Many different cell types, including immune cells, cancer cells, and vascular smooth muscle cells make use of specialized dot-like structures, known as invadosomes, to drill holes into the underyling extracellular matrix, linking them to cellular motility, matrix remodeling and tissue invasion. Invadosomes are highly dynamic structures and are mainly found in motile cells that need to cross tissue boundaries. They are composed of an actin-rich core surrounded by proteins regulating the process of invadosome formation. At present we are almost ignorant when it comes to the nanoscale architecture of these dynamic structures. In this project we take advantage of two opportunities: 1) the properties of small Camelid nanobodies and 2) superresolution microscopy. Nanobodies represent a powerful instrument to knock out specific protein functions in cells. Moreover, because of their small size they meet the requirements for high resolution imaging. Over the years we have assembled a portfolio of nanobodies targeting constituents that represent different stages of invadosome formation. Using these molecules we cannot only perturb invadosome formation, but at the same time use them for imaging at very high resolution. This project brings together a consortium of 4 research groups, each contributing their unique expertise. This consortium will address key questions in the field pertaining to the structure and function of invadosomes and their regulation.