One-dimensional topography underlies three-dimensional fibrillar cell migration

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.

Figures

Figure 1.

Generation of micropatterned PVA films. (A) Schematic of a single PVA molecule conjugated to a glass coverslip through 3-(amino)propyl-trimethyloxysilane (APTMS) and glutaraldehyde (GA). (B) PVA-coated surfaces (1) are photoablated using an LSM 510 NLO META system (Carl Zeiss, Inc.). (2) ROI template generated by the software dictates the ablation pattern. (3) Protein (red) added to the dish adsorbs only to ablated sites. (C) Phase image of four different ROI templates used in close proximity. (D) Serial ablation with μPP to generate patterns of fibrinogen (green dashes), vitronectin (red squares), and FN (blue circles) within micrometers of each other. The DIC image is shown in the bottom left. Bars: (C) 20 µm; and (D) 5 µm.

Figure 2.

1D topography functionally mimics 3D fibrillar matrix. (A) Rapid migration of NIH-3T3 fibroblasts through a 3D cell-derived matrix. (B) Cytoskeletal alignment (green, actin) along oriented FN fibers (red). (C) Fibroblasts plated on single ∼1.5-µm lines mimic this 3D phenotype and migrate continuously. (D) Cell migration rates on 2D surfaces and 1D fibrillar lines at different FN ligand densities versus 3D cell-derived matrix. (E) HK migration over 2D, 3D matrix, and 1D fibrillar lines. (F) HK migration rates on substrates in E. Bars: (A, B, and E) 20 µm; and (C) 10 µm. *, P < 0.01 versus 2D; ‡, P < 0.05 versus 0.5 and 1,000 FN. Error bars indicate SEM.

Figure 3.

Coordinated protrusion–retraction cycles and unique linear adhesions are associated with 1D migration. (A–C) Coordination between leading edge protrusion (red), cell body movement (green), and tail retraction (blue) in directionally migrating cells on 2D (A), in 3D matrix (B), and along a 1D line (C). In A, protrusion is reduced (filled arrowhead) until the tail retracts. As retraction begins (open arrowhead), protrusion resumes. Both 3D matrix and 1D migration display efficient protrusion–retraction cycles. (D) Confocal images showing localization of FAK, phospho-FAK397, α5-integrin, and activated β1-integrin during 1D fibrillar migration and a 2D control. (E, top) Adhesions containing GFP-vinculin using TIRF (green) are shown. Kymograph analysis (middle) of GFP-vinculin adhesions during 1D migration. Red boxes (bottom) show leading and trailing edges. Red, yellow, and green lines indicate slopes of vinculin movement within adhesions at the leading edge, under the cell body, and at the tail, respectively. Dashed lines indicate the cell’s initial position. (F) Widefield image of vinculin (top) shows elongated leading lamella of rapidly migrating cells. TIRF reveals vinculin in a front to rear gradient spanning leading to trailing edge. (G) Line scan (dashed line in F) of the cell in F shows the gradient pattern (green) and close proximity of adhesions to the leading edge (LE). Arrows indicate the direction of migration. Bars, 10 µm.

Figure 4.

Regulation of cell migration by fiber width and dimensionality. (A) Triplets (bracketed) of 1, 2.5, 5, 15, and 20 µm-wide lines used for cell migration tracking. (B) Biphasic effect of fiber width on fibrillar migration rate. (C) TIRF images of GFP-vinculin containing adhesions on a 2.5-µm line show two parallel sets of adhesions. The arrow indicates the direction of migration. (D) Fibroblasts migrating with a 3D/1D phenotype along multiple 1-µm lines. (E) 1D to 2D ECM transition induces spreading of the leading edge (arrow) and formation of many cell processes (arrowheads). The red box indicates ROI in time lapse below. (F) Rac siRNA knockdown in 2D induces a uniaxial phenotype lacking the single linear adhesion seen in 1D and does not increase migration rate. *, P < 0.05 versus all conditions except multilines; +, P < 0.05 versus 15–40 µm. Con, control. Error bars indicate SEM. Bars, 10 µm.

Figure 5.

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.