Ron A. Hoebe

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins.

BACKGROUND The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression. METHODS In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses. RESULTS Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression. CONCLUSIONS These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics. GENERAL SIGNIFICANCE Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.

Re-scan confocal microscopy: scanning twice for better resolution

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Difference in volume of X- and Y-chromosome-bearing bovine sperm heads matches difference in DNA content

BACKGROUND To investigate the possibilities of sperm head volume as a sorting criterion for gender preselection, we determined the magnitude of the difference in volume of X- and Y-chromosome-bearing bull sperm heads. MATERIALS AND METHODS Bovine sperm heads were sorted on the basis of their DNA content in X- and Y-chromosome-bearing fractions, using an existing flow-cytometric technique. Images of sperm heads of both populations were recorded using Differential Interference Contrast (DIC) microscopy. After reconstructing the DIC images, the area and the optical thickness of sperm heads of both populations were determined. RESULTS We found a difference in volume of X- and Y-bearing bovine sperm heads matching the difference in DNA content (3.5-4%). CONCLUSIONS Our findings indicate that volume can be used as a criterion to distinguish X- and Y-chromosome-bearing sperm, making development of a technique to sort X- and Y-chromosome-bearing sperm based on head volume theoretically possible. A strong advantage of such a technique over the existing technique based on DNA content would be that X- and Y-chromosome-bearing sperm cells could thus be sorted without subjecting them to any staining.

Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: Potentials and limitations

Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.

Largest contour segmentation: A tool for the localization of spots in confocal images

An accurate determination of the 3-D positions of multiple spots in images obtained by confocal microscopy is essential for the investigation of the spatial distribution of specific components or processes in biological specimens. The position of the centroid, as an estimator for the position of a spot, can be calculated on the basis of all voxels that belong to the domain of the spot. For this calculation a domain that defines which voxels belong to the spot must be delimited. To create a boundary for a domain we developed a 3-D image segmentation procedure: the largest contour segmentation (LCS). This procedure is based on an iterative region-growing procedure around each local maximum of intensity. By means of this procedure the position of each spot was determined accurately and automatically. Qualities of the procedure were evaluated by means of simulated test-images as well as 3-D images of real biological specimens.

Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1(R132H), a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1(R132H) that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1(R132H) mutation exhibited less IDH-mediated production of NADPH, such that after exposure to ionizing radiation (IR), there were higher levels of reactive oxygen species, DNA double-strand breaks, and cell death compared with IDH1 wild-type cells. These effects were reversed by the IDH1(R132H) inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate was sufficient to reduce IDH-mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well-described DNA hypermethylation phenotype in IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1(R132H) inhibitors in these patients may limit irradiation efficacy in this setting.

Induction of chromosome aberrations in unirradiated chromatin after partial irradiation of a cell nucleus.

Purpose : It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. Materials and methods : Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X q domain) were labelled with 125 I-iododeoxyuridine (125 IdUrd) in a period of S-phase when the vast majority of the X q domain was not replicating. DNA damage from 125 I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125 I induced DSB in the immediate vicinity of the 125 I atom. Chromosome aberrations involving what is essentially the 125 I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125 IdUrd at a later period in S-phase when the X q domain replicates, yielding a labelled X q domain. Results : The 125 I-free X q domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125 I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125 I-free X q domain was approximately half of that observed in the 125 I-labelled X q domain. Conclusions : The involvement of the 125 I-free X q domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125 I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.

Application and detection of IdUrd and CldUrd as two independent cell-cycle markers

Publisher Summary This chapter discusses application and detection of IdUrd and CldUrd as two independent cell-cycle markers. For analysis of a large range of cell kinetics processes, in particular those involving time-dependent phenomena occurring during changes in the dynamics of cell proliferation or during DNA synthesis activity at the subcellular level, the BrdUrd method is not sufficiently versatile. Studies of that type can only be carried out effectively by using two or more independent DNA replication labels. One way of approaching this problem is by application of tritiated thymidine in combination with bromodeoxyuridine (BrdUrd), but that requires the reintroduction of autoradiography techniques. An additional disadvantage of this approach, in particular for studies at the subcellular level, is the limited spatial resolution of autoradiography. These handicaps can be avoided by the introduction of a double-labeling procedure based on the application of a combination of two nonradioactive DNA precursors that are incorporated by cells in vivo. The methods for DNA double-labeling and for staining of nuclei are an extension of the methods for the immunocytochemical detection of BrdUrd.

Towards digital photon counting cameras for single-molecule optical nanoscopy

BackgroundOptical nanoscopy based on separation of single molecules by stochastic switching and subsequent localization allows surpassing the diffraction limit of light. The growing pursuit towards live-cell imaging using nanoscopy demands advancements in both science and technology.ResultsIn this article, we provide an overview of the technological advancements in the development of scientific cameras used for nanoscopy. We discuss the prospects of novel digital photon counting cameras based on a single-photon avalanche diode (SPAD) array camera for optical nanoscopy. Numerical simulations are used to evaluate and compare different scientific cameras for their performance towards single-molecule identification and localization.ConclusionA SPAD array camera with single-photon sensitivity and zero read-out noise allows for the detection of extremely weak signals at ultra-fast imaging speeds. With temporal resolution in the order of micro-seconds, a SPAD array camera offers great potential for live-cell imaging with super-resolution.

Four‐dimensional telomere analysis in recordings of living human cells acquired with Controlled Light Exposure Microscopy

Telomeres are the complex end structures that confer functional integrity and positional stability to human chromosomes. Telomere research has long been dominated by length measurements and biochemical analyses. Recently, interest has shifted towards the role of their three‐dimensional organization and dynamics within the nuclear volume. In the mammalian interphase nucleus, there is increasing evidence for a telomeric configuration that is non‐random and is cell cycle and cell type dependent. This has functional implications for genome stability. Objective and reproducible representation of the spatiotemporal organization of telomeres, under different experimental conditions, requires quantification by reliable automated image processing techniques. In this paper, we describe methods for quantitative telomere analysis in cell nuclei of living human cells expressing telomere‐binding fusion proteins. We present a toolbox for determining telomere positions within the nucleus with subresolution accuracy and tracking telomeres in 4D controlled light exposure microscopy (CLEM) recordings. The use of CLEM allowed for durable imaging and thereby improved segmentation performance considerably. With minor modifications, the underlying algorithms can be expanded to the analysis of other intranuclear features, such as nuclear bodies or DNA double stranded break foci.