title: Inverting adherent cells for visualizing ECM interactions at the basal cell side.
abstract: Interactions with the extracellular matrix (ECM) govern a wide range of cellular functions, including survival, migration and invasion. However, in adherent cells these interactions occur primarily on the basal cell side, making them inaccessible to high-resolution, surface-scanning imaging techniques such as atomic force microscopy (AFM) or scanning electron microscopy (SEM). Here we describe a fast and reliable method for inverting adherent cells, exposing the basal cell membrane for direct analysis by AFM or SEM in combination with fluorescence microscopy. Cells including their matrix adhesion sites remain intact during the inversion process and are transferred together with the complete array of basally associated ECM proteins. Molecular features of ECM proteins, such as the characteristic 67 nm collagen D-periodicity, are well preserved after inversion. To demonstrate the versatility of the method, we compared basal interactions of fibroblasts with fibrillar collagen I and fibronectin matrices. While fibroblasts remodel the fibronectin layer exclusively from above, they actively invade even thin collagen layers by contacting individual collagen nanofibrils both basally and apically through a network of cellular extensions. Cell-matrix entanglement coincides with enhanced cell spreading and flattening, indicating that nanoscale ECM interactions govern macroscopic changes in cell morphology. The presented cell inversion technique can thus provide novel insight into nanoscale cell-matrix interactions at the basal cell side.
authors: DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology-KIT, Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe, Germany.
authors: Tetyana
authors: T
authors: Gudzenko
authors: Clemens M
authors: CM
authors: Franz
mesh_terms: D002448:Cell Adhesion; D002460:Cell Line; D002468:Cell Physiological Phenomena; D003094:Collagen / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008856:Microscopy, Fluorescence
chemical_list: D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D003094:Collagen