title: Live cell imaging of endosomal trafficking in fungi.
abstract: Endosomes are multipurpose membranous carriers important for endocytosis and secretion. During membrane trafficking, endosomes transport lipids, proteins, and even RNAs. In highly polarized cells such as fungal hyphae, they shuttle bidirectionally along microtubules mediated by molecular motors like kinesins and dynein. For in vivo studies of these highly dynamic protein/membrane complexes, advanced fluorescence microscopy is instrumental. In this chapter, we describe live cell imaging of endosomes in two distantly related fungal model systems, the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans. We provide insights into live cell imaging of dynamic endosomal proteins and RNA, dual-color detection for colocalization studies, as well as fluorescence recovery after photobleaching (FRAP) for quantification and photo-activated localization microscopy (PALM) for super-resolution. These methods described in two well-studied fungal model systems are applicable to a broad range of other organisms.
authors.affiliation: Institute for Microbiology, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine University Düsseldorf, Universitätsstr. 1, Build. 26.12, D-40225, Düsseldorf, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Baumann
authors.forename: Norio
authors.initials: N
authors.lastname: Takeshita
authors.forename: Nathalie
authors.initials: N
authors.lastname: Grün
authors.forename: Reinhard
authors.initials: R
authors.lastname: Fischer
authors.forename: Michael
authors.initials: M
authors.lastname: Feldbrügge
mesh_terms: D001692:Biological Transport; D011992:Endosomes / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D005658:Fungi / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D018715:Microscopy, Video / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D011485:Protein Binding; D012331:RNA, Fungal / Q000378:metabolism; D012984:Software
chemical_list: D005656:Fungal Proteins; D012331:RNA, Fungal