Direkte Messung der Protrusionskraft wandernder Zellen

Abstract

Cellular migration is a fundamental process in nature. It is very important, for instance, in wound healing and metastasis. Therefore the movement of cells is in the focus of scientific research for some time and many of the molecular details have been discovered in recent years. A still open question is the actual mechanism generating the protrusion force at the leading edge of a cell. One reason is the lack of direct data measuring force and velocity of the leading edge. I have measured, for the first time, the protrusion force of a well defined local area of the lamellipodium. In this work, an AFM (�Atomic Force Microscope�) � cantilever was oriented perpendicular to the substrate and used as a flexible obstacle, which is hit by the cell. The deflection of the cantilever, which is proportional to the force exerted by the cantilever and the cell, can be determined by video-microscopy.A completely new set-up was constructed to host the cantilever and to position it only 100nm above the glass-substrate with sufficient stability, the possibility to move the sample laterally and vertically by piezo control, and to monitor the sample by transillumination optical microscopy.A protocol was established to cultivate keratocyte cells used in this study. Keratocytes are a primary epithelial cell-line from rainbow trout, which show fast movement at nearly constant velocity.For control measurements fluorescence microscopy, atomic force microscopy and reflection-interference-contrast-microscopy were used.Maximum measured protrusion forces were in the range of 1.2nN for the lamellipodium, measured traction forces for whole cells were around 40nN. From the deflection data of the cantilever force-velocity diagrams could be generated which allows us to compare our results with theoretical model describing lamellar protrusion. The data do not coincide with the elastic brownian ratchet, which has been designed for another system. There is a good agreement with the end-motor model, which proposes the existence of an active motor molecule at the end of the actin filaments, which has not been discovered yet.