AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei.

BACKGROUND: Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. METHODS: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). RESULTS: Nuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. CONCLUSIONS: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. GENERAL SIGNIFICANCE: A new outlook to studying internal nuclei structure is proposed.

Experimental evidence for logarithmic fractal structure of botanical trees.

The area-preserving rule for botanical trees by Leonardo da Vinci is discussed in terms of a very specific fractal structure, a logarithmic fractal. We use a method of the numerical Fourier analysis to distinguish the logarithmic fractal properties of the two-dimensional objects and apply it to study the branching system of real trees through its projection on the two-dimensional space, i.e., using their photographs. For different species of trees (birch and oak) we observe the Q^{-2} decay of the spectral intensity characterizing the branching structure that is associated with the logarithmic fractal structure in two-dimensional space. The experiments dealing with the side view of the tree should complement the area preserving Leonardo's rule with one applying to the product of diameter d and length l of the k branches: d_{i}l_{i}=kd_{i+1}l_{i+1}. If both rules are valid, then the branch's length of the next generation is sqrt[k] times shorter than previous one: l_{i}=sqrt[k]l_{i+1}. Moreover, the volume (mass) of all branches of the next generation is a factor of d_{i}/d_{i+1} smaller than previous one. We conclude that a tree as a three-dimensional object is not a logarithmic fractal, although its projection onto a two-dimensional plane is. Consequently, the life of a tree flows according to the laws of conservation of area in two-dimensional space, as if the tree were a two-dimensional object.

Observation of nucleic acid and protein correlation in chromatin of HeLa nuclei using small-angle neutron scattering with D_{2}O-H_{2}O contrast variation.

The small-angle neutron scattering (SANS) on HeLa nuclei demonstrates the bifractal nature of the chromatin structural organization. The border line between two fractal structures is detected as a crossover point at Q_{c}≈4×10^{-2}nm^{-1} in the momentum transfer dependence Q^{-D}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the large scale structural organizations of nucleic acids (NA) and proteins. Both NA and protein structures have a mass fractal arrangement with the fractal dimension of D≈2.5 at scales smaller than 150 nm down to 20 nm. Both NA and proteins show a logarithmic fractal behavior with D≈3 at scales larger than 150 nm up to 6000 nm. The combined analysis of the SANS and atomic force microscopy data allows one to conclude that chromatin and its constitutes (DNA and proteins) are characterized as soft, densely packed, logarithmic fractals on the large scale and as rigid, loosely packed, mass fractals on the smaller scale. The comparison of the partial cross sections from NA and proteins with one from chromatin as a whole demonstrates spatial correlation of two chromatin's components in the range up to 900 nm. Thus chromatin in HeLa nuclei is built as the unified structure of the NA and proteins entwined through each other. Correlation between two components is lost upon scale increases toward 6000 nm. The structural features at the large scale, probably, provide nuclei with the flexibility and chromatin-free space to build supercorrelations on the distance of 10^{3} nm resembling cycle cell activity, such as an appearance of nucleoli and a DNA replication.

Bifractal structure of chromatin in rat lymphocyte nuclei.

The small-angle neutron scattering (SANS) on the rat lymphocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. The scattering intensity from rat lymphocyte nuclei is described by power law Q^{-D} with fractal dimension approximately 2.3 on smaller scales and 3 on larger scales. The crossover between two fractal structures is detected at momentum transfer near 10^{-1}nm^{-1}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the structural organizations of nucleic acids (NA) and proteins. Both chromatin components show bifractal behavior with logarithmic fractal structure on the large scale and volume fractal with slightly smaller than 2.5 structure on the small scale. Scattering intensities from chromatin, protein component, and NA component demonstrate an extremely extensive range of logarithmic fractal behavior (from 10^{-3} to approximately 10^{-1}nm^{-1}). We compare the fractal arrangement of rat lymphocyte nuclei with that of chicken erythrocytes and the immortal HeLa cell line. We conclude that the bifractal nature of the chromatin arrangement is inherent in the nuclei of all these cells. The details of the fractal arrangement-its range and correlation/interaction between nuclear acids and proteins are specific for different cells and is related to their functionality.

Switch of fractal properties of DNA in chicken erythrocytes nuclei by mechanical stress.

The small-angle neutron scattering (SANS) on the chicken erythrocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. Use of the contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals the differences in the DNA and protein arrangements inside the chromatin substance. It is the DNA that serves as a framework that constitutes the bifractal behavior showing the mass fractal properties with D=2.22 at a smaller scale and the logarithmic fractal behavior with D≈3 at a larger scale. The protein spatial organization shows the mass fractal properties with D≈2.34 throughout the whole nucleus. The borderline between two fractal levels can be significantly shifted toward smaller scales by centrifugation of the nuclei disposed on the dry substrate, since nuclei suffer from mechanical stress transforming them to a disklike shape. The height of this disk measured by atomic force microscopy (AFM) coincides closely with the fractal borderline, thus characterizing two types of the chromatin with the soft (at larger scale) and rigid (at smaller scale) properties. The combined SANS and AFM measurements demonstrate the stress induced switch of the DNA fractal properties from the rigid, but loosely packed, mass fractal to the soft, but densely packed, logarithmic fractal.

Additive scaling law for structural organization of chromatin in chicken erythrocyte nuclei.

Small-angle neutron scattering (SANS) on nuclei of chicken erythrocytes demonstrates the cubic dependence of the scattering intensity Q^{-3} in the range of momentum transfer Q∈10^{-3}-10^{-2}nm^{-1}. Independent spin-echo SANS measurements give the spin-echo function, which is well described by the exponential law in a range of sizes (3×10^{2})-(3×10^{4}) nm. Both experimental dependences reflect the nature of the structural organization of chromatin in the nucleus of a living cell, which corresponds to the correlation function γ(r)=ln(ξ/r) for r<ξ, where ξ=(3.69±0.07)×10^{3} nm, the size of the nucleus. It has the specific scaling property of the logarithmic fractal γ(r/a)=γ(r)+ln(a), i.e., the scaling down by a gives an additive constant to the correlation function, which distinguishes it from the mass fractal, which is characterized by multiplicative constant.