Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

Abstract

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.

Structures and functions in the crowded nucleus: new biophysical insights

“…Diffusion of macromolecules is slowed in the nucleus ( [64,65], reviewed in [66]) probably due to collosions with chromatin and other large obstacles or to viscoelasticity, but nevertheless most macromolecules and multiprotein complexes can explore the entire nuclear volume [67]. Large particles and macromolecules show anomalous diffusion in the nucleus, a lesss-than-linear increase of mean-square displacement with time like that seen in crowded solutions [68].…”

Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy

“…In biological systems, however, and in particular in cells, diffusive motions are often found to deviate from simple Fickian diffusion 1 . This deviation can take dif-ferent forms, from an apparent size-dependent viscosity of the medium to a non-Gaussian distribution of displacements or to a mean-squared displacement that is not directly proportional to time [2][3][4][5][6][7][8][9][10][11][12][13] . Similar effects can be recreated in vitro, using for example polymer solutions [14][15][16] , gels [17][18][19][20] or colloidal suspensions 21 .…”

Chromosome dynamics, molecular crowding, and diffusion in the interphase cell nucleus: a Monte Carlo lattice simulation study

“…By following fluorescent tags with light microscopy, the compaction/decompaction, and mobility of individual loci (Müller et al 2001) and vice versa, the mobility of fluorescent probes of various size has been studied (Bancaud et al 2009;Dross et al 2009;Görisch et al 2004Görisch et al , 2005aGuigas and Weiss 2008;Wachsmuth et al 2000). However, current stateof-the-art techniques limit the resolution of such measurements to about 200 nm, with some exceptions from single-particle tracking (Siebrasse and Kubitscheck 2009), or superresolution techniques such as STED (Klar and Hell 1999), which, however, require particular fluorophores and have rather low sensitivity.…”