Internal organisation of the nucleus: assembly of compartments by macromolecular crowding and the nuclear matrix model

Many and possibly all macromolecules in the nucleus are segregated into discrete compartments, but the current model that this is achieved by a fibrillar nuclear matrix which structures the nuclear interior and compartments is not consistent with all experimental observations, as reviewed here. New results are presented which suggest that macromolecular crowding forces play a crucial role in the assembly of at least two compartments, nucleoli and PML bodies, and an in vitro system in which crowding assembles macromolecular complexes into structures which resemble nuclear compartments is described. Crowding forces, which are strong in the nucleus due to the high macromolecule concentration (in the range of 100 mg/ml), vastly increase the association constants of intermolecular interactions and can segregate different macromolecules into discrete phases. The model that they play a role in compartmentalisation of the nucleus is generally consistent with the properties of compartments, including their spherical or quasispherical form and their dynamic and mobile nature.     

Self-association of polynucleosome chains by macromolecular crowding

The crowding of macromolecules in the cell nucleus, where their concentration is in the range of 100 mg/ml, is predicted to result in strong entropic forces between them. Here the effects of crowding on polynucleosome chains in vitro were studied to evaluate if these forces could contribute to the packing of chromatin in the nucleus in vivo. Soluble polynucleosomes approximately 20 nucleosomes in length formed fast-sedimenting complexes in the presence of inert, volume-occupying agents poly(ethylene glycol) (PEG) or dextran. This self-association was reversible and consistent with the effect of macromolecular crowding. In the presence of these crowding agents, polynucleosomes formed large assemblies as seen by fluorescence microscopy after labelling DNA with the fluorescent stain DAPI, and formed rods and sheets at a higher concentration of crowding agent. Self-association caused by crowding does not require exogenous cations. Single, approximately 800 nucleosome-long chains prepared in 100 muM Hepes buffer with no added cations, labelled with the fluorescent DNA stain YOYO-1, and spread on a polylysine-coated surface formed compact 3-D clusters in the presence of PEG or dextran. This reversible packing of polynucleosome chains by crowding may help to understand their compact conformations in the nucleus. These results, together with the known collapse of linear polymers in crowded milieux, suggest that entropic forces due to crowding, which have not been considered previously, may be an important factor in the packing of nucleosome chains in the nucleus.     

The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells

The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements.     

Structure of metaphase chromosomes: a role for effects of macromolecular crowding

In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ~30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100-200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 μM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ~30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.     

Intracellular distribution of selenium and the growth of mammary cells in culture

Retention of Se in CMT-13 cells increased with an increase in the concentration of selenite in the incubation medium, the duration of exposure, and the density of the culture. The enhanced toxicity of selenite coincided with a proportional increase in Se in both the cytoplasm and nucleus. About 90% of the accumulated Se was isolated with cytoplasmic macromolecules. Increased nuclear Se retention correlated with increased cytoplasmic Se retention. Greater quantities of cytosolic Se-containing proteins (74, 55, 41, 34, and 28 kDa) and a nuclear Se-containing protein (56 kDa) were detected as the quantity of Se within CMT-13 cells increased. These findings suggest that cellular retention and distribution of Se are determinanants of the degree of cellular growth inhibition caused by this trace element.     

Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.     

Nuclear functions of the Arf guanine nucleotide exchange factor BRAG2

BRAG2 is a guanine nucleotide exchange factor for the GTPase Arf6 that cycles between the cytoplasm and nucleus in a CRM1/exportin1-dependent manner. Despite its presence in the nucleus, nuclear functions have not previously been described. Here, we show that depletion of endogenous BRAG2 by RNAi leads to an increased number of Cajal bodies (CBs), and altered structure of nucleoli, as indicated by less focal fibrillarin staining. This result was surprising given that nuclear BRAG2 is diffusely distributed throughout the nucleoplasm and is not concentrated within nucleoli at steady state. However, we found that ectopic expression of the nuclear GTPase PIKE/AGAP2 causes both BRAG2 and the CB marker coilin to accumulate in nucleoli. Neither the GTPase activity of PIKE nor the nucleotide exchange activity of BRAG2 is required for this nucleolar concentration. Increased levels of exogenous BRAG2 in nucleoli result in a redistribution of fibrillarin to the nucleolar periphery, supporting a role for BRAG2 in regulating nucleolar architecture. These observations suggest that, in addition to its role in endocytic regulation at the plasma membrane, BRAG2 also functions within the nucleus.     

Effects of macromolecular crowding on refolding of recombinant human brain-type creatine kinase

In this study, we quantitatively measured the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000), dextran 70, and calf thymus DNA (CT DNA), on the refolding and aggregation of recombinant human brain-type creatine kinase (rHBCK) denatured by guanidine hydrochloride (GdnHCl). The results showed that there is more aggregation in the presence of either a single crowding agent or in a mixture of crowding agents than in the absence of crowding agents, especially in the presence of a mixture containing CT DNA and PEG 2000 (or dextran 70). In the presence of high concentrations of PEG 2000 (100 g/L), dextran 70 (100 g/L), and CT DNA (15 g/L), the refolding yield remarkably decreased from 70% to 20%, 52% and 57%, respectively. A remarkable decrease in the refolding yield and rate with mixed crowding agent containing CT DNA and PEG 2000 (or dextran 70) was also observed. In comparison to refolding in the presence of 100 g/L PEG 2000, the refolding yields and rates improved in the presence of a mixture of PEG 2000 and dextran 70. We speculate that the crowding agents can favor both correct folding and misfolding/aggregation of denatured-rHBCK. Though it is not known what combination of crowding agents most accurately reflects the physiological environment within a cell, we believe our study could contribute to the understanding of protein folding and the factors that contribute to proper conformation and function in the intracellular environment.     

Mixed macromolecular crowding accelerates the refolding of rabbit muscle creatine kinase: implications for protein folding in physiological environments

The effects of four single macromolecular crowding agents, Ficoll 70, dextran 70, polyethylene glycol (PEG) 2000, and calf thymus DNA (CT DNA), and three mixed crowding agents containing both CT DNA and polysaccharide (or PEG 2000) on the refolding of guanidine hydrochloride-denatured rabbit muscle creatine kinase (MM-CK) have been examined by activity assay. When the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under these conditions increases 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of MM-CK by a mixed crowding agent containing CT DNA and dextran 70 (or PEG 2000) is also observed. Further folding kinetics analyses show that these three mixed crowding agents remarkably accelerate the refolding of MM-CK, compared with single crowding agents. Aggregation of MM-CK in the presence of any of the three mixed crowding agents is less serious than that in the presence of a single crowding agent at the same concentration but more serious than that in the absence of crowding agents. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than single crowding agents.     

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

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.     

Development of macroporous poly(ethylene glycol) hydrogel arrays within microfluidic channels

The mass transport of solutes through hydrogels is an important design consideration in materials used for tissue engineering, drug delivery, and protein arrays used to quantify protein concentration and activity. We investigated the use of poly(ethylene glycol) (PEG) as a porogen to enhance diffusion of macromolecules into the interior of polyacrylamide and PEG hydrogel posts photopatterned within microfluidic channels. The diffusion of GST-GFP and dextran-FITC into hydrogels was monitored and effective diffusion coefficients were determined by fitting to the Fickian diffusion equations. PEG-diacrylate (M(r) 700) with porogen formed a macroporous structure and permitted significant penetration of 250 kDa dextran. Proteins copolymerized in these macroporous hydrogels retained activity and were more accessible to antibody binding than proteins copolymerized in nonporous gels. These results suggest that hydrogel macroporosity can be tuned to regulate macromolecular transport in applications such as tissue engineering and protein arrays.     

Protein aggregation in crowded environments

The generic tendency of proteins to aggregate into non-functional, and sometimes cytotoxic, structures poses a universal problem for all types of cell. This tendency is greatly exacerbated by the high total concentration of macromolecules found within most intracellular compartments, a phenomenon referred to as macromolecular crowding. This review discusses the quantitative effects of crowding on protein aggregation and the role of molecular chaperones in combating this problem.     

大分子拥挤对DNA结构的影响

大分子拥挤(macromolecular crowding effect)代表了细胞内高度拥挤状态,其源于非特异性容积排斥效应,是细胞内与pH、离子强度等同等重要的生理因素。生物大分子介导的拥挤环境对于DNA-DNA、DNA-蛋白质的相互作用以及DNA高级结构、细胞核或核区结构的稳定具有重要作用。在拥挤环境中,大分子总浓度的增加将增强溶质的浓缩倾向,从而降低溶液的自由能。拥挤效应是胞内大分子环境的总体反映,具有高度的缓冲性,保证了胞内反应的稳定进行及细胞功能的正常行使。     

Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy

Aqueous environments in living cells are crowded, with up to >50 wt% small and macromolecule-size solutes. We investigated quantitatively one important consequence of molecular crowding--reduced diffusion of biologically important solutes. Fluorescence correlation spectroscopy (FCS) was used to measure the diffusion of a series of fluorescent small solutes and macromolecules. In water, diffusion coefficients (D(o)w) were (in cm2/s x 10(-8)): rhodamine green (270), albumin (52), dextrans (75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5 kb) and polystyrene nanospheres (5.4, 20 nm diameter; 2.3, 100 nm). Aqueous-phase diffusion (Dw) in solutions crowded with Ficoll-70 (0-60 wt%) was reduced by up to 650-fold in an exponential manner: Dw = D(o)w exp (-[C]/[C]exp), where [C]exp is the concentration (in wt%) of crowding agent reducing D(o)w by 63%. FCS data for all solutes and Ficoll-70 concentrations fitted well to a model of single-component, simple (non-anomalous) diffusion. Interestingly [C]exp were nearly identical (11+/-2 wt%, SD) for diffusion of the very different types of macromolecules in Ficoll-70 solutions. However, [C]exp was dependent on the nature of the crowding agent: for example, [C]exp for diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500 kDa dextran. Our results indicate that molecular crowding can greatly reduce aqueous-phase diffusion of biologically important macromolecules, and demonstrate a previously unrecognized insensitivity of crowding effects on the size and characteristics of the diffusing species.     

Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions

The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation.     

Crowding,Entropic Forces,and Confinement: Crucial Factors for Structures and Functions in the Cell Nucleus

The view of the cell nucleus as a crowded system of colloid particles and that chromosomes are giant self-avoiding polymers is stimulating rapid advances in our understanding of its structure and activities, thanks to concepts and experimental methods from colloid, polymer, soft matter, and nano sciences and to increased computational power for simulating macromolecules and polymers. This review summarizes current understanding of some characteristics of the molecular environment in the nucleus, of how intranuclear compartments are formed, and of how the genome is highly but precisely compacted, and underlines the crucial, subtle, and sometimes unintuitive effects on structures and reactions of entropic forces caused by the high concentration of macromolecules in the nucleus.     

Packing of the polynucleosome chain in interphase chromosomes: evidence for a contribution of crowding and entropic forces

In the crowded intranuclear environment, entropic depletion forces between macromolecules are expected to be strong. A review of simulations of linear polymers leads to several predictions about probable conformations of a polynucleosome chain in these conditions. These include a globular conformation, variable compaction due to different local rigidity or curvature of the mosaic of isochores, satellite sequences, and nucleosomes containing different histone variants, and the possibility that chromosomes represent separate phases like those seen in heterogeneous particle mixtures by experiment and simulation. Experimental results which show that macromolecular crowding alone, in the absence of exogenous cations, can stabilise interphase chromosomes and cause self-association of polynucleosome chains are presented. Together, these considerations suggest that macromolecular crowding and entropic forces are major factors in packing polynucleosome chains in vivo.     

Effect of molecular crowding on DNA polymerase activity

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.