Date Published: July 19, 2017
Publisher: Public Library of Science
Author(s): Mahdie Mollazade, Thibault Tabarin, Philip R. Nicovich, Alexander Soeriyadi, Daniel J. Nieves, J. Justin Gooding, Katharina Gaus, Etienne Dague.
Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision.
The nanoscale organization of the extracellular environment influences cellular behaviors such as adhesion, migration and differentiation . In particular, cell adhesion and spreading are highly sensitive to the nanoscale spatial organization of adhesive ligands such as the tripeptide arginine-glycine-aspartic acid (RGD) found in some extracellular matrix (ECM) proteins . Using patterned surfaces with well-defined chemistries, previous work identified a critical spacing of adhesive ligands in the range of 30–80 nm that controlled effective adhesion, spreading of cells and formation of focal adhesions . However, the actual distribution of adhesive ligands was often not directly measured and had to be inferred from the design of the surface modifications. For example, electron microscopy or atomic force microscopy (AFM)  can determine the underlying topology of engineered surfaces. However, it has been challenging to directly map the nanoscale distribution of ligands with these techniques so that the ligand presentation can be linked to the organization of cell surface receptors in cells that adhere to these surfaces  .
The phase separation of block copolymers  can be exploited to create nanodomains with varying diameters and interdomain spacings that are on the biologically relevant scale of tens of nanometers . For example, a polystyrene-poly(ethylene oxide) (PS-PEO) copolymer system  was used to engineer a controlled cellular microenvironment for mesenchymal stem cells that contained nanodomains decorated with cell adhesion peptides . We have produced surfaces where vertical cylinders of PEO in thin films of PS-PEO:PS mixtures on glass coverslips formed nanodomains  that were easily detectable as ~ 3 nm indentations with AFM (Fig 1A–1C, Figure A (B-C) in S1 File). Changing the PS-PEO to PS ratio resulted in different nearest neighbor distances (NND) between the domain centers, i.e., 58 (± 14) nm, 38 (± 3) nm and 31 (± 1) nm for 25:75 PS-PEO:PS, 50:50 PS-PEO:PS and 100:0 PS-PEO:PS surfaces, respectively, while the domain diameters were maintained at ~10 nm (Table 1). The prior functionalization of the PEO group with a maleimide group enabled the attachment of cysteine-terminated RGD containing peptides, CGRGDSK, conjugated to the fluorophore AlexaFluor647 (RGD-AF647) to the surface. This enables dSTORM imaging and the localization of RGD-AF647 molecules with an average localization precision of 16 nm (Figure B (D) in S1 File).
The nanoscale distribution of adhesive ligands is key to the formation and maturation of focal adhesions. Therefore, materials whereby the distribution of adhesive ligands, such as the RGD peptide, can be controlled at the nanometer scale are important tools for probing focal adhesion formation. However, directly mapping ligand distribution has remained challenging. We were motivated to explore whether dSTORM and an appropriate analysis could be used for mapping even closely spaced RGD nanodomains, thus providing a route to RGD ligand mapping in the presence of cells seeded on the engineered surface.