Simultaneous detection of multiple targets for ultrastructural immunocytochemistry.

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

Transcription-dependent rearrangements of actin and nuclear myosin I in the nucleolus.

Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

Actin complexes in the cell nucleus: new stones in an old field.

Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.

LR White is preferable to Unicryl for immunogold detection of fixation-sensitive nuclear antigens.

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.

The nucleoskeleton: a permanent structure of cell nuclei regardless of their transcriptional activity.

Nuclear matrix or nucleoskeleton is thought to provide structural basis for intranuclear order. However, the nature of this structure is still uncertain because of numerous technical difficulties in its visualization. To reveal the "real" morphology of the nucleoskeleton, and to identify possible sources of structural artifacts, three methods of nucleoskeleton preparations were compared. The nucleoskeleton visualized by all these techniques consists of identical elements: nuclear lamina and an inner network comprising core filaments and the "diffuse" nucleoskeleton. We then tested if the nucleoskeleton is a stable structure or a transient transcription-dependent structure. Incubation with transcription inhibitors (alpha-amanitin, actinomycin D, and DRB) for various periods of time had no obvious effect on the morphology of the nucleoskeleton. A typical nucleoskeleton structure was observed also in a physiological model-in transcriptionally inactive mouse 2-cell embryos and in active 8- to 16-cell embryos. Our data suggest that the nucleoskeleton is a permanent structure of the cell nucleus regardless of the nuclear transcriptional state, and the principal architecture of the nucleoskeleton is identical throughout the interphase.

Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA.

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction.

Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190 kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo, p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.

Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells.

Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.