title: Vacuolar structures can be identified by AFM elasticity mapping.
abstract: Fluid-filled organelles like vesicles, endosomes and pinosomes are inevitable parts of cellular signalling and transport. Endothelial cells, building a barrier between blood and tissue, can form vacuolar organelles. These structures are implicated in upregulated fluid transport across the endothelium under inflammatory conditions. Vacuolar organelles have been described by transmission electron microscopy so far. Here, we present a method that images and mechanically characterizes intracellular structures in whole cells by atomic force microscopy (AFM). After crosslinking the cellular proteins with the fixative glutaraldehyde, plasma membrane depressions become observable, which are scattered around the cell nucleus. Nanomechanical analysis identifies them as spots of reduced stiffness. Scanning electron microscopy confirms their pit-like appearance. In addition, fluorescence microscopy detects an analogous pattern of protein-poor spots, thereby confirming mechanical rigidity to arise from crosslinked proteins. This AFM application opens up a mechanical dimension for the investigation of intracellular organelles.
authors.affiliation: Institute of Physiology II, University of Muenster, Münster, Germany. chrth@uni-muenster.de
authors.forename: Christoph
authors.initials: C
authors.lastname: Riethmüller
authors.forename: Tilman E
authors.initials: TE
authors.lastname: Schäffer
authors.forename: Ferry
authors.initials: F
authors.lastname: Kienberger
authors.forename: Werner
authors.initials: W
authors.lastname: Stracke
authors.forename: Hans
authors.initials: H
authors.lastname: Oberleithner
mesh_terms: D002478:Cells, Cultured; D004548:Elasticity; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D014617:Vacuoles / Q000648:ultrastructure*