Quantifying Nanoscale Viscosity and Structures of Living Cells Nucleus from Mobility Measurements

Abstract

Understanding the mobility of nano-objects in the eukaryotic cell nucleus, at multiple length-scales, is essential for dissecting nuclear structure–function relationships both in space and in time. Here, we demonstrate, using single-molecule fluorescent correlation spectroscopies, that motion of inert probes (proteins, polymers, or nanoparticles) with diameters ranging from 2.6 to 150 nm is mostly unobstructed in a nucleus. Supported by the analysis of electron tomography images, these results advocate the ∼150 nm-wide interchromosomal channels filled with the aqueous diluted protein solution. The nucleus is percolated by these channels to allow various cargos to migrate freely at the nanoscale. We determined the volume of interchromosomal channels in the HeLa cell nucleus to 237 ± 61 fL, which constitutes 34% of the cell nucleus volume. The volume fraction of mobile proteins in channels equals 16% ± 4%, and the concentration is 1 mM.

Intracellular Crowding by Bio‐Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis

“…This technique uses the random diffusion of small molecule fluorophores within a single confocal sampling volume to determine local viscosity, which allows for changes in intracellular viscosity to be measured. [ 41,42 ] Using calcein AM as a small molecule tracer and live cell indicator, the relative viscosity of the cytosol of MCF10A cells was measured with and without polymer crosslinking. With this technique, the relative viscosity of the cytosol in the SPAAC condition had a threefold increase compared to the nontransfected control and the lipofectamine‐only control ( Figure a).…”

“…This technique uses the random diffusion of small molecule fluorophores within a single confocal sampling volume to determine local viscosity, which allows for changes in intracellular viscosity to be measured. [41,42] Using calcein AM as a small molecule tracer and live cell indicator, the relative viscosity of the cytosol of MCF10A cells was measured with and without polymer crosslinking.…”

Microrheology of isotropic and liquid-crystalline phases of hard rods by dynamic Monte Carlo simulations

“…The striking evidence of this apparently obvious conclusion is given by the analysis of the microviscosity in the Sm phase. Its non-monotonic dependence on the tracer size along the phase director suggests that the effect of phase ordering should not be neglected and that empirical scaling laws, merely based on geometrical considerations, might not be especially accurate when assessing MR in nanostructured fluids [28,29,30]. The impact of long-ranged order is also fully appreciated when calculating the microviscosity experienced by the probe particle at low frequencies, that is when 𝜂 MR ∼ 𝐺 ′′ (𝜔 → 0)∕𝜔.…”

“…Nevertheless, this intriguing scenario does not necessarily reproduce what experiments indicate [27]. Other works have used empirical expressions to fit the experimental data [28,29], applied to different systems, including polymers [28] and living cells [30]. To the best of our knowledge, none of these expressions incorporate the effect of long-range ordering, which, as we will show here, cannot a priori be neglected.…”

Ion Complexation Explains Orders of Magnitude Changes in the Equilibrium Constant of Biochemical Reactions in Buffers Crowded by Nonionic Compounds

“…The molecular crowding phenomenon is also studied because a high concentration of cosolutes can alter molecules’ structures, diffusion coefficients, and binding rates. 7 − 11 The crowded environment is frequently recreated artificially to understand biochemical reactions occurring inside living cells. 12 , 13 Cosolutes used in solutions mimicking cells’ interior have to be chemically inactive.…”