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Published January 31, 2012 | public
Journal Article

Dynamics of Nuclear Protein Exploration Revealed by Intracellular Single Particle Tracking PALM

Abstract

Cellular regulation of eukaryotic cells involves molecular interactions of factors diffusing within the cellular volume. Understanding the gene expression regulation requires thus elucidating the spatio-temporal dynamics of intranuclear proteins at the single molecule level. However, live cell imaging of single molecules in eukaryotic cells has remained mostly restricted to processes occurring in the plasma membrane, readily accessible by TIRF microscopy as opposed to intra-nuclear processes. We report an intracellular single particle tracking method using photoactivated localization microscopy that enables the study of protein dynamics inside live eukaryotic cells. So far single particle tracking PALM (sptPALM) (Manley et al, 2008) has been restricted to cellular systems for which imaging can be performed using total internal reflection microscopy (TIRF), and believed to be limited to slow diffusing systems (∼ 0.1 um2/s). Here we demonstrate an approach that reduces the background of out-of-focus fluorophores by a tight control of the photoactivation, thus allowing the detection and characterization of single protein dynamics directly in the nucleus of living cells. Applying this method to several nuclear proteins, we captured a wide range of diffusive behaviors from very rapid diffusion (> 10 μm2/s) to bound chromatin associated states (< 0.1 μm2/s). We measured the single molecule dynamics for a diverse set of proteins, from free fluorophores (Dendra2) with no known interactions in the nucleoplasm, to DNA binding (c-Myc), RNA binding (Fibrillarin), and protein-protein interacting complexes (p-TEFb). We observe that, overall, nuclear exploration is not governed by a unique nucleoplasmic geometry but rather a protein-specific variable. Our approach provides a versatile tool for single molecule in vivo studies in eukaryotes.

Additional Information

© 2011 Biophysical Society. Published by Elsevier Inc. Available online 14 February 2012.

Additional details

Created:
August 19, 2023
Modified:
October 23, 2023