In situ 3D visualization of biomineralization matrix proteins.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.

Nuclear proteins are often given a concise title that captures their function, such as 'transcription factor,' 'polymerase' or 'nuclear-receptor.' However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology.

Nuclear protein extraction from frozen porcine myocardium

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Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5 ± 0.7% of total protein; mean±SE, n = 9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofilament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosine-phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the â-agonist dobutamine. Upon treatment, PY-STAT3 increased 30.2 ± 8.5-fold in total homogenates, but only 6.9 ± 2.1-fold (n = 4, P = 0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks (AU)

[Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

Do we need molecular tomography of a cell and how can it be achieved?

1. The spatial relationship between intracellular molecules and their local concentrations are two critical parameters required for a better understanding of protein-protein interactions in the cell. 2. Determination of the local concentration of proteins in individual cells using more sophisticated techniques and determination of the spatial relationship between a molecular platform and its partners is essential for allow us to obtain more convincing and concrete scientific conclusions. 3. As a reasonable goal, development of molecular tomography of the cell is proposed.

Organization of higher-level chromatin structures (chromomere, chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization.

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.