Search

Geometry of cell to solid substrate interface was studied by optical techniques and through localization of the cell adhesion sites by molecular biological techniques. Measurement of cell-substrate distance with fluorescence interference contrast (FLIC) microscopy performed on various extracellular matrix (ECM) protein-coated silicon chip has yielded in a large range between 15-100nm, depending on the size as well as on the conformation of the ligand. The cellular morphology and the efficiency of adhesion were specific to each ECM protein. Focal contacts in fibroblasts localized by vinculin tagged with green fluorescent protein (GFP) observed by FLIC microscopy did not exhibit the expected sharp close cell-substrate adhesion. The classical stripes of vinculin clustering in response to fibronectin induced ruffling of the membrane parallel to but not exactly at the focal contacts. The cells in neuronal culture with smooth membrane recruited vinculin mainly at cell periphery. Sites of ligand-receptor interaction was visualized by tagging ß1 integrin subunit with GFP and correlated simultaneously with FLIC microscopy. The presence of grainy distribution of ß1 integrin in fibroblasts adhered to fibronectin corresponded to the region where the membrane was held at dominating cell-substrate separation. The point contact-like dots of the fusion protein did not induce any membrane deformation in cells of neuronal culture. Focal contacts in fibroblasts were observed concurrently by total internal reflection aqueous fluorescence (TIRAF) microscopy and interference reflection microscopy (IRM) to compare the cell-substrate distance analysis by each technique. Dark patches interpreted as sites of close contact in these images precisely matched the vinculin distribution localized by tagging with the fluorescence protein. Calculation of the cell-substrate distance at focal contacts by both techniques reveal sensitivity to local variations in optical parameters, which explains the discrepancies at these sites from the estimations by FLIC microscopy. Die Adhäsion lebender Zellen auf Siliziumchips wurde mit fluoreszenzmikroskopischen Techniken studiert. Die Lage der zellulären Proteine Vinculin und Integrin aus der Adhäsionsregion wurde durch Fusion der Gene mit dem Green Fluorescent Protein sichtbar gemacht. Der Abstand der Zellmembran von der Unterlage wurde durch Interferometrie mit lipidgebundenen Fluoreszenzfarbstoffen bestimmt. Überraschenderweise konnte kein Zusammenhang zwischen der Verteilung von Vinculin und Integrin und dem Abstand zwischen Zellmembran und Substrat festgestellt werden. Vergleichende Abstandsmessungen mit etablierten Methoden wie der Interferenzreflektionsmikroskopie zeigten, daß diese wegen ihrer Abhängigkeit von unsicheren optischen Parametern leicht zu Fehlinterpretationen führen.