Keywords: b. cell polarity; r. cell-cell interactions
Planar Cell Polarity (PCP) polarizes cells along an axis parallel to the tissue plane, and this results in organized cell polarity across entire tissues. PCP includes both positive and negative feedback mechanisms to cause the polarization of two core protein complexes. However, the interdependency between molecular players and lack of genetic techniques has left the role of feedback mechanism mired in mystery. To uncover a more detailed mechanism of polarization we have developed a novel genetic ‘Velcro’ approach to force molecular players in the pathway to cell-cell junctions. This method allows us to parse the specific steps of polarization and differentiate between the role of positive and negative feedback in PCP. It also lets us ask the important evolutionary question of whether cells within PCP retain the single cell system of polarization when we use this tool together with a mutant that removes cell-cell PCP communication. Using the novel ‘Velcro’ tool, we have shown by proof of concept that forcing a single component, Dsh, to cell-cell junctions at the clonal boundary we can reverse cell polarity.
We can dissect the role of clustering as the mechanistic function underlying positive feedback by expressing clustering mutants of Dsh, Pk and Fmi. We have engineered flies expressing proteins that are oligomerization deficient and will position them using ‘Velcro’. This will determine if self-assemblies are required for polarization. Finally, we can test if this is also required for cell autonomous polarization. These experiments will reveal the role of positive feedback during cellular polarization and if PCP retains the ability of single cells to use this positive feedback to polarize autonomously.
We will also tackle the proposed long-range negative feedback and role of ‘mutual’ exclusion in the segregation of proximal and distal complexes in PCP. Using our novel ‘Velcro’ technique, we are testing if proximal proteins such as Vang and Pk are excluded when Dsh is forced to localize asymmetrically at clonal boundaries. We also will ask if this exclusion requires clustering by introducing oligomerization deficient Dsh mutants. Second, we will ask if this exclusion can happen cell autonomously by removing cell-cell connections with the Fmi mutant. Furthermore, we will investigate the minimal requirements for this exclusion by removing other molecular players such as Fz, Dgo, Vang, and Pk. Lastly, we will attempt to perform the same exclusion experiments by forcing Pk, the proximal counterpart to Dsh. We will evaluate if distal complexes are excluded in alignment with the idea of ‘reverse’ mutual exclusion. Ultimately this will complement the first set of experiments and reveal the role of long-range negative feedback in PCP signaling. With these tools and experiments, we aim to address the previously inaccessible questions concerning the contributions of positive and negative feedback in PCP.