t that distinct signals arise from cell-matrix adhesions during different stages of their maturation. Results Design of the RGD-gold patterns In order to restrict the growth of cell-matrix adhesions during cell adhesion to a defined size, gold square patterns in the micrometer and sub-micrometer range were applied to glass surfaces with a nanostencil technique. Before seeding fibroblasts, Cys-containing RGD peptide was bound to the gold squares to provide restricted and integrin-specific cell attachment, and the area surrounding the gold squares was passivated with poly-L-lysine covalently grafted to polyehthylene glycol. Four different patterns with gold squares ranging from 2506250 nm2, 5006500 nm2, 161 mm2 to 262 mm2 were prepared. To ensure that cells on all patterns had the same areal density of adhesive ligand to attach, the patterns were designed in a way that the surface area of RGD-coupled 
gold was fixed to 14726663” 25% of the total substrate area. This was achieved by spacing the gold squares at a distance corresponding to their side length. Control experiments showed that gold surfaces were functionally saturated with RGD peptide under the coating conditions used. Thus, the only difference HC-030031 web between the four patterns used is the distribution, not the amount or density, of adhesive RGD-gold ligand. Scanning electron microscope images of the patterns are shown in Fig. 1B. For simplicity, the four different substrates are called 250 nm, 500 nm, 1000 nm and 2000 nm patterns in the following paragraphs. For positive and negative controls, cells were plated on plain RGD-gold or on plain PLL-g-PEG passivated glass surfaces, respectively. Restriction of cell-matrix adhesions to RGD-coupled gold areas Mouse embryo fibroblasts were plated on the RGDcoupled and PLL-g-PEG-passivated gold patterns in medium containing 3% fibronectin-depleted FCS. Cells were allowed to attach and spread for 4 hours, subsequently fixed and stained for vinculin. Some cytoplasmic staining in the perinuclear area was seen on all substrates. On plain RGD-coupled gold surface, specific staining for vinculin was additionally found in elongated, up to 10 mm long cell-matrix adhesions primarily at the cell border, with radial orientation towards the cell center. Such a distribution is indistinguishable from what is observed for the same cells on tissue culture plastic. On the 2000 nm and 1000 nm patterns, anti-vinculin labeled cell-matrix adhesions were located over the RGD-coupled gold squares. More intense staining was usually detected for ” adhesion sites in the cell periphery; however, they were also present in more central regions. On the 2000 nm patterns, often two vinculin-positive, elongated foci could be discerned on individual RGD-gold squares. In most cases, they ran either parallel or diagonal to the sides of the squares, and their orientation was roughly centripetal with respect to the cell nucleus. On the 1000 nm patterns, a single cell-matrix adhesion practically filled the area of each RGD-gold square. Interestingly, however, many peripheral adhesions exhibited a vinculin-positive short “tail”that extended over the margin of the respective 1 mm2 square, pointing again towards the cell center. The directionality of cell-matrix adhesions on both the 2000 nm and 1000 nm patterns indicated that they were subjected to centripetal cytoskeletal force by actin contractility, a typical feature of classical focal adhesions. In contrast, on the September 2011 | Volume 6 |
Heme Oxygenase heme-oxygenase.com
Just another WordPress site
