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.

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 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.

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.

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.

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+.

Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition.

Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.

Condensin in Chromatid Cohesion and Segregation.

After replication of genomic DNA during the S phase, 2 chromatids hold together longitudinally. When cells enter mitosis, the paired sister chromatids start to condense and then segregate into individual chromatids except for the centromeric region. Upon attachment of microtubules to the kinetochore, subsequent pulling of the 2 sister chromatids by the spindles towards opposite poles results in 2 completely separated chromatids. Besides more than 100 kinds of kinetochore proteins, several key proteins such as cohesin, separase, shugoshin, and condensin contribute to chromatid cohesion and segregation. Among these proteins, condensin, a protein complex composed of 5 subunits discovered 2 decades ago, has been extensively studied in terms of the maintenance of chromosome morphology as its major function. Recent studies on condensin uncovered its role in chromatid cohesion and segregation, which will be reviewed in this article.

Detection of histidine oxidation in a monoclonal immunoglobulin gamma (IgG) 1 antibody.

Although oxidation of methionine and tryptophan are known as popular chemical modifications that occur in monoclonal antibody (mAb) molecules, oxidation of other amino acids in mAbs has not been reported to date. In this study, oxidation of the histidine residue in a human immunoglobulin gamma (IgG) 1 molecule was discovered for the first time by mass spectrometry. The oxidation of a specific histidine located at the CH2 domain of IgG1 occurred under light stress, but it was not observed under heat stress. With the forced degradation study using several reactive oxygen species, the singlet oxygen was attributed to a reactive source of the histidine oxidation. The reaction mechanism of the histidine oxidation was proposed on the basis of the mass spectrometric analysis of IgG1 oxidized in deuterium oxide and hydrogen heavy oxide.

Condensin II alleviates DNA damage and is essential for tolerance of boron overload stress in Arabidopsis.

Although excess boron (B) is known to negatively affect plant growth, its molecular mechanism of toxicity is unknown. We previously isolated two Arabidopsis thaliana mutants, hypersensitive to excess B (heb1-1 and heb2-1). In this study, we found that HEB1 and HEB2 encode the CAP-G2 and CAP-H2 subunits, respectively, of the condensin II protein complex, which functions in the maintenance of chromosome structure. Growth of Arabidopsis seedlings in medium containing excess B induced expression of condensin II subunit genes. Simultaneous treatment with zeocin, which induces DNA double-strand breaks (DSBs), and aphidicolin, which blocks DNA replication, mimicked the effect of excess B on root growth in the heb mutants. Both excess B and the heb mutations upregulated DSBs and DSB-inducible gene transcription, suggesting that DSBs are a cause of B toxicity and that condensin II reduces the incidence of DSBs. The Arabidopsis T-DNA insertion mutant atr-2, which is sensitive to replication-blocking reagents, was also sensitive to excess B. Taken together, these data suggest that the B toxicity mechanism in plants involves DSBs and possibly replication blocks and that plant condensin II plays a role in DNA damage repair or in protecting the genome from certain genotoxic stressors, particularly excess B.

The effect of magnesium ions on chromosome structure as observed by helium ion microscopy.

One of the few conclusions known about chromosome structure is that Mg2+ is required for the organization of chromosomes. Scanning electron microscopy is a powerful tool for studying chromosome morphology, but being nonconductive, chromosomes require metal/carbon coating that may conceal information about the detailed surface structure of the sample. Helium ion microscopy (HIM), which has recently been developed, does not require sample coating due to its charge compensation system. Here we investigated the structure of isolated human chromosomes under different Mg2+ concentrations by HIM. High-contrast and resolution images from uncoated samples obtained by HIM enabled investigation on the effects of Mg2+ on chromosome structure. Chromatin fiber information was obtained more clearly with uncoated than coated chromosomes. Our results suggest that both overall features and detailed structure of chromatin are significantly affected by different Mg2+ concentrations. Chromosomes were more condensed and a globular structure of chromatin with 30 nm diameter was visualized with 5 mM Mg2+ treatment, while 0 mM Mg2+ resulted in a less compact and more fibrous structure 11 nm in diameter. We conclude that HIM is a powerful tool for investigating chromosomes and other biological samples without requiring metal/carbon coating.

Chromosome interior observation by focused ion beam/scanning electron microscopy (FIB/SEM) using ionic liquid technique.

Attempts to elucidate chromosome structure have long remained elusive. Electron microscopy is useful for chromosome structure research because of its high resolution and magnification. However, biological samples such as chromosomes need to be subjected to various preparation steps, including dehydration, drying, and metal/carbon coating, which may induce shrinkage and artifacts. The ionic liquid technique has recently been developed and it enables sample preparation without dehydration, drying, or coating, providing a sample that is closer to the native condition. Concurrently, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed, allowing the investigation and direct analysis of chromosome interiors. In this study, we investigated chromosome interiors by FIB/SEM using plant and human chromosomes prepared by the ionic liquid technique. As a result, two types of chromosomes, with and without cavities, were visualized, both for barley and human chromosomes prepared by critical point drying. However, chromosome interiors were revealed only as a solid structure, lacking cavities, when prepared by the ionic liquid technique. Our results suggest that the existence and size of cavities depend on the preparation procedures. We conclude that combination of the ionic liquid technique and FIB/SEM is a powerful tool for chromosome study.

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.

The nuclear scaffold protein SAF-A is required for kinetochore-microtubule attachment and contributes to the targeting of Aurora-A to mitotic spindles.

Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization.

Effects of ionic strength and sugars on the aggregation propensity of monoclonal antibodies: influence of colloidal and conformational stabilities.

PURPOSE: To develop a general strategy for optimizing monoclonal antibody (MAb) formulations. METHODS: Colloidal stabilities of four representative MAbs solutions were assessed based on the second virial coefficient (B 2) at 20°C and 40°C, and net charges at different NaCl concentrations, and/or in the presence of sugars. Conformational stabilities were evaluated from the unfolding temperatures. The aggregation propensities were determined at 40°C and after freeze-thawing. The electrostatic potential of antibody surfaces was simulated for the development of rational formulations. RESULTS: Similar B 2 values were obtained at 20°C and 40°C, implying little dependence on temperature. B 2 correlated quantitatively with aggregation propensities at 40°C. The net charge partly correlated with colloidal stability. Salts stabilized or destabilized MAbs, depending on repulsive or attractive interactions. Sugars improved the aggregation propensity under freeze-thaw stress through improved conformational stability. Uneven and even distributions of potential surfaces were attributed to attractive and strong repulsive electrostatic interactions. CONCLUSIONS: Assessment of colloidal stability at the lowest ionic strength is particularly effective for the development of formulations. If necessary, salts are added to enhance the colloidal stability. Sugars further improved aggregation propensities by enhancing conformational stability. These behaviors are rationally predictable according to the surface potentials of MAbs.

Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency.

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 11 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

Creation of a binuclear purple copper site within a de novo coiled-coil protein.

Although various kinds of metal binding proteins have been constructed by de novo design, the creation of a binuclear metal binding site remains especially challenging. The purple copper site in subunit II of COX, referred to as the Cu(A) site, has two copper ions bridged by two Cys residues. We constructed the Cu(A) site consisting of two Cys and two His residues in a de novo designed four-helical coiled-coil protein. The protein bound two copper ions and exhibited a purple color, with relatively intense absorption bands at 488 and 530 nm in the UV-vis spectrum. The EPR spectrum displayed unresolved hyperfine splittings in the g(∥) region, which was similar to the native or engineered Cu(A) site with an A(∼480)/A(∼530) > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu(2)S(2) core structure, with two typical N(His)-Cu bonds per core at 1.90 Å, two S (Cys)-Cu bonds at 2.21 Å, and the Cu-Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.

Sensitivity enhancement of fiber-laser-based stimulated Raman scattering microscopy by collinear balanced detection technique.

We propose the collinear balanced detection (CBD) technique for noise suppression in fiber laser (FL)-based stimulated Raman scattering (SRS) microscopy. This technique reduces the effect of laser intensity noise at a specific frequency by means of pulse splitting and recombination with a time delay difference. We experimentally confirm that CBD can suppress the intensity noise of second harmonic (SH) of Er-FL pulses by 13 dB.The measured noise level including the thermal noise is higher by only ~1.4 dB than the shot noise limit. To demonstrate SRS imaging, we use 4-ps SH pulses and 3-ps Yb-FL pulses, which are synchronized subharmonically with a jitter of 227 fs. The effectiveness of the CBD technique is confirmed through SRS imaging of a cultured HeLa cell.