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Molecular Mechanisms of Dynamic and Spatial Control of Eph Receptors clustering

Periodic Reporting for period 1 - DynaSpaCER (Molecular Mechanisms of Dynamic and Spatial Control of Eph Receptors clustering)

Reporting period: 2016-03-01 to 2018-02-28

The aim of DynaSpaCER was to understand how cell communicate when touching each other. Cellular surface is covered in proteins called receptors that upon contact recognize their matching pair (ligand), start reorganizing forming aggregates and undergo to a chemical modification that leads to signal transduction from one cell to the other. All these events happen to a length and time scale that is not easily accessible and currently there is not a unique technology that on its own could give a clear picture of how the proteins on the surface are reorganized during the interaction of two cells. In order to overcome this challenge during the project we develop a combined approach mixing advanced techniques ranging from DNA nanotechnology to high-resolution microscopies via standard biochemical techniques and computational tools to get an insight of what are the molecular interactions involved at the cellular membrane and what kind of signals are triggered. As a model biological system, we investigated a particular kind of protein (Eph receptors) that has been show to be present in high quantity on the outer membrane of some type of cancer cells. The project explored new ways to target receptor signaling at the nanoscale and has established a basis to open new avenues of investigation of fundamental mechanisms in membrane biology and of knowledge-based drug development.
During the period of the project, we developed a way to recreate the signalling geometry at the cell-cell contact interface with some synthetic structures made out of DNA that have a dimension comparable of the starting aggregates. Further, we investigated how other proteins interact at the membrane when the receptor form clusters. In particular we focused on two aspects:
1) The activation and clustering of the receptor upon stimulation with just one ligand per receptor or if they can form pair.
2) The interplay between the receptor activation and assembly with the cortical actin, a mesh lying very close to membrane that is able to segregate receptors and other membrane proteins in confined areas that could be used as hubs for signaling.
We also started using genetic engineering techniques to make the cancer cell for the experiments produce a fluorescent version of the receptor. We started exploring the possibility of combine the DNA structures to control protein organization on the cell surface by studying their mechanical properties. Finally we used computational simulations to validate the outcome of our super-resolution imaging method.
Our results of receptor organization were consistent with a model that has been hypothesised in a scientific article by our lab. We plan to publish our results in an open access leading scientific journal by the end of 2018.
The results supported our working hypothesis that the signaling and the formation of aggregates at cell-cell contact are controlled by how the receptors move on the membrane. We integrated different biophysical and cell biology techniques in order to grasp the fine regulation of the shape and size of receptor clusters and their signaling induced by ligand and cortical actin organization. The outcome of the project has the potential to enable new lines of research in drug delivery, cell biology, molecular diagnostics and imaging.
Graphical overview of the project techniques and target lenghtscales