Improved Determination of Subnuclear Position Enabled by Three-Dimensional Membrane Reconstruction
| Autor: | Paolo Colombi, Yao Zhao, Megan C. King, Peter K. Koo, Simon G. J. Mochrie, Sarah M. Schreiner |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Nuclear Envelope Biophysics Locus (genetics) Computational biology Biology Endoplasmic Reticulum Models Biological 03 medical and health sciences Mice 0302 clinical medicine Imaging Three-Dimensional Schizosaccharomyces Nuclear volume Animals Organelle Shape Computational Tool Fluorescent Dyes Genetics Alternative methods Endoplasmic reticulum membrane Endoplasmic reticulum Chromatin 030104 developmental biology Membrane NIH 3T3 Cells 030217 neurology & neurosurgery |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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