Alterations in the chromatin packaging, driven by transcriptional activity, revealed by AFM.

BACKGROUND: The gene expression differs in the nuclei of normal and malignant mammalian cells, and transcription is a critical initial step, which defines the difference. The mechanical properties of transcriptionally active chromatin are still poorly understood. Recently we have probed transcriptionally active chromatin of the nuclei subjected to mechanical stress, by Atomic Force Microscopy (AFM) [1]. Nonetheless, a systematic study of the phenomenon is needed. METHODS: Nuclei were deformed and studied by AFM. Non-deformed nuclei were studied by fluorescence confocal microscopy. Their transcriptional activity was studied by RNA electrophoresis. RESULTS: The malignant nuclei under the study were stable to deformation and assembled of 100-300 nm beads-like units, while normal cell nuclei were prone to deformation. The difference in stability to deformation of the nuclei correlated with DNA supercoiling, and transcription-depended units were responsive to supercoils breakage. The inhibitors of the topoisomerases I and II disrupted supercoiling and made the malignant nucleus prone to deformation. Cell nuclei treatment with histone deacetylase inhibitors (HDACIs) preserved the mechanical stability of deformed malignant nuclei and, at the same time, made it possible to observe chromatin decondensation up to 20-60 nm units. The AFM results were supplemented with confocal microscopy and RNA electrophoresis data. CONCLUSIONS: Self-assembly of transcriptionally active chromatin and its decondensation, driven by DNA supercoiling-dependent rigidity, was visualized by AFM in the mechanically deformed nuclei. GENERAL SIGNIFICANCE: We demonstrated that supercoiled DNA defines the transcription mechanics, and hypothesized the nuclear mechanics in vivo should depend on the chromatin architecture.

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.

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.

Detection of inflammatory processes during various diseases by the method of flow cytofluorometry.

Oxidative (respiratory) burst is an important manifestation of inflammation. Precise quantitative assessment of this reaction by flow cytometry made it possible to record and evaluate the severity of the inflammatory processes in a wide spectrum of diseases including diphtheria, hepatitis, pneumonia, bronchial asthma, arthritis, vasculitis, postoperative complications, tuberculosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and myocardial infarction. This approach can be employed as a highly sensitive method of detection of inflammatory reactions and monitoring of their course in various pathological processes.