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One-dimensional topography underlies three-dimensional fibrillar cell migration.

Doyle AD, Wang FW, Matsumoto K, Yamada KM - J. Cell Biol. (2009)

Bottom Line: We use a novel micropatterning technique termed microphotopatterning (microPP) to identify functions for 1D fibrillar patterns in 3D cell migration.In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome.We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. adoyle@mail.nih.gov

ABSTRACT
Current concepts of cell migration were established in regular two-dimensional (2D) cell culture, but the roles of topography are poorly understood for cells migrating in an oriented 3D fibrillar extracellular matrix (ECM). We use a novel micropatterning technique termed microphotopatterning (microPP) to identify functions for 1D fibrillar patterns in 3D cell migration. In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome. We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.

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Cytoskeletal regulation by ECM dimensionality. (A) Actin (green) stress fibers (arrows) extend from the leading to the trailing edge, whereas stabilized acetylated tubulin (red) is tightly compacted toward the front of the cell. (B) Cells in 3D matrix (blue) show high levels of stabilized Glu-tubulin (green) directed toward the leading edge, which contains mostly Tyr-tubulin (red). The graph shows the Glu/Tyr tubulin ratio in 2D, 3D, and 1D. (C) After blebbistatin treatment, Glu-tubulin remains concentrated in an axonlike structure (arrowheads) polarized along the matrix, whereas nocodazole disrupts this architecture. (D) Comparison of effects of blebbistatin (Bleb) and nocodazole (Noc) on cell migration in 2D, 3D, and 1D conditions. (E) Summary of the effects of ECM topography/dimensionality on cell morphology and migration. Asterisks indicate centrosomes. (B and D) *, P < 0.05 versus 2D. Error bars indicate SEM. Bars, 10 µm.
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fig5: Cytoskeletal regulation by ECM dimensionality. (A) Actin (green) stress fibers (arrows) extend from the leading to the trailing edge, whereas stabilized acetylated tubulin (red) is tightly compacted toward the front of the cell. (B) Cells in 3D matrix (blue) show high levels of stabilized Glu-tubulin (green) directed toward the leading edge, which contains mostly Tyr-tubulin (red). The graph shows the Glu/Tyr tubulin ratio in 2D, 3D, and 1D. (C) After blebbistatin treatment, Glu-tubulin remains concentrated in an axonlike structure (arrowheads) polarized along the matrix, whereas nocodazole disrupts this architecture. (D) Comparison of effects of blebbistatin (Bleb) and nocodazole (Noc) on cell migration in 2D, 3D, and 1D conditions. (E) Summary of the effects of ECM topography/dimensionality on cell morphology and migration. Asterisks indicate centrosomes. (B and D) *, P < 0.05 versus 2D. Error bars indicate SEM. Bars, 10 µm.

Mentions: Immunostaining of actin and MT cytoskeletal networks in 1D revealed linear organization (Fig. 5 A). On fibrillar patterns, stress fibers stretched from leading to trailing edge parallel to the matrix, whereas MTs, implicated in establishing cell polarity, became localized in parallel arrays extending into the lamellipodium. Staining for detyrosinated Glu-tubulin, a posttranslational modification of tubulin associated with MT stabilization, revealed large quantities of stabilized MTs throughout 1D- and 3D-migrating cells and accumulated in an anterior bundle (Fig. 5 B), as shown by an elevated Glu/Tyr-tubulin ratio in both. Western blotting further confirmed this difference between 2D and 3D (Fig. S3 B). This distribution resembles the stabilized MTs of neurons required for axon maintenance (Shea, 1999), suggesting that it may help maintain uniaxial polarity and migration along a matrix fibril. Surprisingly, the centrosome as marked by pericentrin was oriented toward the rear of fibroblasts migrating in 3D and 1D (Fig. S3 C), as was recently discovered for epithelial cells and in zebrafish (Pouthas et al., 2008). Similar but less pronounced results were obtained for Golgi orientation (unpublished data). This inconsistency with frontal centrosome and Golgi orientation in 2D wound-healing assays (Gundersen and Bulinski, 1988; Etienne-Manneville and Hall, 2001) suggests distinct mechanisms of polarity in single cell migration on 1D and 3D ECM fibrils.


One-dimensional topography underlies three-dimensional fibrillar cell migration.

Doyle AD, Wang FW, Matsumoto K, Yamada KM - J. Cell Biol. (2009)

Cytoskeletal regulation by ECM dimensionality. (A) Actin (green) stress fibers (arrows) extend from the leading to the trailing edge, whereas stabilized acetylated tubulin (red) is tightly compacted toward the front of the cell. (B) Cells in 3D matrix (blue) show high levels of stabilized Glu-tubulin (green) directed toward the leading edge, which contains mostly Tyr-tubulin (red). The graph shows the Glu/Tyr tubulin ratio in 2D, 3D, and 1D. (C) After blebbistatin treatment, Glu-tubulin remains concentrated in an axonlike structure (arrowheads) polarized along the matrix, whereas nocodazole disrupts this architecture. (D) Comparison of effects of blebbistatin (Bleb) and nocodazole (Noc) on cell migration in 2D, 3D, and 1D conditions. (E) Summary of the effects of ECM topography/dimensionality on cell morphology and migration. Asterisks indicate centrosomes. (B and D) *, P < 0.05 versus 2D. Error bars indicate SEM. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654121&req=5

fig5: Cytoskeletal regulation by ECM dimensionality. (A) Actin (green) stress fibers (arrows) extend from the leading to the trailing edge, whereas stabilized acetylated tubulin (red) is tightly compacted toward the front of the cell. (B) Cells in 3D matrix (blue) show high levels of stabilized Glu-tubulin (green) directed toward the leading edge, which contains mostly Tyr-tubulin (red). The graph shows the Glu/Tyr tubulin ratio in 2D, 3D, and 1D. (C) After blebbistatin treatment, Glu-tubulin remains concentrated in an axonlike structure (arrowheads) polarized along the matrix, whereas nocodazole disrupts this architecture. (D) Comparison of effects of blebbistatin (Bleb) and nocodazole (Noc) on cell migration in 2D, 3D, and 1D conditions. (E) Summary of the effects of ECM topography/dimensionality on cell morphology and migration. Asterisks indicate centrosomes. (B and D) *, P < 0.05 versus 2D. Error bars indicate SEM. Bars, 10 µm.
Mentions: Immunostaining of actin and MT cytoskeletal networks in 1D revealed linear organization (Fig. 5 A). On fibrillar patterns, stress fibers stretched from leading to trailing edge parallel to the matrix, whereas MTs, implicated in establishing cell polarity, became localized in parallel arrays extending into the lamellipodium. Staining for detyrosinated Glu-tubulin, a posttranslational modification of tubulin associated with MT stabilization, revealed large quantities of stabilized MTs throughout 1D- and 3D-migrating cells and accumulated in an anterior bundle (Fig. 5 B), as shown by an elevated Glu/Tyr-tubulin ratio in both. Western blotting further confirmed this difference between 2D and 3D (Fig. S3 B). This distribution resembles the stabilized MTs of neurons required for axon maintenance (Shea, 1999), suggesting that it may help maintain uniaxial polarity and migration along a matrix fibril. Surprisingly, the centrosome as marked by pericentrin was oriented toward the rear of fibroblasts migrating in 3D and 1D (Fig. S3 C), as was recently discovered for epithelial cells and in zebrafish (Pouthas et al., 2008). Similar but less pronounced results were obtained for Golgi orientation (unpublished data). This inconsistency with frontal centrosome and Golgi orientation in 2D wound-healing assays (Gundersen and Bulinski, 1988; Etienne-Manneville and Hall, 2001) suggests distinct mechanisms of polarity in single cell migration on 1D and 3D ECM fibrils.

Bottom Line: We use a novel micropatterning technique termed microphotopatterning (microPP) to identify functions for 1D fibrillar patterns in 3D cell migration.In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome.We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. adoyle@mail.nih.gov

ABSTRACT
Current concepts of cell migration were established in regular two-dimensional (2D) cell culture, but the roles of topography are poorly understood for cells migrating in an oriented 3D fibrillar extracellular matrix (ECM). We use a novel micropatterning technique termed microphotopatterning (microPP) to identify functions for 1D fibrillar patterns in 3D cell migration. In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome. We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.

Show MeSH
Related in: MedlinePlus