title: Monitoring phosphatidylinositol 3-phosphate in multivesicular endosome biogenesis.
abstract: The endocytic pathway comprises a variety of intracellular compartments that regulate sorting of internalized plasma membrane constituents as well as extracellular material. A major sorting station on this route is the early endosome, where internalized receptors destined for degradation are trafficked from the limiting membrane into the interior of the endosome by formation of intraluminal vesicles (ILVs). This invagination and budding process leads to the biogenesis of multivesicular endosomes (MVEs). The formation of ILVs depends on the sequential action of protein complexes that are partly recruited in a phosphatidylinositol 3-phosphate (PtdIns3P)-dependent manner. The underlying mechanisms of the biogenesis of MVEs are still not completely understood and it is therefore of great interest to study the sorting of PtdIns3P in this process. We are describing several methods to track these sorting events by both light and electron microscopy and combination of both methods.
authors.affiliation: Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
authors.forename: Catherine Sem
authors.initials: CS
authors.lastname: Wegner
authors.affiliation: Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
authors.forename: Kay Oliver
authors.initials: KO
authors.lastname: Schink
authors.affiliation: Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
authors.forename: Harald
authors.initials: H
authors.lastname: Stenmark
authors.affiliation: Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway. Electronic address: Andreas.Brech@rr-research.no.
authors.forename: Andreas
authors.initials: A
authors.lastname: Brech
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D004853:Epoxy Resins / Q000737:chemistry; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008867:Microtomy; D057146:Multivesicular Bodies / Q000378:metabolism* / Q000648:ultrastructure; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D021381:Protein Transport; D016707:Tissue Fixation
chemical_list: D004853:Epoxy Resins; D018129:Phosphatidylinositol Phosphates; C485184:enhanced green fluorescent protein; C055525:phosphatidylinositol 3-phosphate; D049452:Green Fluorescent Proteins; C004875:EPON; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: CLEM; EGFR; ESCRT; Immuno-electron microscopy; Multivesicular endosomes; Pre-embedding labeling; Receptor sorting