Quantitative analysis of endosome tubulation and microdomain organization mediated by the WASH complex

Sorting of cargoes in endosomes occurs through their concentration into sorting platforms, called microdomains, from which transport intermediates are formed. The WASH complex localizes to such endosomal microdomains and triggers localized branched actin nucleation by activating the Arp2/3 complex. These branched actin networks are required for both the lateral compartmentalization of endosome membranes into distinct microdomains and for the fission of transport intermediates from these sorting platforms. In this chapter, we provide experimental protocols to study these two aspects of WASH physiology. We first describe how to image the dynamic membrane tubules resulting from the defects of WASH-mediated fission. We then describe how to study quantitatively the microdomain localization of WASH in live and fixed cells. Since microdomains are below the resolution limit of conventional light-microscopy techniques, this required the development of specific image analysis pipelines, which are detailed. The guidelines presented in this chapter can apply to other endomembrane microdomains beyond WASH in order to increase our understanding of trafficking in molecular and quantitative terms.