (C-E) Immunofluorescence staining for phosphorylated Erk in crest cells cultured for 24 hours on LN-1, E8 and E1' at 10 µg/ml. All images were acquired with the same exposure duration. (F-H) Adhesion, spreading and migration on LN-1, E1' and E8 in the presence of the MEK inhibitor PD98059. In all assays, cells were incubated with 5 µg/ml PD98059 throughout the duration of the assay and, in the adhesion and spreading assays, cells were preincubated in suspension with the drug for 10 minutes prior to the assay. (I-K) Morphology and migration of cells over LN-1, E1' and E8 in the presence of 5 µg/ml PD98059. (L-N) Cell survival and morphology in the presence of PD98059. Cells were cultured for up to 24 hours on their substrate to allow complete spreading and the drug was added at 5 µg/ml for 4 hours.

 

Figure 9
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  		Fig. 9. (A-C) Model of the neural crest cell response to LN isoforms. (A) On LN-1, both cell-binding domains (CBD) situated at both extremities of the {alpha}1 chain interact with the {alpha}1ß1 integrin. Erk signaling activated upon binding to the E1' CBD promotes cell survival and moderate, random migration, whereas FAK signaling after binding to the E8 CBD induces cell spreading and rapid, oriented migration. Cooperation between signals elicited by E1' and E8 is believed to promote sustained cell migration. On LN-10/11 (B), there is only one CBD located in the C-terminus of the {alpha}5 chain. This CBD is recognized by the {alpha}1ß1 integrin in crest cells and, in a manner similar to the E8 CBD of LN-1, it promotes cell spreading and rapid, oriented migration, presumably also via FAK signaling. However, because Erk signaling is not activated upon binding of LN-10/11, migration is supported only transiently and no survival is promoted. Finally, on LN-5 (C), the CBD located in the C-terminus of the {alpha}3 chain is not recognized by the {alpha}1ß1 integrin but instead by the {alpha}6ß1 integrin. However, because the {alpha}6ß1 integrin is poorly active in crest cells, interaction with LN-5 is reduced and results only in limited migration. Again, because no CBD exists in the N-terminus of the {alpha}3 chain, Erk signaling is not activated, resulting in a poor cell survival.

 

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© The Company of Biologists Ltd 2006