title: Methods for Single-Cell Pulse-Chase Analysis of Nuclear Components.
abstract: Pulse-chase methods offer powerful tools for following the evolution of a biological system over time, but are usually limited to ensemble measurements of the average behavior of very large numbers of cells. Here we describe three methods ranging from a true pulse-chase, through selective regional photoactivation, to pharmacological induction of an altered protein state, which can be applied to time-dependent studies at the single-cell level. These methods are exemplified by experimental protocols to follow region-selective nuclear envelope targeting of nascent phospholipids, a nascent nuclear lamin protein (lamin B1), and an immature lamin precursor (prelamin A).
authors.affiliation: Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK.
authors.forename: Marek
authors.initials: M
authors.lastname: Drozdz
authors.affiliation: Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK. david.vaux@path.ox.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vaux
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015870:Gene Expression; D017930:Genes, Reporter; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D009687:Nuclear Proteins / Q000378:metabolism; D021381:Protein Transport; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D009687:Nuclear Proteins
keywords: Nucleoplasmic reticulum; Photoactivatable fluorescent proteins; Pulse-chase; Secondary ion mass spectrometry; Single-cell assays; Stable isotope labeling; Super-resolution light microscopy