3D Protein Dynamics in the Cell Nucleus. BIOPHYSICAL JOURNAL, January 2017

By Anand P. Singh1, Remi Galland2,3, Megan L. Finch-Edmondson1,4, Gianluca Grenci1, Jean-Baptiste Sibarita2,3, Vincent Studer2,3, Virgile Viasnoff1,5,6, and Timothy E. Saunders1,5,7

Biophysical Journal. January 2017. 112(1). 133-142. doi: http://dx.doi.org/10.1016/j.bpj.2016.11.3196

Abstract

 

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3)fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.

 

 

1Mechanobiology Institute, National University of Singapore, Singapore
2Institut Interdisciplinaire de Neurosciences, University of Bordeaux, France
3Centre National de la Recherche Scientifique UMR 5297, University of Bordeaux, France
4Department of Physiology, National University of Singapore, Singapore
5Department of Biological Sciences, National University of Singapore, Singapore
6Centre National de la Recherche Scientifique UMI 3639, France
7Institute for Molecular and Cell Biology, A*Star, Singapore