Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

Rolf Harkes; Veer I P Keizer; Marcel J M Schaaf; Thomas Schmidt

doi:10.1371/journal.pone.0141080

Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

PLoS One. 2015 Nov 10;10(11):e0141080. doi: 10.1371/journal.pone.0141080. eCollection 2015.

Authors

Rolf Harkes¹, Veer I P Keizer², Marcel J M Schaaf², Thomas Schmidt¹

Affiliations

¹ Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
² Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands.

Abstract

Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49±0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Algorithms*
Animals
Bacterial Proteins / analysis
Bacterial Proteins / genetics
COS Cells
Cell Nucleus / chemistry*
Cell Nucleus / ultrastructure
Chlorocebus aethiops
Diffusion
Imaging, Three-Dimensional / instrumentation
Imaging, Three-Dimensional / methods*
Luminescent Proteins / analysis
Luminescent Proteins / genetics
Microscopy, Fluorescence / instrumentation
Microscopy, Fluorescence / methods*
Motion
Receptors, Glucocorticoid / analysis*
Receptors, Glucocorticoid / genetics
Recombinant Fusion Proteins / analysis
Time Factors
Transfection

Substances

Bacterial Proteins
Luminescent Proteins
Receptors, Glucocorticoid
Recombinant Fusion Proteins
yellow fluorescent protein, Bacteria

Grants and funding

This work is part of the research programme of the Foundation for Fundamental Research on Matter (FOM), which is part of the Netherlands Organisation for Scientific Research (NWO) (www.fom.nl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.