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

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-fi...

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Bibliographic Details
Published in:PloS one 2015-11, Vol.10 (11), p.e0141080-e0141080
Main Authors: Harkes, Rolf, Keizer, Veer I P, Schaaf, Marcel J M, Schmidt, Thomas
Format: Article
Language:English
Subjects:
Accuracy
Algorithms
Animals
Bacterial Proteins - analysis
Bacterial Proteins - genetics
Biology
Cameras
Cell Nucleus - chemistry
Cell Nucleus - ultrastructure
Cercopithecus aethiops
COS Cells
Data processing
Deoxyribonucleic acid
Diffusion
DNA
Glucocorticoid receptors
Glucocorticoids
Imaging systems
Imaging, Three-Dimensional - instrumentation
Imaging, Three-Dimensional - methods
Innovations
Laboratories
Luminescent Proteins - analysis
Luminescent Proteins - genetics
Membranes
Methods
Microscopy
Microscopy, Fluorescence - instrumentation
Microscopy, Fluorescence - methods
Motion
Nuclei
Nuclei (cytology)
Physics
Properties
Proteins
Quantum dots
Receptor mechanisms
Receptors
Receptors, Glucocorticoid - analysis
Receptors, Glucocorticoid - genetics
Recombinant Fusion Proteins - analysis
Recovery (Medical)
Spectrum analysis
Three-dimensional imaging
Time Factors
Transcription factors
Transfection
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Description
Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0141080