PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components Grande, M.A.; van der Kraan, K.; van Steensel, B.; Schul, W.; de The, H.; van der Voort, H.T.M.; de Jong, L.; van Driel, R. Link to publicationCitation for published version (APA): Grande, M. A., van der Kraan, K., van Steensel, B., Schul, W., de The, H., van der Voort, H. T. M., ... van Driel, R. (1996). PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components. Journal of Cellular Biochemistry, 63, 280-291. 3.0.CO;2-T" class="link">https://doi.org/10.1002/(SICI)1097-4644(19961201)63:33.0.CO;2-T General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, 'TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RARa gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protcin, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bod...

SummaryIn this paper, we compare the performance of three iterative methods for image restoration: the Richardson-Lucy algorithm, the iterative constrained Tikhonov-Miller algorithm (ICTM) and the Carrington algorithm. Both the ICTM and the Carrington algorithm are based on an additive Gaussian noise model, but differ in the way they incorporate the non-negativity constraint. Under low light-level conditions, this additive (Gaussian) noise model is a poor description of the actual photon-limited image recording, compared with that of a Poisson process. The RichardsonLucy algorithm is a maximum likelihood estimator for the intensity of a Poisson process. We have studied various methods for determining the regularization parameter of the ICTM and the Carrington algorithm and propose a (Gaussian) prefiltering to reduce the noise sensitivity of the Richardson-Lucy algorithm. The results of these algorithms are compared on spheres convolved with a point spread function and distorted by Poisson noise. Our simulations show that the Richardson-Lucy algorithm, with Gaussian prefiltering, produces the best result in most of the tests. Finally, we show an example of how restoration methods can improve quantitative analysis: the total amount of fluorescence inside a closed object is measured in the vicinity of another object before and after restoration.

The relationship between cell shape and function has long been of interest. However, although the behaviour of the cytoskeleton during the cell cycle has been studied extensively variations in the shape and three-dimensional substructure of the nucleus are less well documented. The spatial distribution of chromatin has previously been studied by a mathematical analysis of the optical densities of stained nuclei, allowing an indirect derivation of the three-dimensional distribution of chromatin. More direct information on chromatin organization can be obtained from electron-microscopic serial sections, although this is very laborious. Using an iterative deconvolution algorithm, Agard and Sedat achieved a degree of optical sectioning in conventional fluorescence microscopy and reconstructed the three-dimensional arrangement of polytene chromosomes. We report here on the three-dimensional structure of cultured mammalian cells as visualized by confocal scanning laser microscopy (CSLM). The exceptionally short depth of field of this imaging technique provides direct optical sectioning which, together with its higher resolution, makes CSLM extremely useful for studying the three-dimensional morphology of biological structures.

SummaryWe report three-dimensional (3D) microscopy with nearly isotropic resolution in the l/5 ¹ l/10 range. Our approach combines 4Pi-confocal two-photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F-actin fibers in mouse fibroblast cells. A comparison with unrestored twophoton confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.

The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image restoration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a significant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.