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

Izeddin, Ignacio and Récamier, Vincent and Cissé, Ibrahim I. and Bosanac, Lana and Boudarene, Lydia and Proux, Florence and Dugast-Darzacq, Claire and Bénichou, Olivier and Voituriez, Raphaël and Dahan, Maxime and Darzacq, Xavier (2012) Dynamics of Nuclear Protein Exploration Revealed by Intracellular Single Particle Tracking PALM. Biophysical Journal, 102 (3, S1). 385a. ISSN 0006-3495. doi:10.1016/j.bpj.2011.11.2108. https://resolver.caltech.edu/CaltechAUTHORS:20201211-153659268

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.bpj.2011.11.2108DOIArticle
ORCID:
AuthorORCID
Izeddin, Ignacio0000-0002-8476-3915
Cissé, Ibrahim I.0000-0002-8764-1809
Boudarene, Lydia0000-0002-6204-4936
Proux, Florence0000-0002-2603-4888
Dugast-Darzacq, Claire0000-0001-8602-3534
Bénichou, Olivier0000-0002-3178-9046
Voituriez, Raphaël0000-0001-8746-5713
Dahan, Maxime0000-0002-4943-2491
Darzacq, Xavier0000-0003-2537-8395
Additional Information:© 2011 Biophysical Society. Published by Elsevier Inc. Available online 14 February 2012.
Issue or Number:3, S1
DOI:10.1016/j.bpj.2011.11.2108
Record Number:CaltechAUTHORS:20201211-153659268
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201211-153659268
Official Citation:Ignacio Izeddin, Vincent Récamier, Ibrahim I. Cissé, Lana Bosanac, Lydia Boudarene, Florence Proux, Claire Dugast-Darzacq, Olivier Bénichou, Raphael Voituriez, Maxime Dahan, Xavier Darzacq, Dynamics of Nuclear Protein Exploration Revealed by Intracellular Single Particle Tracking PALM, Biophysical Journal, Volume 102, Issue 3, Supplement 1, 2012, Page 385a, ISSN 0006-3495, https://doi.org/10.1016/j.bpj.2011.11.2108. (http://www.sciencedirect.com/science/article/pii/S0006349511034564)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:107043
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:11 Dec 2020 23:53
Last Modified:16 Nov 2021 18:59

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