Mechanismen Aktin-vermittelter Krafterzeugung, Morphodynamik und Motilität einzelner Zellen
Bioinformatik und Theoretische Biologie
Zellbiologie
Zusammenfassung der Projektergebnisse
Cell crawling is essential for embyogenesis, wound healing or tumor metastasis. Lamellipodia are flat membrane protrusions formed during migration processes, in particular on rigid, flat surfaces or along extracellular matrix fibers. Actin polymerization at the leading edges of lamellipodia assembles an actin filament network inside them that is capable of generating protrusion forces, but how exertion of these forces relates to the assembly efficiency of such actin networks or their density, and how, conversely, thickness (or height) and density of such networks affect rates of actin polymerization, had remained poorly understood. In this project, we had thus set out to explore such relationships by combinations of experimental manipulation of actin network parameters on one hand (usually employing state-ofthe-art genome editing approaches targeting key lamellipodial actin filament regulators), force measurements of protruding lamellipodia by atomic force microscopy on the other hand, as well as mathematical modelling. These experiments allowed us to establish critical parameters of these actin networks for the extent of force exertion, such as how the “amount” of actin filaments present in lamellipodia affects effective protrusion. The project started off with the reduction of actin filament numbers formed per unit lamellipodial area, which became possible through the functional characterization of FMNL subfamily formins. At project start, we had already known that reduction of FMNL2 expression reduces the rate of lamellipodium protrusion and efficiency or of cell migration. This observation was extended to its close relative FMNL3 and combined inactivation of both. Reduction of protrusion rates upon FMNL2/3 removal were initially thought to derive from deceleration of the growth of individual actin filaments in the lamellipodium, as had been found at the individual filament level by biochemical experiments in vitro. However, when inspecting the actin polymerization rate of these FMNL2/3-deficient lamellipodia, we found, to our surprise, that actin assembly rates were not changed at the level of collectively polymerising actin networks. Instead, FMNL2/3 removal suppressed the generation (nucleation) and elongation of individual filaments, thus reducing the “density” of actin filaments throughout the lamellipodium, as evidenced by intensity measurements of lamellipodial actin filaments employing fluorescently-labelled phalloidin or by ultrastructural analyses using electron microscopy. Most strikingly, the reduction of actin filament densities in these FMNL2/3-free lamellipodia not only reduced protrusion rates, but also significantly suppressed the exertion of protrusion forces by roughly 75%, as assessed by atomic force microscopy. Notably, reduction of protrusion rates without rates of actin network assembly must be compensated by increased rearward flow, because both flow and protrusion add up to actin assembly rate in the lamellipodium. This was also confirmed experimentally. But why these cells respond to the actin density reduction with decreased protrusion velocity, but constant network assembly rate, had originally remained unclear. This could finally be explained by mathematical modelling: Indeed, the relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network did explain the experimental results. FMNL subfamily formins act as filament nucleators and elongators with differential rates. Although downregulation of their activity suggested some effect on network assembly rates, the effects on F-actin quantities per network area were much more substantial. We could explain this conundrum by considering that the lamellipodium, albeit flat, constitutes a 3D-volume. The assumption of a constant F-actin volume density, achieved through thinning of the lamellipodium indeed entails an increase in tension and flow velocity upon filament number reduction. We could also confirm the assumption on constant volume density through lamellipodial thinning experimentally. We thus proposed a lamellipodial adaptation mechanism to the reduction of actin filaments generated throughout the structure that mostly reduces protrusion forces and velocity, but keeps overall actin assembly rates roughly constant. At the same time, and finally, we established that excess actin filament generation in lamellipodia, and thus too high filament packing and density of actin networks is also counterproductive for protrusion because of interfering with the amounts of available, polymerizable actin monomer. This was assessed e.g. by state-of-the-art photomanipulation approaches such as photoactivation (PA) of PA-EGFP-tagged actin employed to determine the extent of actin monomer translocation to protruding leading edges.
Projektbezogene Publikationen (Auswahl)
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(2017) Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly. Mol Biol Cell, 28(10):1311-1325
Dimchev, G., Steffen, A., Kage, F., Dimchev, V., Pernier, J., Carlier, M.-F., Rottner, K.
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(2017) FMNL formins boost lamellipodial force generation. Nat Commun, 8:14832
Kage, F., Winterhoff, M., Dimchev, V., Mueller, J., Thalheim, T., Freise, A., Brühmann, S., Kollasser, J., Block, J., Dimchev, G., Geyer, M., Schnittler, H.-J., Brakebusch, C., Stradal, T.E., Carlier, M.-F., Sixt, M., Käs, J., Rottner, K.
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(2018) Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators. J Vis Exp, (135)
Dimchev, G., Rottner, K.
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(2018) On the relation between filament density, force generation and protrusion rate in mesenchymal cell motility. Mol Biol Cell, 29(22):2674-2686
Dolati, S., Kage, F., Mueller, J., Müsken, M., Kirchner, M., Dittmar, G., Sixt, M., Rottner, K., Falcke, M.
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(2020) Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation. J Cell Sci, bioRxiv
Dimchev, G., Amiri, B., Humphries, A.C., Schaks, M., Dimchev, V., Stradal, E.B., Faix, J., Krause, M., Way, M., Falcke, M., Rottner, K.
