title: Improved Determination of Subnuclear Position Enabled by Three-Dimensional Membrane Reconstruction.
abstract: Many aspects of chromatin biology are influenced by the nuclear compartment in which a locus resides, from transcriptional regulation to DNA repair. Further, the dynamic and variable localization of a particular locus across cell populations and over time makes analysis of a large number of cells critical. As a consequence, robust and automatable methods to measure the position of individual loci within the nuclear volume in populations of cells are necessary to support quantitative analysis of nuclear position. Here, we describe a three-dimensional membrane reconstruction approach that uses fluorescently tagged nuclear envelope or endoplasmic reticulum membrane marker proteins to precisely map the nuclear volume. This approach is robust to a variety of nuclear shapes, providing greater biological accuracy than alternative methods that enforce nuclear circularity, while also describing nuclear position in all three dimensions. By combining this method with established approaches to reconstruct the position of diffraction-limited chromatin markers-in this case, lac Operator arrays bound by lacI-GFP-the distribution of loci positions within the nuclear volume with respect to the nuclear periphery can be quantitatively obtained. This stand-alone image analysis pipeline should be of broad practical utility for individuals interested in various aspects of chromatin biology, while also providing, to our knowledge, a new conceptual framework for investigators who study organelle shape.
authors.affiliation: Department of Applied Physics, Yale University, New Haven, Connecticut.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Cell Biology, Yale School of Medicine, Yale University, New Haven, Connecticut.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Schreiner
authors.affiliation: Department of Physics, Yale University, New Haven, Connecticut.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Koo
authors.affiliation: Department of Cell Biology, Yale School of Medicine, Yale University, New Haven, Connecticut.
authors.forename: Paolo
authors.initials: P
authors.lastname: Colombi
authors.affiliation: Department of Cell Biology, Yale School of Medicine, Yale University, New Haven, Connecticut. Electronic address: megan.king@yale.edu.
authors.forename: Megan C
authors.initials: MC
authors.lastname: King
authors.affiliation: Department of Applied Physics, Yale University, New Haven, Connecticut; Department of Physics, Yale University, New Haven, Connecticut. Electronic address: simon.mochrie@yale.edu.
authors.forename: Simon G J
authors.initials: SG
authors.lastname: Mochrie
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D051379:Mice; D008954:Models, Biological; D041681:NIH 3T3 Cells; D009685:Nuclear Envelope / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000166:cytology
chemical_list: D005456:Fluorescent Dyes