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.

Differential intra-host infection kinetics in Aedes aegypti underlie superior transmissibility of African relative to Asian Zika virus.

IMPORTANCE: The recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell-level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency.

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.

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.

Chromosome inner structure investigation by electron tomography and electron diffraction in a transmission electron microscope.

Our understanding of the inner structure of metaphase chromosomes remains inconclusive despite intensive studies using multiple imaging techniques. Transmission electron microscopy has been extensively used to visualize chromosome ultrastructure. This review summarizes recent results obtained using two transmission electron microscopy-based techniques: electron tomography and electron diffraction. Electron tomography allows advanced three-dimensional imaging of chromosomes, while electron diffraction detects the presence of periodic structures within chromosomes. The combination of these two techniques provides results contributing to the understanding of local structural organization of chromatin fibers within chromosomes.

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.

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.

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.

Parameters affecting the accuracy of nanoparticle shape and size measurement in 3D.

While electron tomography can be used to visualize objects at nanoscale, it is difficult to perform reproducible quantitative measurements. Here we measure the shape and size of nanoparticles (NPs) in three dimensions (3D) using electron tomography. We evaluated the accuracy of maximum Feret diameter (Feretmax), minimum Feret diameter (Feretmini) and volume of NPs measurements from reconstructed 3D images which were obtained from data acquired with varied electron dose. We perform both simulations and experiment to clarify what factors effect on the accuracy of the NP shape measurement. Based on the results, suitable reconstruction methods and threshold for binarization were evaluated. We also report comparison results obtained on exactly the same samples in two different laboratories.

Calcium depletion destabilises kinetochore fibres by the removal of CENP-F from the kinetochore.

The attachment of spindle fibres to the kinetochore is an important process that ensures successful completion of the cell division. The Ca2+ concentration increases during the mitotic phase and contributes microtubule stability. However, its role in the spindle organisation in mitotic cells remains controversial. Here, we investigated the role of Ca2+ on kinetochore fibres in living cells. We found that depletion of Ca2+ during mitosis reduced kinetochore fibre stability. Reduction of kinetochore fibre stability was not due to direct inhibition of microtubule polymerisation by Ca2+-depletion but due to elimination of one dynamic component of kinetochore, CENP-F from the kinetochore. This compromised the attachment of kinetochore fibres to the kinetochore which possibly causes mitotic defects induced by the depletion of Ca2+.

Calcium ions function as a booster of chromosome condensation.

Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division. The depletion of chromosome scaffold proteins does not prevent chromosome condensation despite structural defects. This suggests that other factors contribute to condensation. Here we investigated the contribution of divalent cations, particularly Ca2+, to chromosome condensation in vitro and in vivo. Ca2+ depletion caused defects in proper mitotic progression, particularly in chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca2+. Chromosomes had compact globular structures when exposed to Ca2+ and expanded fibrous structures without Ca2+. Therefore, we have clearly demonstrated a role for Ca2+ in the compaction of chromatin fibres.