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This is a post-peer-review, pre-copyedit version of an article published in Chromosome Research. The final authenticated version is available online at: http://dx.doi.org/10.1007/s10577-021-09648-3., 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.

Wako, T., Yoshida, A., Kato, J. et al. Human metaphase chromosome consists of randomly arranged chromatin fibres with up to 30-nm diameter. Sci Rep 10, 8948 (2020). https://doi.org/10.1038/s41598-020-65842-z., 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.

Yusuf, M., Kaneyoshi, K., Fukui, K. et al. Use of 3D imaging for providing insights into high-order structure of mitotic chromosomes. Chromosoma 128, 7–13 (2019). https://doi.org/10.1007/s00412-018-0678-5., 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.

Ohmido N, Iwata A, Kato S, Wako T, Fukui K (2018) Development of a quantitative pachytene chromosome map and its unification with somatic chromosome and linkage maps of rice (Oryza sativa L.). PLOS ONE 13(4): e0195710. https://doi.org/10.1371/journal.pone.0195710., A quantitative pachytene chromosome map of rice (Oryza sativa L.) was developed using imaging methods. The map depicts not only distribution patterns of chromomeres specific to pachytene chromosomes, but also the higher order information of chromosomal structures, such as heterochromatin (condensed regions), euchromatin (decondensed regions), the primary constrictions (centromeres), and the secondary constriction (nucleolar organizing regions, NOR). These features were image analyzed and quantitatively mapped onto the map by Chromosome Image Analyzing System ver. 4.0 (CHIAS IV). Correlation between H3K9me2, an epigenetic marker and formation and/or maintenance of heterochromatin, thus was, clearly visualized. Then the pachytene chromosome map was unified with the existing somatic chromosome and linkage maps by physically mapping common DNA markers among them, such as a rice A genome specific tandem repeat sequence (TrsA), 5S and 45S ribosomal RNA genes, five bacterial artificial chromosome (BAC) clones, four P1 bacteriophage artificial chromosome (PAC) clones using multicolor fluorescence in situ hybridization (FISH). Detailed comparison between the locations of the DNA probes on the pachytene chromosomes using multicolor FISH, and the linkage map enabled determination of the chromosome number and short/long arms of individual pachytene chromosomes using the chromosome number and arm assignment designated for the linkage map. As a result, the quantitative pachytene chromosome map was unified with two other major rice chromosome maps representing somatic prometaphase chromosomes and genetic linkages. In conclusion, the unification of the three rice maps serves as an indispensable basic information, not only for an in-depth comparison between genetic and chromosomal data, but also for practical breeding programs.

Takata H, Madung M, Katoh K, Fukui K (2018) Cdk1-dependent phosphorylation of KIF4A at S1186 triggers lateral chromosome compaction during early mitosis. PLOS ONE 13(12): e0209614. https://doi.org/10.1371/journal.pone.0209614., 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-depen-dent 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.

Phengchat, R., Takata, H., Uchiyama, S. et al. Calcium depletion destabilises kinetochore fibres by the removal of CENP-F from the kinetochore. Sci Rep 7, 7335 (2017). https://doi.org/10.1038/s41598-017-07777-6., 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⁺.

Shinozaki, N., Hashimoto, R., Fukui, K. et al. Efficient generation of single domain antibodies with high affinities and enhanced thermal stabilities. Sci Rep 7, 5794 (2017). https://doi.org/10.1038/s41598-017-06277-x., Single domain antibodies (sdAbs), made of natural single variable regions of camelid or cartilaginous fish antibodies, or unpaired variable regions of mouse or human IgGs, are some of the more promising biologic modalities. However, such conventional sdAbs have difficulties of either using unwieldy animals for immunization or having high affinity deficiencies. Herein, we offer a versatile method to generate rabbit variable domain of heavy chain (rVH) derived sdAbs with high affinities (K D values of single digit nM or less) and enhanced thermal stabilities (equal to or even higher than those of camelid derived sdAbs). It was found that a variety of rVH binders, including those with high affinities, were efficiently acquired using an rVH-displaying phage library produced at a low temperature of 16 °C. By a simple method to introduce an additional disulfide bond, their unfolding temperatures were increased by more than 20 °C without severe loss of binding affinity. Differential scanning calorimetry analysis suggested that this highly efficient thermal stabilization was mainly attributed to the entropic contribution and unique thermodynamic character of the rVHs.

Poonperm R, Takata H, Uchiyama S, Fukui K (2017) Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold. PLOS ONE 12(8): e0183298. https://doi.org/10.1371/journal.pone.0183298., Kinesin family member 4 (KIF4) and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1) is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis.

Li H, Tsuchimoto S, Harada K, Yamasaki M, Sakai H, Wada N, Alipour A, Sasai T, Tsunekawa A,Tsujimoto H, Ando T, Tomemori H, Sato S, Hirakawa H, Quintero VP, Zamarripa A, Santos P, Hegazy A, Ali AM and Fukui K (2017) GeneticTracing of Jatropha curcas L. from Its Mesoamerican Origin to the World. Front. Plant Sci. 8:1539.doi: 10.3389/fpls.2017.01539., Jatropha curcas L. (Jatropha), a shrub species of the family Euphorbiaceae, has been recognized as a promising biofuel plant for reducing greenhouse gas emissions. However, recent attempts at commercial cultivation in Africa and Asia have failed because of low productivity. It is important to elucidate genetic diversity and relationship in worldwide Jatropha genetic resources for breeding of better commercial cultivars. Here, genetic diversity was analyzed by using 246 accessions from Mesoamerica, Africa and Asia, based on 59 simple sequence repeat markers and eight retrotransposonbased insertion polymorphism markers. We found that central Chiapas of Mexico possesses the most diverse genetic resources, and the Chiapas Central Depression could be the center of origin. We identified three genetic groups in Mesoamerica, whose distribution revealed a distinct geographic cline. One of them consists mainly of accessions from central Chiapas. This suggests that it represents the original genetic group. We found two Veracruz accessions in another group, whose ancestors might be shipped from Port of Veracruz to the Old World, to be the source of all African and Asian Jatropha. Our results suggest the human selection that caused low productivity in Africa and Asia, and also breeding strategies to improve African and Asian Jatropha. Cultivars improved in the productivity will contribute to expand mass commercial cultivation of Jatropha in Africa and Asia to increase biofuel production, and finally will support in the battle against the climate change.

Phengchat, R., Takata, H., Morii, K. et al. Calcium ions function as a booster of chromosome condensation. Sci Rep 6, 38281 (2016). https://doi.org/10.1038/srep38281., 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.

Elena Krayukhina, Kouhei Tsumoto, Susumu Uchiyama, Kiichi Fukui. Effects of Syringe Material and Silicone Oil Lubrication on the Stability of Pharmaceutical Proteins. Journal of Pharmaceutical Sciences, Volume 104, Issue 2, 2015, Pages 527-535. ttps://doi.org/10.1002/jps.24184., Currently, polymer-based prefillable syringes are being promoted to the pharmaceutical market because they provide an increased break resistance relative to traditionally used glass syringes. Despite this significant advantage, the possibility that barrel material can affect the oligomeric state of the protein drug exists. The present study was designed to compare the effect of different syringe materials and silicone oil lubrication on the protein aggregation. The stability of a recombinant fusion protein, abatacept (Orencia), and a fully human recombinant immunoglobulin G1, adalimumab (Humira), was assessed in silicone oil-free (SOF) and silicone oil-lubricated 1-mL glass syringes and polymer-based syringes in accelerated stress study. Samples were subjected to agitation stress, and soluble aggregate levels were evaluated by size-exclusion chromatography and verified with analytical ultracentrifugation. In accordance with current regulatory expectations, the amounts of subvisible particles resulting from agitation stress were estimated using resonant mass measurement and dynamic flow-imaging analyses. The amount of aggregated protein and particle counts were similar between unlubricated polymer-based and glass syringes. The most significant protein loss was observed for lubricated glass syringes. These results suggest that newly developed SOF polymer-based syringes are capable of providing biopharmaceuticals with enhanced physical stability upon shipping and handling.

Susumu Uchiyama, Kazuki Kawahara, Yuki Hosokawa, Shunsuke Fukakusa, Hiroya Oki, Shota Nakamura, Yukiko Kojima, Masanori Noda, Rie Takino, Yuya Miyahara, Takahiro Maruno, Yuji Kobayashi, Tadayasu Ohkubo, Kiichi Fukui. Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition. Journal of Biological Chemistry, Volume 290, Issue 49, 2015, Pages 29461-29477. https://doi.org/10.1074/jbc.M115.670794., 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.

Robinson I., Schwenke J., Yusuf M., et al. Towards single particle imaging of human chromosomes at SACLA. Journal of Physics B: Atomic, Molecular and Optical Physics, 48, 24, 244007. https://doi.org/10.1088/0953-4075/48/24/244007., Single particle imaging (SPI) is one of the front-page opportunities which were used to motivate the construction of the first x-ray free electron lasers (XFELs). SPI's big advantage is that it avoids radiation damage to biological samples because the diffraction takes place in femtosecond single shots before any atomic motion can take place in the sample, hence before the onset of radiation damage. This is the 'diffract before destruction' theme, destruction being assured from the high x-ray doses used. This article reports our collaboration's first attempt at SPI using the SACLA XFEL facility in June 2015. The report is limited to experience with the instrumentation and examples of data because we have not yet had time to invert them to images.

Poonperm, R., Takata, H., Hamano, T. et al. Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins. Sci Rep 5, 11916 (2015). https://doi.org/10.1038/srep11916., Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.

Takemoto, K., Matsuda, T., Sakai, N. et al. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep 3, 2629 (2013). https://doi.org/10.1038/srep02629., Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

Alipour, A., Tsuchimoto, S., Sakai, H. et al. Structural characterization of copia-type retrotransposons leads to insights into the marker development in a biofuel crop, Jatropha curcasL.. Biotechnol Biofuels 6, 129 (2013). https://doi.org/10.1186/1754-6834-6-129., Background: Recently, Jatropha curcas L. has attracted worldwide attention for its potential as a source of biodiesel. However, most DNA markers have demonstrated high levels of genetic similarity among and within jatropha populations around the globe. Despite promising features of copia-type retrotransposons as ideal genetic tools for gene tagging, mutagenesis, and marker-assisted selection, they have not been characterized in the jatropha genome yet. Here, we examined the diversity, evolution, and genome-wide organization of copia-type retrotransposons in the Asian, African, and Mesoamerican accessions of jatropha, then introduced a retrotransposon-based marker for this biofuel crop. Results: In total, 157 PCR fragments that were amplified using the degenerate primers for the reverse transcriptase (RT) domain of copia-type retroelements were sequenced and aligned to construct the neighbor-joining tree. Phylogenetic analysis demonstrated that isolated copia RT sequences were classified into ten families, which were then grouped into three lineages. An in-depth study of the jatropha genome for the RT sequences of each family led to the characterization of full consensus sequences of the jatropha copia-type families. Estimated copy numbers of target sequences were largely different among families, as was presence of genes within 5 kb flanking regions for each family. Five copia-type families were as appealing candidates for the development of DNA marker systems. A candidate marker from family Jc7 was particularly capable of detecting genetic variation among different jatropha accessions. Fluorescence in situ hybridization (FISH) to metaphase chromosomes reveals that copia-type retrotransposons are scattered across chromosomes mainly located in the distal part regions. Conclusion: This is the first report on genome-wide analysis and the cytogenetic mapping of copia-type retrotransposons of jatropha, leading to the discovery of families bearing high potential as DNA ma

SPIE BiOS, 2013, San Francisco, California, United States, Yasuyuki Ozeki, Yoichi Otsuka, Shuya Sato, Hiroyuki Hashimoto, Wataru Umemura, Kazuhiko Sumimura, Norihiko Nishizawa, Kiichi Fukui, Kazuyoshi Itoh, "Label-free observation of tissues by high-speed stimulated Raman spectral microscopy and independent component analysis," Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 858806 (22 February 2013); https://doi.org/10.1117/12.2002772, We have developed a video-rate stimulated Raman scattering (SRS) microscope with frame-by-frame wavenumber tunability. The system uses a 76-MHz picosecond Ti:sapphire laser and a subharmonically synchronized, 38-MHz Yb fiber laser. The Yb fiber laser pulses are spectrally sliced by a fast wavelength-tunable filter, which consists of a galvanometer scanner, a 4-f optical system and a reflective grating. The spectral resolution of the filter is ~ 3 cm-1. The wavenumber was scanned from 2800 to 3100 cm-1 with an arbitrary waveform synchronized to the frame trigger. For imaging, we introduced a 8-kHz resonant scanner and a galvanometer scanner. We were able to acquire SRS images of 500 x 480 pixels at a frame rate of 30.8 frames/s. Then these images were processed by principal component analysis followed by a modified algorithm of independent component analysis. This algorithm allows blind separation of constituents with overlapping Raman bands from SRS spectral images. The independent component (IC) spectra give spectroscopic information, and IC images can be used to produce pseudo-color images. We demonstrate various label-free imaging modalities such as 2D spectral imaging of the rat liver, two-color 3D imaging of a vessel in the rat liver, and spectral imaging of several sections of intestinal villi in the mouse. Various structures in the tissues such as lipid droplets, cytoplasm, fibrous texture, nucleus, and water-rich region were successfully visualized.

Sachihiro Matsunaga, Hideaki Takata, Akihiro Morimoto, Kayoko Hayashihara, Tsunehito Higashi, Kouhei Akatsuchi, Eri Mizusawa, Mariko Yamakawa, Mamoru Ashida, TomokoM. Matsunaga, Takachika Azuma, Susumu Uchiyama, Kiichi Fukui. RBMX: A Regulator for Maintenance and Centromeric Protection of Sister Chromatid Cohesion. Cell Reports, Volume 1, Issue 4, 2012, Pages 299-308. https://doi.org/10.1016/j.celrep.2012.02.005., Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.

Ma N., Matsunaga S., Morimoto A., et al. The nuclear scaffold protein SAF-A is required for kinetochore-microtubule attachment and contributes to the targeting of Aurora-A to mitotic spindles. Journal of Cell Science, 124, 3, 394. https://doi.org/10.1242/jcs.063347., 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.

Matsunaga, Sachihiro and Fukui, Kiichi. "The chromosome peripheral proteins play an active role in chromosome dynamics" , vol. 1, no. 2, 2010, pp. 157-164. https://doi.org/10.1515/bmc.2010.018, The chromosome periphery is a chromosomal structure that covers the surface of mitotic chromosomes. The structure and function of the chromosome periphery has been poorly understood since its first description in 1882. It has, however, been proposed to be an insulator or barrier to protect chromosomes from subcellular substances and to act as a carrier of nuclear and nucleolar components to direct their equal distribution to daughter cells because most chromosome peripheral proteins (CPPs) are derived from the nucleolus or nucleus. Until now, more than 30 CPPs were identified in mammalians. Recent immunostaining analyses of CPPs have revealed that the chromosome periphery covers the centromeric region of mitotic chromosomes in addition to telomeres and regions between two sister chromatids. Knockdown analyses of CPPs using RNAi have revealed functions in chromosome dynamics, including cohesion of sister chromatids, kinetochore-microtubule attachments, spindle assembly and chromosome segregation. Because most CPPs are involved in various subcellular events in the nucleolus or nuclear at interphase, a temporal and spatial-specific knockdown method of CPPs in the chromosome periphery will be useful to understand the function of chromosome periphery in cell division.

Kawahara, K., Nakamura, S., Katsu, Y., Motooka, D., Hosokawa, Y., Kojima, Y., Matsukawa, K., Takinowaki, H., Uchiyama, S., Kobayashi, Y., Fukui, K. & Ohkubo, T. (2010). Acta Cryst. F66, 1067-1070., In higher eukaryotes, the condensin complex, which mainly consists of two structural maintenance of chromosomes (SMC) subunits, SMC2 (CAP-E) and SMC4 (CAP-C), plays a critical role in the formation of higher order chromosome structures during mitosis. Biochemical and electron-microscopic studies have revealed that the SMC2 and SMC4 subunits dimerize through the interaction of their hinge domains, forming a characteristic V-shaped hetero-dimer. However, the details of their function are still not fully understood owing to a lack of structural information at the atomic level. In this study, the human SMC2 hinge domain with short coiled coils was cloned, expressed, purified and crystallized in the orthorhombic space group C222 in native and SeMet-derivatized forms. Because of the poor diffraction properties of these crystals, the mutant Leu68→SeMet was designed and crystallized in order to obtain the experimental phases. The SeMet-derivatized crystals of the mutant belonged to space group P3212, with unit-cell parameters a = b = 128.8, c = 91.4 Å. The diffraction data obtained from a crystal that diffracted to 2.4 Å resolution were suitable for SAD phasing. © 2010 International Union of Crystallography All rights reserved.

Kayoko Hayashihara, Susumu Uchiyama, Shigeru Shimamoto, Shouhei Kobayashi, Miroslav Tomschik, Hidekazu Wakamatsu, Daisuke No, Hiroki Sugahara, Naoto Hori, Masanori Noda, Tadayasu Ohkubo, Jordanka Zlatanova, Sachihiro Matsunaga, Kiichi Fukui. The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA. Journal of Biological Chemistry, Volume 285, Issue 9, 2010, Pages 6498-6507. https://doi.org/10.1074/jbc.M109.092833., In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 M NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷-Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined byNMRrevealed that the globular domain (Met¹⁵³-Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.

Ozeki Y., Kitagawa Y., Sumimura K., et al. Stimulated raman scattering microscope with shot noise limited sensitivity using subharmonically synchronized laser pulses. Optics Express, 18, 13, 13708. https://doi.org/10.1364/OE.18.013708., We propose and demonstrate the use of subharmonically synchronized laser pulses for low-noise lock-in detection in stimulated Raman scattering (SRS) microscopy. In the experiment, Yb-fiber laser pulses at a repetition rate of 38 MHz are successfully synchronized to Ti:sapphire laser pulses at a repetition rate of 76 MHz with a jitter of <8 fs by a two-photon detector and an intra-cavity electro-optic modulator. By using these pulses, high-frequency lock-in detection of SRS signal is accomplished without high-speed optical modulation. The noise level of the lock-in signal is found to be higher than the shot noise limit only by 1.6 dB. We also demonstrate high-contrast, 3D imaging of unlabeled living cells.

Shusei Sato, Hideki Hirakawa, Sachiko Isobe, Eigo Fukai, Akiko Watanabe, Midori Kato, Kumiko Kawashima, Chiharu Minami, Akiko Muraki, Naomi Nakazaki, Chika Takahashi, Shinobu Nakayama, Yoshie Kishida, Mitsuyo Kohara, Manabu Yamada, Hisano Tsuruoka, Shigemi Sasamoto, Satoshi Tabata, Tomoyuki Aizu, Atsushi Toyoda, Tadasu Shin-i, Yohei Minakuchi, Yuji Kohara, Asao Fujiyama, Suguru Tsuchimoto, Shin'ichiro Kajiyama, Eri Makigano, Nobuko Ohmido, Nakako Shibagaki, Joyce A. Cartagena, Naoki Wada, Tsutomu Kohinata, Alipour Atefeh, Shota Yuasa, Sachihiro Matsunaga, Kiichi Fukui, Sequence Analysis of the Genome of an Oil-Bearing Tree, Jatropha curcas L., DNA Research, Volume 18, Issue 1, February 2011, Pages 65–76, https://doi.org/10.1093/dnares/dsq030., The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ∼95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.

Hieu M. Dang, Gen Omura, Toshiyuki Omano, Masatomo Yamagiwa, Shin'ichiro Kajiyama, Yasuyuki Ozeki, Kazuyoshi Itoh, and Kiichi Fukui "Label-free imaging by stimulated parametric emission microscopy reveals a difference in hemoglobin distribution between live and fixed erythrocytes," Journal of Biomedical Optics 14(4), 040506 (1 July 2009). https://doi.org/10.1117/1.3207151, We demonstrate that stimulated parametric emission (SPE) microscopy enables label-free, 3-D visualization of internal hemoglobin distribution of live mouse and chicken erythrocytes with high sensitivity. Change in hemoglobin distribution in chicken erythrocytes before and after ethanol fixation is clearly visualized. © 2009 Society of Photo-Optical Instrumentation Engineers.

Ozeki Y., Dake F., Kajiyama S., et al. Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy. Optics Express, 17, 5, 3651. https://doi.org/10.1364/OE.17.003651., We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.

Wataru Watanabe, Sachhiro Matsunaga, Tsunehito Higashi, Kiichi Fukui, and Kazuyoshi Itoh "In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation," Journal of Biomedical Optics 13(3), 031213 (1 May 2008). https://doi.org/10.1117/1.2939401, Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein. © 2008 Society of Photo-Optical Instrumentation Engineers.

Shusei Sato, Yasukazu Nakamura, Takakazu Kaneko, Erika Asamizu, Tomohiko Kato, Mitsuteru Nakao, Shigemi Sasamoto, Akiko Watanabe, Akiko Ono, Kumiko Kawashima, Tsunakazu Fujishiro, Midori Katoh, Mitsuyo Kohara, Yoshie Kishida, Chiharu Minami, Shinobu Nakayama, Naomi Nakazaki, Yoshimi Shimizu, Sayaka Shinpo, Chika Takahashi, Tsuyuko Wada, Manabu Yamada, Nobuko Ohmido, Makoto Hayashi, Kiichi Fukui, Tomoya Baba, Tomoko Nakamichi, Hirotada Mori, Satoshi Tabata, Genome Structure of the Legume, Lotus japonicus, DNA Research, Volume 15, Issue 4, August 2008, Pages 227–239, https://doi.org/10.1093/dnares/dsn008, The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.

SPIE BiOS, 2008, San Jose, California, United States, Wataru Watanabe, Tomoko Shimada, Sachihiro Matsunaga, Daisuke Kurihara, Shin-ichi Arimura, Nobuhiro Tsutsumi, Kiichi Fukui, Kazuyoshi Itoh, "Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein," Proc. SPIE 6860, Multiphoton Microscopy in the Biomedical Sciences VIII, 68601B (15 February 2008); https://doi.org/10.1117/12.768745., We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

Ma N., Matsunaga S., Takata H., et al. Nucleolin functions in nucleolus formation and chromosome congression. Journal of Cell Science, 120, 12, 2091. https://doi.org/10.1242/jcs.008771., A complex structure, designated the chromosome periphery, surrounds each chromosome during mitosis. Although several proteins have been shown to localize to the chromosome periphery, their functions during mitosis remain unclear. Here, we used a combination of high-resolution microscopy and RNA-interference-mediated depletion to study the functions of nucleolin, a nucleolar protein localized at the chromosome periphery, in interphase and mitosis. During mitosis, nucleolin was localized in the peripheral region including the vicinity of the outer kinetochore of chromosomes. Staining with an antibody specific for nucleolin phosphorylated by CDC2 revealed that nucleolin was also associated with the spindle poles from prometaphase to anaphase. Nucleolin depletion resulted in disorganization of the nucleoli at interphase. Furthermore, nucleolin-depleted cells showed a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. The misaligned chromosomes showed syntelic kinetochore-microtubule attachments with reduced centromere stretching. Taken together, our results indicate that nucleolin is required for nucleolus formation, and is also involved in chromosome congression and spindle formation.

Isobe K., Ozeki Y., Kawasumi T., et al. Highly sensitive spectral interferometric fourwave mixing microscopy near the shot noise limit and its combination with two-photon excited fluorescence microscopy. Optics Express, 14, 23, 11204. https://doi.org/10.1364/OE.14.011204., We present spectral interferometric four-wave mixing (FWM) microscopy with a nearly shot-noise limited sensitivity and with the capability of separating FWM signals from fluorescence signals. We analyze the requirements for obtaining the shot-noise limited sensitivity and experimentally achieve the sensitivity that is only 4-dB lower than the shotnoise limit. Moreover, we show that only FWM signals can be extracted through the Fourier filtering even when the FWM spectrum is overlapped and overwhelmed by the fluorescence spectrum. We demonstrate simultaneous acquisition of FWM and two-photon excited fluorescence images of fluorescent monodispersed polystyrene microspheres.

Lasers and Applications in Science and Engineering, 2006, San Jose, California, United States, Wataru Watanabe, Tomoko Shimada, Sachihiro Matsunaga, Hiroshi Ishii, Tsunehito Higashi, Kiichi Fukui, Kazuyoshi Itoh, "Nanosurgery of sub-cellular organelles in living cells using a femtosecond laser oscillator," Proc. SPIE 6108, Commercial and Biomedical Applications of Ultrafast Lasers VI, 610804 (28 February 2006); https://doi.org/10.1117/12.645474., Femtosecond laser pulses in the near-infrared region have potential applications in nanosurgery in cell biology. Femtosecond laser pulses can be used to selectively disrupt and dissect intracellular organelles in living cells. We report on disruption of fluorescence-labeled nucleus and mitochondria in living HeLa cells using a femtosecond Ti:sapphire laser oscillator with a repetition rate of 76 MHz. We examined the effectiveness of the restaining method in combination with fluorescence recovery after photobleaching (FRAP) analysis to discern disruption or bleaching.

Lin, L., Nakano, H., Uchiyama, S., Fujimoto, S., Matsunaga, S., Nakamura, S., Kobayashi, Y., Ohkubo, T. & Fukui, K. (2005). Acta Cryst. F61, 414-416., A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6322, with unit-cell parameters a = b = 53.69, c = 159.2 Å. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 Å. A complete data set from crystals of the SeMet-substituted protein was also obtained. © 2005 International Union of Crystallography All rights reserved.

Shimada T., Watanabe W., Matsunaga S., et al. Intracellular disruption of mitochondria in a living HeLa cell with a 76-MHz femtosecond laser oscillator. Optics Express, 13, 24, 9869. https://doi.org/10.1364/OPEX.13.009869., Femtosecond laser pulses can be used to selectively disrupt and dissect intracellular organelles. We report on disruption of mitochondria in living HeLa cells using a femtosecond laser oscillator with a repetition rate of 76 MHz. We studied the laser parameters used for disruption. The longterm viability of the cells after disruption of a single mitochondrion was confirmed by the observation of cell division, indicating that intracellular disruption of organelles using a femtosecond laser oscillator can be performed without compromising the long-term cell viability.

Susumu Uchiyama, Shouhei Kobayashi, Hideaki Takata, Takeshi Ishihara, Naoto Hori, Tsunehito Higashi, Kayoko Hayashihara, Takefumi Sone, Daisuke Higo, Takashi Nirasawa, Toshifumi Takao, Sachihiro Matsunaga, Kiichi Fukui. Proteome Analysis of Human Metaphase Chromosomes. Journal of Biological Chemistry, Volume 280, Issue 17, 2005, Pages 16994-17004. https://doi.org/10.1074/jbc.M412774200., DNA is packaged as chromatin in the interphase nucleus. Dining mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite > 1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into sis groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein frame-work for future research on this topic.

European Conferences on Biomedical Optics 2005, 2005, Munich, Germany, Wataru Watanabe, Tomoko Shimada, Kazuyoshi Itoh, Sachihiro Matsunaga, Kiichi Fukui, "Femtosecond laser manipulation of subcellular organelles in living cells," Proc. SPIE 5863, Therapeutic Laser Applications and Laser-Tissue Interactions II, 58630B (26 August 2005); https://doi.org/10.1117/12.633079., Manipulation—that is, inactivation and movement—of subcellular organelles in living cells is important to understand cell dynamics. We review disruption of subcellular organelles in living cells by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of organelles in living cells was experimentally confirmed by restaining of organelles. Femtosecond laser based nanosurgery has the possibility to remove or manipulate organelles in living cells and may lead to elucidation of functional interactions between organelles, protein transportation, and inter-cell communication.