Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing

Abstract

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 mum when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.

Figure 1

Nanoparticle spacing gradients. Variations in gold nanoparticle spacing along a substrate by using a PS(1780)-b-P[2VP(HAuCl₄)_0.2](520) diblock copolymer solution as a function of (a) polymer concentration; substrate retraction velocity (v=12 mm/min), and (b) retraction velocity; polymer solution of 2 mg/mL. (c) Three examples of particle spacing gradients ranging from ~80 to ~250 nm, for three different molecular weight diblock copolymers, and varying substrate retraction velocities [Substrate 1: ■ (PS(990)-b-P[2VP(HAuCl₄)_0.5] (385) (c = 3 mg/mL, v = 40 mm/min to 8 mm/min), Substrate 2: PS(1826)-b-P[2VP(HAuCl₄)_0.5] (523) (c = 2 mg/mL, v = 40 mm/min to 8 mm/min) and Substrate 3: PS(5355)-b-P[4VP(HAuCl₄)_0.5] (714) (c = 1 mg/mL, v = 30 mm/min to 8 mm/min)]. (d) Particle spacing gradients with varying gradient strength of (19 +/- 1), (30 +/- 3) and (50 +/- 7) nm/mm. Gradients were generated from a PS(990)-b-P[2VP(HAuCl₄)_0.3](385) diblock copolymer solution of 5 mg/mL. Retraction procedure: 1) 3 mm constant retraction velocity of 40 mm/min; 2) 0.5, 1 or 2 mm gradual deceleration of retraction velocity from 40 to 8 mm/min; 3) constant retraction velocity of 8 mm/min. All scale bars: 300 nm. Error bars = s.d. of measured particle spacing. (e) Particle spacing for two conversely oriented gradients derived from a PS(1780)-b-P[2VP(HAuCl₄)_0.2](520) diblock copolymer solution of 2 mg/mL on one substrate plotted against substrate position. The retraction velocity was accelerated from 8 to 40 mm/min for the first gradient (0-5 mm) and decreased from 40 to 8 mm/min for the second gradient (6-10 mm) on a single substrate. The asterisks indicate the insets displaying SEM micrographs of the respective substrate positions. Insets show SEM micrographs of the substrate positions indicated by different asterisks (all scale bars: 300 nm)(error bars = Stdev of measured particle spacing).

Figure 2

Biofunctionalized particle spacing gradient. (a) Scheme of the biofunctionalized substrate; (b), (c) Scanning electron micrographs show parts of critical point-dried MC3T3 osteoblasts plated for 21 h on biofunctionalized nanopatterns of ~60 nm particle separation. The inset in (c) shows a close-up of ultra-small cellular protrusions with a diameter of ~10 to ~20 nm and a length of ~30 to ~50 nm, interacting selectively with the c(-RGDfK-) functionalized gold nanoparticles which are adhesion patches for single integrin receptors. (d) SE micrograph with a tilt angle of 40° depicts ultra-small cellular protrusions interacting with integrin adhesion sites. (e) Cells were plated for 21 h on biofunctionalized particle spacing gradient substrates with c(-RGDfK-) patch spacing from 50 – 80 nm, between a 3 and 5 mm substrate position. Top: Stitched phase-contrast micrograph presenting the adhesion of cells to the different areas of the substrate. Bottom: Close-up of MC3T3 cells on patch spacing, at substrate areas offering ~50 nm, ~60 nm, ~70 and ~80 nm patch spacing. Scale bars: (b) 500 nm; (c) 200 nm (Inset: 100 nm); (d) 100 nm; (e) 100 μm.

Figure 3

Projected area of cells adhering along gradients covering various ligand patch spacing, strengths, and adhesion times. Ligand patch spacing ( ) and projected cell area (□) of cells plated after (a) 2.5 h (248 cells analysed); (b) 8 h (159 cells analysed); and (c) 23 h (200 cells analysed) as a function of substrate position, with a gradient strength of Δ15 nm/mm each. Control experiments with non-functionalized nanogold particles ( ) and particle that were functionalized with RGE-peptides ( ) proved the specificity of cell responses due to c(-RGDfK-) functionalization. Also the total number of cells that could be detected on the substrate with these controls was very little, i.e. couple of tens. Ligand patch spacing ( ) and projected cell area (□) 23 h after cell plating as a function of substrate position, when the spacing range of c(-RGDfK-) patches covers (d) 45-65 nm, gradient strength Δ80 nm/mm; and (e) 80-110 nm, gradient strength Δ15 nm/mm.

Figure 4

Morphological analysis of MC3T3 osteoblasts in contact with ligand patch spacing gradients. (a) GPR-value (■) and ligand patch spacing ( ) as a function of substrate position for cells adhering to a gradient with a strength of Δ15 nm/mm. Cell polarization angle distribution of MC3T3 osteoblasts adhering to areas which consist of (b) a constant ligand patch spacing of ~50 nm and (c) a gradient as documented in (a) (0° = cells oriented along the gradient; 90° = cells oriented perpendicular to the gradient). REF fibroblast migration paths on areas which present (d) a constant ligand patch spacing of ~60 nm; and (e) a ligand patch gradient with a strength of Δ25 nm/mm covering 60 to 110 nm spacing. For migration studies cells were cultured for 13 h on the respective substrates and then imaged every 10 min. for 12 h.