Localization and quantitative distribution of a chromatin structural protein Topoisomerase II on plant chromosome using HVTEM and UHVTEM.

Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

Higher-order structure of barley chromosomes observed by electron tomography.

The higher order structure of the metaphase chromosome has been an enigma for over a century and several different models have been presented based on results obtained by a variety of techniques. Some disagreements in the results between methods have possibly arisen from artifacts caused during sample preparation such as staining and dehydration. Therefore, we treated barley chromosomes with ionic liquid to minimize the effects of dehydration. We also observed chromosomes on a film with holes to keep pristine chromosome structure from being flattened as seen when placed on a continuous support film. A chromosome placed over a hole in a thin carbon film was mounted on a tomography holder, and its structure was observed in three dimensions (3D) using electron tomography. We found that there are periodic structures with 300-400 nm pitch along the axis in barley chromosomes. The pitch sizes are larger than those observed in human chromosomes.

Insight into magnesium ions effect on chromosome banding and ultrastructure.

Magnesium ion (Mg2+ ) plays a fundamental role in chromosome condensation which is important for genetic material segregation. Studies about the effects of Mg2+ on the overall chromosome structure have been reported. Nevertheless, its effects on the distribution of heterochromatin and euchromatin region have yet to be investigated. The aim of this study was to evaluate the effects of Mg2+ on the banding pattern and ultrastructure of the chromosome. Chromosome analysis was performed using the synchronized HeLa cells. The effect of Mg2+ was evaluated by subjecting the chromosomes to three different solutions, namely XBE5 (containing 5 mM Mg2+ ) as a control, XBE (0 mM Mg2+ ), and 1 mM EDTA as cations-chelator. Chromosome banding was carried out using the GTL-banding technique. The ultrastructure of the chromosomes treated with and without Mg2+ was further obtained using SEM. The results showed a condensed chromosome structure with a clear banding pattern when the chromosomes were treated with a buffer containing 5 mM Mg2+ . In contrast, chromosomes treated with a buffer containing no Mg2+ and those treated with a cations-chelator showed an expanded and fibrous structure with the lower intensity of the banding pattern. Elongation of the chromosome caused by decondensation resulted in the band splitting. The different ultrastructure of the chromosomes treated with and without Mg2+ was obvious under SEM. The results of this study further emphasized the role of Mg2+ on chromosome structure and gave insights into Mg2+ effects on the banding distribution and ultrastructure of the chromosome.

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human-Arabidopsis Hybrid Cell Line.

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human-Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.

Molecular organization of recombinant human-Arabidopsis chromosomes in hybrid cell lines.

Although plants and animals are evolutionarily distant, the structure and function of their chromosomes are largely conserved. This allowed the establishment of a human-Arabidopsis hybrid cell line in which a neo-chromosome was formed by insertion of segments of Arabidopsis chromosomes into human chromosome 15. We used this unique system to investigate how the introgressed part of a plant genome was maintained in human genetic background. The analysis of the neo-chromosome in 60- and 300-day-old cell cultures by next-generation sequencing and molecular cytogenetics suggested its origin by fusion of DNA fragments of different sizes from Arabidopsis chromosomes 2, 3, 4, and 5, which were randomly intermingled rather than joined end-to-end. The neo-chromosome harbored Arabidopsis centromeric repeats and terminal human telomeres. Arabidopsis centromere wasn't found to be functional. Most of the introgressed Arabidopsis DNA was eliminated during the culture, and the Arabidopsis genome in 300-day-old culture showed significant variation in copy number as compared with the copy number variation in the 60-day-old culture. Amplified Arabidopsis centromere DNA and satellite repeats were localized at particular loci and some fragments were inserted into various positions of human chromosome. Neo-chromosome reorganization and behavior in somatic cell hybrids between the plant and animal kingdoms are discussed.

Calcium ion significance on the maintenance of barley (Hordeum vulgare) chromosome compaction.

Cations, especially calcium ions (Ca2+), is one of the major factors responsible for the chromosome higher-order structure formation. The effects of cations on the human chromosomes have already been evaluated, however, whether the presence of similar effects on plant chromosomes has not been reported to date. Thus, in this study, we investigated the role of Ca2+ on the barley (Hordeum vulgare L.) chromosome structure. Barley chromosomes were isolated from the meristematic tissue within the germinated roots. The roots were subjected to enzymatic treatment, fixed, and drop on the cover glass to spread the chromosomes out. Some chromosomes were treated with BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) to chelate Ca2+. Chromosome samples were then observed by fluorescence microscopy and scanning electron microscopy (SEM). The disperse structure of the chromosome was observed after BAPTA treatment. Chromosomes showed less condensed structure due to Ca2+ chelation. The high-resolution of SEM provided a more detailed visualization of chromosome ultrastructure under different calcium ion conditions. This study revealed the calcium ion effect on chromosome structure is important regardless of the organisms, suggesting a similar mechanism of chromosome condensation through humans and plants.

Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy.

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.

Imaging approaches for chromosome structures.

This review describes image analyses for chromosome visible structures, focusing on the chromosome imaging system CHIAS (Chromosome Image Analyzing System). CHIAS is the first comprehensive imaging system for the analysis and characterization of plant chromosomes. A simulation method for human vision for capturing band positive regions was developed and used for the image analysis of large plant chromosomes with bands. Applying this method to C-banded Crepis chromosomes enabled recognition of band positive regions as seen by human vision. Furthermore, a new image parameter, condensation pattern was developed and successfully applied to identify small plant chromosomes such as rice and brassicas. Condensation profile (CP) derived from condensation pattern was also effective in developing quantitative chromosome maps. The result was quantitative chromosomal maps of several plants with small chromosomes, including Arabidopsis, diploid brassicas, rapeseed, rice, spinach, and sugarcane. In the final chapter, various applications of imaging techniques to the analysis of pachytene chromosomes, improved visibility of multicolor FISH images, 3D reconstruction of a human chromosome based on cross-section images obtained by a FIB/SEM, automatic extraction of chromosomal regions by machine learning, etc. are described.

Imaging the inner structure of chromosomes: contribution of focused ion beam/scanning electron microscopy to chromosome research.

Visualization of the chromosome ultrastructure has revealed new insights into its structural and functional properties. The use of new methods for revealing not only the surface but also the inner structure of the chromosome has been emerged. Some methods have long been used, such as conventional transmission electron microscopy (TEM). Although it has indispensably contributed to the revelation of the ultrastructure of the various biological samples, including chromosomes, some challenges have also been encountered, such as laborious sample preparation, limited view areas, and loss of information on some parts due to ultramicrotome sectioning. Therefore, a more advanced method is needed. Scanning electron microscopy (SEM) is also advantageous in the surface visualization of chromosome samples. However, it is limited by accessibility to gain the inner structure information. Focused ion beam/scanning electron microscopy (FIB/SEM) provides a way to investigate the inner structure of the samples in a direct slice-and-view manner to observe the ultrastructure of the inner part of the sample continuously and further construct a three-dimensional image. This method has long been used in the material science field, and recently, it has also been applied to biological research, such as in showing the inner structure of chromosomes. This review article presents the contributions of this new method to chromosome research and its recent developments in the inner structure of chromosome and discusses its current and potential applications to the high-resolution imaging of chromosomes.

Higher-Order Structure of Human Chromosomes Observed by Electron Diffraction and Electron Tomography.

It is well known that two DNA molecules are wrapped around histone octamers and folded together to form a single chromosome. However, the nucleosome fiber folding within a chromosome remains an enigma, and the higher-order structure of chromosomes also is not understood. In this study, we employed electron diffraction which provides a noninvasive analysis to characterize the internal structure of chromosomes. The results revealed the presence of structures with 100­200 nm periodic features directionally perpendicular to the chromosome axis in unlabeled isolated human chromosomes. We also visualized the 100­200 nm periodic features perpendicular to the chromosome axis in an isolated chromosome whose DNA molecules were specifically labeled with OsO4 using electron tomography in 300 keV and 1 MeV transmission electron microscopes.

Application of the Chromosome Image Analyzing System (CHIAS) for Straightening Cation-treated Bent Chromosomes.

Divalent cations, mainly calcium and magnesium ions, are known to play a major role in the maintenance of chromosomes. The depletion of both ions using ethylenediaminetetraacetic acid (EDTA) results in a bent chromosome structure with extended arms and dispersed chromatin fibers. The importance of divalent cations for the maintenance of chromosome structure has been reported previously; nevertheless, previous studies were limited to qualitative data only. Straightening the bent image of the chromosome would provide quantitative data. Thus, this study aimed to evaluate the effects of cation depletion by the application of the Chromosome Image Analyzing System (CHIAS) to straighten bent chromosomes. Human HeLa chromosomes were treated with EDTA as a known chelating agent in order to investigate the importance of divalent cations on the maintenance of chromosome structure. Chromosomes were stained and directly observed with a fluorescence microscope. Images were then analyzed using CHIAS. The results revealed that EDTA-treated chromosomes showed longer arms than those without EDTA treatment, and most of them tended to bend-out. By straightening the image using CHIAS, the bent chromosomes were successfully straightened. The average lengths of the chromosomes treated with and without EDTA were 4.97 and 0.96 µm, respectively. These results signify the advantages of CHIAS for chromosome analysis and highlight the fundamental effects of cations on chromosome condensation.

Human metaphase chromosome consists of randomly arranged chromatin fibres with up to 30-nm diameter.

During cell division, mitotic chromosomes assemble and are equally distributed into two new daughter cells. The chromosome organisation of the two chromatids is essential for even distribution of genetic materials. Although the 11-nm fibre or nucleosome structure is well-understood as a fundamental fibrous structure of chromosomes, the reports on organisation of 30-nm basic chromatin fibres have been controversial, with debates on the contribution of 30-nm or thicker fibres to the higher order inner structure of chromosomes. Here, we used focused ion beam/scanning electron microscopy (FIB/SEM) to show that both 11-nm and 30-nm fibres are present in the human metaphase chromosome, although the higher-order periodical structure could not be detected under the conditions employed. We directly dissected the chromosome every 10-nm and observed 224 cross-section SEM images. We demonstrated that the chromosome consisted of chromatin fibres of an average diameter of 16.9-nm. The majority of the chromatin fibres had diameters between 5 and 25-nm, while those with 30-nm were in the minority. The reduced packaging ratio of the chromatin fibres was detected at axial regions of each chromatid. Our results provide a strong basis for further discussions on the chromosome higher-order structure.

Impact of Diabetes Perceptions on Medication Adherence in Japan.

Background: Patients' perception of diabetes mellitus is one of the psychosocial factors influencing diabetic behavior. This patients' perception of the disease is a mental image formed from the experience of patients with type 2 diabetes mellitus and reportedly reflects the aspect of recuperation. We investigated the relationship between changes in the patients' perception of the disease and medication adherence, as influenced by the active involvement of community pharmacists. Methods: A prospective cohort study that used patient registry based in community pharmacies was conducted in patients with type 2 diabetes using oral antidiabetic agents at a pharmacy in Ishikawa Prefecture in Japan. Patients responded to the questionnaire at the time of enrollment and at the end of the one-year intervention period. The pharmacist confirmed the patient's medication status and treatment problems via telephone calls at least once every two weeks for one year. Main outcome measures: Type 2 diabetes patients' perception of the disease related to medication adherence. Results: The study enrolled 113 patients. Among the seven diabetes image factors, "Living an orderly life" and "Feeling of fear" were significantly associated with medication adherence. "Feeling of neglect of health" was significantly associated at the subscale level. Conclusion: All the three factors related to medication adherence indicated self-care ability. To enhance the self-care ability of the patient, pharmacists should assist in self-care interventions for the patients.

3D observation of chromosome scaffold structure using a 360° electron tomography sample holder.

The chromosome scaffold is considered to be a key structure of the mitotic chromosome. It plays a vital role in chromosome condensation, shaping the X-shaped structure of the mitotic chromosome, and also provides flexibility for chromosome movement during cell division. However, it remains to be elucidated how the chromosome scaffold organizes the mitotic chromosome and how it supports shaping the structure of the chromosome during metaphase. Here we present a new technique that enables the observation of the chromosome scaffold structure in metaphase chromosomes from any direction, by transferring an isolated chromosome to a 360° rotational holder for electron tomography (ET). The chromosome was stained with immunogold-labeled condensin complex, one of the major chromosome scaffold proteins and then observed in three dimensions using ET. Using the locations of gold nanoparticles to visualize the underlying structure, the tomograms we obtained reveal the patterns of chromosome scaffold organization, which appears to consist of a helical structure that serves to organize chromatin loops into the metaphase chromosome.

Reversible Changes of Chromosome Structure upon Different Concentrations of Divalent Cations.

The structural details of chromosomes have been of interest to researchers for many years, but how the metaphase chromosome is constructed remains unsolved. Divalent cations have been suggested to be required for the organization of chromosomes. However, detailed information about the role of these cations in chromosome organization is still limited. In the current study, we investigated the effects of Ca2+ and Mg2+ depletion and the reversibility upon re-addition of one of the two ions. Human chromosomes were treated with different concentrations of Ca2+and Mg2+. Depletion of Ca2+ and both Ca2+ and Mg2+ were carried out using 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ethylenediaminetetraacetic acid (EDTA), respectively. Chromosome structure was examined by fluorescence microscopy and scanning electron microscopy. The results indicated that chromosome structures after treatment with a buffer without Mg2+, after Ca2+ depletion, as well as after depletion of both Mg2+, and Ca2+, yielded fewer compact structures with fibrous chromatin than those without cation depletion. Interestingly, the chromatin of EDTA-treated chromosomes reversed to their original granular diameters after re-addition of either Mg2+ or Ca2+ only. These findings signify the importance of divalent cations on the chromosome structure and suggest the interchangeable role of Ca2+ and Mg2+.

Use of 3D imaging for providing insights into high-order structure of mitotic chromosomes.

The high-order structure of metaphase chromosomes remains still under investigation, especially the 30-nm structure that is still controversial. Advanced 3D imaging has provided useful information for our understanding of this detailed structure. It is evident that new technologies together with improved sample preparations and image analyses should be adequately combined. This mini review highlights 3D imaging used for chromosome analysis so far with future imaging directions also highlighted.

Cdk1-dependent phosphorylation of KIF4A at S1186 triggers lateral chromosome compaction during early mitosis.

Chromosome organization during cell division is achieved through the timely association of proteins with chromatin and is regulated by protein phosphorylation. Kinesin family member 4A (KIF4A) plays an important role in the chromosome organization through the formation of the chromosome scaffold structure. However, the relationship between the function of KIF4A and its phosphorylation remains unclear. Here, we demonstrate that Cdk1-dependent phosphorylation of KIF4A at S1186 is required for chromosome binding and chromosome scaffold formation. The KIF4A mutant, which is not phosphorylated at S1186, was found to localize to the nucleus during interphase but did not accumulate in the chromosome scaffold after nuclear envelope breakdown. In addition, defects in KIF4A phosphorylation were found to disrupt the interaction of KIF4A with the condensin I complex. As a result, the morphology of the chromosomes was observed to be laterally decondensed, without condensin I in the chromosome scaffold. Additionally, a defect in chromosome segregation, chromosome bridge formation, was often observed. Although both KIF4A and condensin I disappeared from the chromosomes, the chromosomal localization of condensin II was not affected. Collectively, our novel results revealed that Cdk1-dependent KIF4A phosphorylation at S1186 is a trigger for chromosomal organization during early mitosis.

Production of a Human Cell Line with a Plant Chromosome.

It is a major challenge in biology to know whether chromosome functions of replication, segregation, gene expression, inheritance, etc. are conserved among evolutionary distant organisms where common structural features are maintained. Establishment of hybrid cell lines between evolutionary distant organisms, such as humans and plants, would be one of the promising synthetic approaches to study the evolutionary conservation of chromosome functions. In this chapter, we describe the protocol for successful establishment of human cell lines with a functional plant chromosome. Systematic analyses of hybrid cells will facilitate the evolutionary study of organisms with respect to chromosome functions. It will also provide a basic platform for genome writing and construction of chromosomal shuttle vectors .