Développement et caractérisation d’une méthode photonique pour créer des distributions spatiales de protéines

Cells are able to sense spatial distribution of proteins and accordingly migrate or extend in the appropriate direction. Understanding cellular responses to modifications in molecular spatial distributions is essential for advances in several fields such as development, immunology and oncology. A particularly complex example is axonal guidance that occurs during the development of the nervous system, which relies on distributions of attractive and repulsive guidance molecules to correctly wire this intricate network. Since several guidance cues collaborate to development of the nervous system, it is particularly difficult to assess the individual contribution of each cue and the signaling cascade each trigger in vivo; therefore methods to reproduce those distributions individually in vitro are necessary to study in detail the effect of each guidance cue. Several methods exist to produce graded distributions of protein that are either soluble or substrate-bound. A few methods making solution gradients are already widely used in several laboratories to perform experiments with the guidance cues that are normally diffusing in vivo. However, current methods allowing the fabrication of substrate-bound gradients are quite complex, which restrict their use to a few laboratories. First, we present a straightforward method exploiting photobleaching of a fluorescently tagged molecule using a visible laser to generating substrate-bound protein patterns: Laser-assisted protein adsorption by photobleaching (LAPAP). This method allows producing complex patterns of protein with micron spatial resolution and high dynamic range. An extensive characterization of the technique was performed and as proof of functionality, axons from dorsal root ganglions cells were guided on laminin peptide gradients. Secondly, LAPAP was improved in order to produce multicomponent patterns by using lasers at different wavelengths and antibodies conjugated to fluorophores corresponding to these wavelengths. Moreover, to speed-up the fabrication process and simplify the device, we developed widefield illumination LAPAP which uses a spatial light modulator, a light emitting diode and a standard microscope to directly print patterns. This patterning method is relatively simple compared to the original LAPAP setup, since it does not involve controlling the laser power or a motorized stage, but only sends an image of the desired pattern to a spatial light modulator. Finally, we used LAPAP to show how it could be used in automated high-content screening assays to quantify the morphological changes resulting from axon growth on gradients of guidance proteins. We produced thousands of laminin-1 gradients of different slopes and analyzed the variations in neurite guidance of neuron-like cells (RGC-5). An image analysis algorithm was developed to process bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the maximal neurite. This data showed that laminin gradients influence the initiation angle of neurite extension of RGC-5, but does not contribute to its turning. We believe that the results presented in this thesis will facilitate the use of substrate- bound protein patterning in typical life science laboratories, since a confocal microscope or a slightly modified standard microscope is the only specialized equipment needed to fabricate patterns by LAPAP. This could increase the number of laboratories working with substrate-bound protein patterns in order to reach the critical mass necessary for major advances in neuroscience.

Keyphrases: protein patterning, axon guidance, photobleaching