Controlled initiation and quantitative visualization of cell interaction dynamics - a novel hybrid microscopy method

This thesis describes the development, validation, and application of a hybrid microscopy technique to study cell-substrate and cell-cell interactions in a controlled and quantitative manner. We studied the spatial and temporal dynamics of the selected membrane molecules CD6 and the activated leukocyte cell adhesion molecule (ALCAM), since they appear to play an important role in T-cell activation in the immune system. To obtain detailed information on interaction dynamics of cell membrane molecules, we combined total internal reflection fluorescence (TIRF) microscopy (using an evanescent field to illuminate the sample) with optical tweezers (OT) to align the interaction site in the focal plane of the microscope objective and to visualize the interactions from the precise timepoint of onset of these processes. We demonstrated that the combination of prism-based TIRF microscopy with optical tweezers yields a versatile hybrid method to study cell-substrate interactions. We described the method developed in detail and presented its application to study the interaction of K562 cells, stably expressing GFP-tagged ALCAM with a CD6-functionalized surface. We showed that K562-ALCAM-GFP cells spread on the surface in accordance with an existing model. Additionally, we described an active redistribution of ALCAM upon contact initiation. We further used the hybrid TIRF-OT method to investigate the CD6-induced cell spreading and recruitment of CD6 towards the contact site in the interaction between Jurkat-CD6-RFP cells and a functionalized surface. We showed that Jurkat-CD6-RFP cells spread on anti-CD6 functionalized surfaces in an active process with a possible role for the actin cytoskeleton. We also measured recruitment of CD6 towards the contact site on anti-CD6 and non-CD6 specific functionalized surfaces. This recruitment is also an active process. Furthermore, we tested three manipulation and three visualization methods to measure cell-cell interactions in a controlled and quantitative manner. Combinations of highly inclined laminated optical sheet (HILO) microscopy with OT and confocal microscopy with a micropipette are both able to induce contact between two cells in a controlled way, while simultaneously monitoring the interaction site in the field of view.