Studying Intracellular Trafficking Pathways with Probabilistic Density Maps

The compartmentalization of cellular functions in complex membranous organelles is a key feature of eukaryotic cells. To cope with the enormous complexity of trafficking pathways that connect these compartments, new approaches need to be considered and introduced into the field of cell biology. We exploit the advantages of the "micropatterning technique," which is to bring cells to adopt a highly reproducible shape, and probabilistic density mapping, which quantifies spatial organization of trafficking compartments, to study regulatory mechanisms of intracellular trafficking. Here, we provide a protocol to analyze and quantify alterations in trafficking compartments upon cellular manipulation. We demonstrate how this approach can be employed to study the regulation of Rab6-labeled transport carriers by the cytoskeleton.