title: In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow.
abstract: No methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of ≈300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hinde
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001562:Benzimidazoles; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D055459:Facilitated Diffusion; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008954:Models, Biological; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D001562:Benzimidazoles; D002843:Chromatin; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride
references.citation: Nature. 2000 Apr 6;404(6778):604-9
references.pmid: 10766243
references.citation: Nature. 2010 Jul 1;466(7302):77-81
references.pmid: 20562858
references.citation: Curr Opin Cell Biol. 2006 Jun;18(3):284-90
references.pmid: 16621498
references.citation: PLoS One. 2009;4(4):e5041
references.pmid: 19347038
references.citation: J Cell Biol. 1997 Jun 30;137(7):1459-68
references.pmid: 9199163
references.citation: Bioessays. 2005 May;27(5):477-87
references.pmid: 15832379
references.citation: Bioconjug Chem. 2000 May-Jun;11(3):301-5
references.pmid: 10821645
references.citation: Annu Rev Biochem. 1998;67:509-44
references.pmid: 9759496
references.citation: Nat Rev Genet. 2007 Feb;8(2):104-15
references.pmid: 17230197
references.citation: Nat Methods. 2007 Nov;4(11):963-73
references.pmid: 17971781
references.citation: Chromosome Res. 2008;16(3):439-49
references.pmid: 18461483
references.citation: J Mol Biol. 2000 May 12;298(4):677-89
references.pmid: 10788329
references.citation: Proc Natl Acad Sci U S A. 1998 May 26;95(11):6043-8
references.pmid: 9600914
references.citation: Mol Biol Cell. 2003 Dec;14(12):4805-12
references.pmid: 12960421
references.citation: Mol Biol Cell. 2004 Oct;15(10):4749-60
references.pmid: 15292455
references.citation: Biophys J. 2004 Jan;86(1 Pt 1):555-64
references.pmid: 14695300
references.citation: J Mol Biol. 1999 Jan 22;285(3):1053-65
references.pmid: 9887267
references.citation: J Cell Biol. 1997 Jul 14;138(1):131-42
references.pmid: 9214387
references.citation: Biophys J. 2009 Jul 22;97(2):665-73
references.pmid: 19619481
references.citation: PLoS One. 2010 May 03;5(5):e10475
references.pmid: 20454622
references.citation: Histochem Cell Biol. 2005 Mar;123(3):217-28
references.pmid: 15830242
references.citation: Cold Spring Harb Symp Quant Biol. 1993;58:777-92
references.pmid: 7525149
references.citation: Trends Cell Biol. 2005 Feb;15(2):84-91
references.pmid: 15695095
references.citation: Opt Express. 2008 Sep 15;16(19):14353-68
references.pmid: 18794971