Erik M. M. Manders

Measurement of co-localization of objects in dual-colour confocal images

A method to measure the degree of co‐localization of objects in confocal dual‐colour images has been developed. This image analysis produced two coefficients that represent the fraction of co‐localizing objects in each component of a dual‐channel image. The generation of test objects with a Gaussian intensity distribution, at well‐defined positions in both components of dual‐channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co‐localization coefficients were determined before degrading the image with background, cross‐talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non‐specific binding and cross‐reactivity of fluorescent probes, optical cross‐talk and photon noise. The degraded images were restored by filtering and cross‐talk correction. The co‐localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co‐localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.

Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.

Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy (CLEM), a simple imaging approach that reduces photobleaching and phototoxicity two- to tenfold, depending on the fluorophore distribution in the object. By spatially controlling the light-exposure time, CLEM reduces the excitation-light dose without compromising image quality. We show that CLEM reduces photobleaching sevenfold in tobacco plant cells expressing microtubule-associated GFP-MAP4 and reduces production of reactive oxygen species eightfold and prolongs cell survival sixfold in HeLa cells expressing chromatin-associated H2B-GFP. In addition, CLEM increases the dynamic range of the fluorescence intensity at least twofold.

Severe Early Life Stress Hampers Spatial Learning and Neurogenesis, but Improves Hippocampal Synaptic Plasticity and Emotional Learning under High-Stress Conditions in Adulthood

Early life stress increases the risk for developing stress-related pathologies later in life. Recent studies in rats suggest that mild early life stress, rather than being overall unfavorable, may program the hippocampus such that it is optimally adapted to a stressful context later in life. Here, we tested whether this principle of “adaptive programming” also holds under severely adverse early life conditions, i.e., 24 h of maternal deprivation (MD), a model for maternal neglect. In young adult male rats subjected to MD on postnatal day 3, we observed reduced levels of adult hippocampal neurogenesis as measured by cell proliferation, cell survival, and neuronal differentiation. Also, mature dentate granule cells showed a change in their dendritic morphology that was most noticeable in the proximal part of the dendritic tree. Lasting structural changes due to MD were paralleled by impaired water maze acquisition but did not affect long-term potentiation in the dentate gyrus. Importantly, in the presence of high levels of the stress hormone corticosterone, even long-term potentiation in the dentate gyrus of MD animals was facilitated. In addition to this, contextual learning in a high-stress environment was enhanced in MD rats. These morphological, electrophysiological, and behavioral observations show that even a severely adverse early life environment does not evolve into overall impaired hippocampal functionality later in life. Rather, adversity early in life can prepare the organism to perform optimally under conditions associated with high corticosteroid levels in adulthood.

The Polycomb group protein EZH2 is upregulated in proliferating, cultured human mantle cell lymphoma

Polycomb group (PcG) proteins are involved in the stable transmittance of the repressive state of their gene targets throughout the cell cycle. Mis‐expression of PcG proteins can lead to proliferative defects and tumorigenesis. There are two separate multimeric PcG protein complexes: an EED–EZH2‐containing complex and a BMI1–RING1‐containing complex. In the normal human follicle mantle, both PcG complexes have mutually exclusive expression patterns. BMI1–RING1 is expressed, but EZH2–EED is not. Here, we studied the expression of both complexes in six cases of mantle cell lymphoma (MCL), which is derived from the follicle mantle. MCL cells can be cultured in vitro and stimulated to proliferation. We found that resting MCL cells expressed BMI1–RING1, but not EZH2–EED, like normal mantle cells. Proliferating MCL cells, however, showed strongly enhanced expression of EZH2. Also, BMI1 and RING1 continued to be expressed in proliferating MCL. This is the first demonstration that EZH2 expression can be upregulated in fresh lymphoma cells. To test whether the enhanced EZH2 expression was causal for the increased proliferation in MCL, we overexpressed EZH2 in two different cell lines. In the B cell‐derived Ramos cell line, EZH2 overexpression caused an increase in the proliferation rate. This suggests a possible causal effect between EZH2 upregulation and increased proliferation in haematopoietic cells.

Spatially confined folding of chromatin in the interphase nucleus

Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10–30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin–chromatin interactions and determine the 3-dimensional organization of the chromatin fiber.

Local UV‐induced DNA damage in cell nuclei results in local transcription inhibition

UV‐induced DNA damage causes cells to repress RNA synthesis and to initiate nucleotide excision repair (NER). NER and transcription are intimately linked processes. Evidence has been presented that, in addition to damaged genes, undamaged loci are transcriptionally inhibited. We investigated whether RNA synthesis from undamaged genes is affected by the presence of UV damage elsewhere in the same nucleus, using a novel technique to UV irradiate only part of a nucleus. We show that the basal transcription/repair factor TFIIH is recruited to the damaged nuclear area, partially depleting the undamaged nuclear area. Remarkably, this sequestration has no effect on RNA synthesis. This result was obtained for cells that are able to carry out NER and for cells deficient in NER. We conclude that cross talk between NER and transcription occurs only over short distances in nuclei of living cells.

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place predominantly at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors.

The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces

Although Candida containing biofilms contribute to the development of oral candidosis, the characteristics of multi-species Candida biofilms and how oral bacteria modulate these biofilms is poorly understood. The aim of this study was to investigate interactions between Candida albicans and either Candida glabrata or Streptococcus mutans in biofilms grown on various surfaces, with or without saliva. Hydroxyapatite (HA), polymethylmetacrylate (PMMA) and soft denture liner (SL) discs were used as substratum. Counts of viable micro-organisms in the accumulating biofilm layer were determined and converted to colony forming units per unit surface area. Confocal laser scanning microscopy was used to characterize biofilms and to quantitate the number of hyphae in each condition tested. Viable counts of C. albicans and C. glabrata per mm(2) decreased in the order HA>PMMA>SL (p<0.05). Biofilms grown on saliva-coated specimens harboured fewer C. glabrata than uncoated specimens (p<0.05). Glucose and the presence of S. mutans suppressed C. albicans hyphal formation. Dual Candida species biofilms did not show competitive interaction between the two species. We conclude that Candida biofilms are significantly affected by saliva, substratum type and by the presence of other micro-organisms.

Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

Confocal Microscopy of Living Cells

If a picture is worth a thousand words, then a movie may be worth a million words. Microcinematography and, later, video microscopy have provided great insight into biological phenomena. One limitation, however, has been the difficulty of imaging in three dimensions. In many cases, observations have been made on cultured cells that are thin to start with or tissue preparations that have been sectioned.