Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50–60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25–35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Expansion Microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known whether the chromatin ultrastructure becomes altered by physical expansion.In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ∼4.2-times physically expanded nuclei corresponding to a maximal resolution of ∼50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution the chromatin structures were observed at a resolution of ∼25-35 nm.WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM we observed that the preservation of the chromatin ultrastructure after expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure.Nevertheless, using expanded nuclei we detected successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunostaining. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation) their ultrastructural arrangement was impaired.

Ultrastructure of the elementary fibril in extended and condensed chromatin and in metaphase chromosomes

The chromatin Ultrastructure of nuclei in ultrathin sections of root meristem in <i>Cucurbita pepo</i> - the species poor in DNA, and in <i>Haemanthus katharinae</i> - rich in DNA was compared. Four combinations of fixation and embedding were used: 1) OsO₄ (pH 7.2), methacrylates; 2) OsO₄ (pH 7.2), Epon; 3) OsO₄ (pH 6.8), Epon; 4) glutaraldehyde (pH 7.2-7.4) with OsO4 postfixation, Epon embedding. Most suitable was OsO₄ fixation (pH 7.2) and embedding in Epon because of the smallest dispersion of dimensions of chromatin fibrils and good visibility of their substructure. Fibrils of condensed chromatin, components of chromocenters (heterochromatin) in <i>Cucurbita pepo</i>, bands of chromonemata visible in the light microscope and metaphase chromosomes in <i>Haemanthus katharinae</i> showed a diameter of about 11.5 nm after OsO₄ fixation, or 14-19 nm after glutaraldehyde. Fibrils of extended chromatin (euchromatin) in <i>Cucurbita pepo</i> are thinner, their diameter is about 10 nm after OsO₄ fixation and Epon embedding, and about 14.5 nm after glutaraldehyde. There are no visible differences in the width of elementary fibrils after OsO₄ fixation and embedding in methacrylates. In both species the fibrils of extended chromatin are built up of one coiled deoxyribonucleohistone (DNH) thread about 2.3 nm in diameter, while the fibrils of condensed chromatin consist of two DNH threads, each about 3 nm in diameter. The diameter of DNH threads is determined neither by the fixative nor by the way of embedding; they are less visible in the material embedded in methacrylates. The results obtained show that the structure of chromatin fibrils depends on the kind of chromatin, but not on DNA content. In <i>Haemanthus katharinae</i>, the species rich in DNA, the greater amount of chromatin appears in condensed form.

Ultrastructural, autoradiographic and electrophoretic examinations of Chara tomentosa spermiogenesis

Ultrastructure of a spermatid nucleus changes many times during spermiogenesis. Condensed chromatin forms irregular clusters during phases I-II, a continuous ring adjacent to a nuclear envelope during phases III-V and a network occupying the whole nucleus during phase VI. In advanced spermiogenesis dense chromatin disappears and short randomly positioned fibrils arise, then long parallel ones are found (phase VIII) which during phase IX form a lamellar structure. In mature spermatozoids (phase X) chromatin becomes extremely condensed. ³H-arginine and ³H-lysine incorporation into spermatids during 2-min incubation is intensive during phases IN, decreases during phases VI, VII and becomes very low during phases VIII-IX. Capillary electrophoresis has shown that during <em>Chara tomentosa</em> spermiogenesis replacement of histones with basic proteins whose mobility is comparable to that of salmon protamines takes place. At the beginning of spermiogenesis core and linker histones are found in spermatids. During early spermiogenesis protamine-like proteins appear and their amount increases in late spermiogenesis when core histones are still present. In mature spermatozoids only protamine-like proteins represented by 3 fractions: 9.1 kDa, 9.6 kDa, 11.2 kDa are found. Disappearance of linker histones following their modification precedes disappearance of core histones. The results indicate that dynamic rearrangement of chromatin ultrastructure and aminoacid incorporation rate during spermiogenesis are reflected in basic nuclear protein changes.

Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1α localization in erythroblasts

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1gt/gt homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.

A New Technique to Reveal DNA “En Bloc” and in Lowicryl Ultrathin Sections: The NaOH-Formaldehyde-MA-Method

This paper deals with the developing of a new simple and repetitive technique to preferentially stain DNA-containing structures at the E.M. level that can be used “en bloc” and on sections. DNA staining techniques are scarce, being the osmium ammine one the only cytochemical method known until now that specifically stains DNA but not the proteins that are complexed with it, having been used to study chromatin ultrastructure. The osmium ammine synthesis is a complex process and, although an improvement has been lately reported, the stain is not comercially available.Glutaraldehyde-fixed Allium cepa L. root meristematic cells, Capsicum annuum L. & Scilla peruviana L. pollen grains were treated with 0.5N NaOH in 4% formaldehyde overnight, rinsed in water and 1% acetic acid, methanol dehydrated, and inmersed in methanol-acetic anhydride (5:1) at 25ºC overnight. Then, the samples were Epon embedded. To perform the technique on sections, some samples were glutaraldehyde or formaldehyde fixed, methanol dehydrated, Lowicryl embedded at -20ºC and ultrathin sections were treated in the same way as in the “en bloc” procedure.

Assaying chromosome arrangement in embryonic interphase nuclei of Drosophila melanogaster by radiation induced interchanges

SummaryDespite recent advances in our understanding of chromatin ultrastructure, little is known of the arrangement of chromosomes during interphase, the portion of the cell cycle associated with somatic gene transcription. An experimental procedure is described which has allowed the determination of the nature of the relative arrangement during interphase of chromosomes in a specific diploid cell type of Drosophila, the salivary gland anlage of the 10–14-h-old embryo. At this stage of development the salivary gland cells have ceased mitotic divisions. Embryos of 10–14 h in age were irradiated with 12000 rads of gamma radiation and then allowed to develop into third instar larvae. The polytene chromosomes of these larvae were examined for radiation-induced interchanges. From the distribution of observed interchanges, three major features of interphase chromosome arrangement were inferred. (1) Each euchromatic chromosomal arm occupies a specific domain within the interphase nucleus which does not appreciably overlap with those of other arms. (2) Within these chromosomal domains DNA folding is very extensive. (3) The heterochromatic regions of each chromosomal arm are sequestered from the euchromatic regions. An additional point of interest concerns the nature of the interchanges observed. No reciprocal interchanges were observed – all appeared to be partial exchanges, possibly subchromatid interchanges involving only one DNA strand from each of the two exchange sites.