Advanced microscopy studies of invadosome rosettes

In this thesis we describe functional microscopy studies of invadosome rosettes. Invadosome rosettes are circular arrays of actin-rich cellular protrusions that locally degrade extracellular matrix. As matrix degradation plays an important role in a range of normal and pathological processes including immune response or metastasis of cancer cells, it has to be tightly regulated. To understand this regulation we asked which signaling pathways are involved in the development of rosettes and showed that these structures are controlled by GPCR signaling with a dominant role for the Gα(i) axis. We found that one of the potent rosettes-inducing GPCR agonists is lysophosphatidic acid – LPA. Moreover, we observed that LPA-stimulated rosettes are extremely dynamic. In order to study their behavior we developed and characterized a tool to stimulate cells with unmatched precision - caged LPA. Caged LPA provides a tight spatio-temporal control of LPA delivery in a microscopy experiment as it releases biologically active compound only upon illumination with a selected wavelength of light. Using caged LPA together with quantitative image analysis algorithms we found a correlation between development of rosettes and the direction of cell migration. Furthermore, using Super Resolution imaging we described the internal architecture of rosettes and found that individual invadosomes do not change their molecular makeup while forming higher order structures. Finally, we developed a new method to measure the lifetime of fluorophores – Single Image FLIM- to further expand the toolbox of functional microscopy techniques to study molecular interactions in dynamic cellular structures such as invadosome rosettes.