Mechanics and Dynamics of Dictyostelium discoideum adhesion
von Nadine Kamprad geb. Höppner
Datum der mündl. Prüfung:2021-05-26
Betreuer:Dr. Marco Tarantola
Gutachter:Prof. Dr. Andreas Janshoff
Gutachter:Dr. Marco Tarantola
EnglischThe amoeba Dictyostelium discoideum (D.d.) is an excellent laboratory model system for studying amoeboid adhesion. In its natural habitat, D.d. lives in the soil and hunts for bacteria, an environment, which is very heterogeneous, where surface properties change rapidly. This phenomenon requires a high degree of flexibility from the adhesion apparatus of D.d.. To block amoeboid adhesion of D.d., first studies used polyethylene glycol (PEG) “brushes” as surface functionalization. In the present study, it was shown that gels of PEG are a good alternative, and it was possible to identify that developmental stage of D.d., axenic background and cytoskeletal fluorescence labelling can reduce the effect of surface passivation. In a second set of experiments conducted in the present work, the focus lay on the adhesion versatility of D.d. to a variety of substrates, which bases on several non-specific driving forces. To specify these forces, model substrates made of silicon were used, which influence DLVO forces based in their layered structure. It was found that Van der Waals forces, hydrophobic effects and electrostatic interactions are involved in D.d. adhesion. Besides membrane- and glycocalyx-based interactions, these forces are complemented by additional structures, individual adhesion bonds and adhesion bond clusters of the transmembrane protein SadA (Substrate Adhesion-deficient A), while D.d. does not possess integrins and thus focal contacts. Furthermore, actin foci, dynamic structures of freshly polymerised actin in the ventral membrane, have been shown to be involved in amoeboid adhesion and migration. In the present work, it was possible to analyse SadA adhesion bonds and actin foci both from a mechanical point of view employing step spectroscopy based on single cell force spectroscopy, but also in a more dynamical approach using reflection interference contrast and total internal reflection microscopy. Several established axenic laboratory strains as well as corresponding mutants deficient for components of actin-adhesome and endocytosis-signalling were analysed. The following two phenomena could were observed: I) reduction of the adhesion forces and step as well as spot density for inhibition of the Arp2/3 complex II) manipulation of adhesion apparatus (anchoring protein TalA, transmembrane protein SadA) increases the number of actin foci, with a simultaneous reduction in adhesion force. Finally, a quantification of the role of actin foci in adhesion and endocytosis was achieved. It was possible to show that actin foci exist independently of clathrin-mediated endocytosis, which also affects the lifetime of actin foci. Furthermore, clathrin-containing structures could also be detected after the appearance of actin foci, which may stabilise adhesive structures. It was shown that D.d. is a very versatile organism that adapts substrate-dependently to the environment by non-specific adhesion. Furthermore, it could also be shown that specific adhesion of D.d. works very similarly to that of integrins.
Keywords: Dictyostelium discoideum; Single Cell Force Spectroscopy; Substrate-Adhesion-deficient A; actin focus; clathrin mediated endocytosis; Arp2/3 complex