We perfomed TFM microscopy experiments. Unfortunately, it turned out that the rigidity of the PAA gel was a limiting technical factor for the study of topological defects. Therefore, we opted for an alternative strategy based on fluorescent reporters that also allows to study the coupling between bacterial adhesion and topological defects. For this purpose, we developed a protocol to perform live microscopy while visualizing adhesion proteins (TIRF setup). We focused on Ag43, which mediates cell-cell adhesion through homophilic interactions. Through the TIRF observations, we could visualize the distribution of the protein Ag43, and, for different conditions (temperature, cell-cell adhesion over-expression), we measured the dynamics of topological defects during microcolony growth.
In the WT strain, we first showed that the rate of topological defect formation increased with growth rate, i.e. with the temperature, while the lifetime of topological defects decreased with growth rate. We then showed that the lifetime of the -1/2-charged defects and the rate of defect generation were positively and negatively correlated to the level of cell-cell adhesion, respectively. Our results also clearly establish that topological defects influence neither the size of microcolony at second layer formation nor the location of the second layer initiation site. However, our results indicate that the rate of defects generation has a direct impact on the shape of the microcolony. The higher this rate, the rounder the micro-colonies. Surprisingly, we observed that topological defects were generated at a lower rate upon overexpression of cell-cell adhesion. Consistently, we observed that micro-colonies with higher cell-cell adhesion are more elongated.
We published the part related to the methodology (Chekli et al. Sci. Rep. 2020). We will soon publish the part related to the coupling between adhesion and defects.
