Summary Project description 2nd funding period
To gain entry into host cells, viruses have developed an exquisite tuning of their chemical and physical
signatures. This entails the direct presentation or exposure of cell receptor binding sites on their surface, as
well as a great variability in size and shape of viral particles. During the current funding period, we focused on
the interplay between integrin-mediated adhesion and clathrin-mediated machinery for regulating
mechanotransduction and signaling for virus particle uptake. By combining live-cell microscopy and surface
chemistry approaches with the development of biosensors for single molecule force measurements, we have
established a method to study the physical interaction of single viral particles with the cell surface.
Unexpectedly, the crucial steps for force generation are initiated by membrane bending depending on particle
size, and do not rely on specific interactions with receptors (integrin 51, JAM-A) and cytoskeleton
contractility. This suggests that a fine regulation of local, rather than global, physical events is crucial for virus
uptake. At the same time, although we established a method to study early events of clathrin-mediated
endocytosis, the challenge to precisely dissect the molecular processes during entry remains. These events
are very difficult to study in detail within complete virus-cell systems because of multiple and confounding
signals that take place within seconds.
The goal of this project is to develop a bottom-up synthetic approach to study the encounter between cells and
viruses. We will dissect the physical, structural and molecular contributions of different molecular players to
the endocytic cell-virus machinery. To this end, we will mimic the surface of virus particles and their physicochemical
properties by decorating metal and different mechanical stiff polymer particles with specific nanoscale
binding motifs that are usually presented during virus entry, will be employed in cell uptake studies. Inspired
by collaborations with Kräusslich, Boulant and Grimm, we will use nanoparticles which mimic the size, shape
and mechanical properties of different viruses. All these will be compared to the established virus particles
together with the respective labs. We will work with Funaya/Schwab and Johnsson on particle visualization.
Just as importantly, we will create synthetic cells by a novel bottom-up approach established in the lab,
consisting of lipid giant vesicles differing in lipid composition in collaboration with Brügger, as well as type and
combination of reconstituted cell receptors and components of the endocytic machinery with Boulant. These
may play the deciding role for initiating membrane bending and virus uptake. In a reverse engineering
approach the quantitative results obtained from the synthetic approach will be confirmed in in vivo situations.
As such, our project will allow us to identify relevant molecular partners and mechanisms, physico-chemical
parameters and the dynamics of the endocytic cell-virus machinery at the nanoscale. This synthetic cell
approach for gaining molecular information on the interaction of pathogens with cells will contribute to and be
tightly coupled also with the work of Frischknecht/Spatz, Lanzer/Tanaka and Schwarz for force measurements
and with Fackler and Ruggieri for the environmental control over cell interactions.