Architecture of a Coat for the Nuclear Pore Membrane

Nuclear pore complexes (NPCs) reside in the nuclear membrane and mediate macromolecules exchange between the nucleus and cytoplasm. Although the protein components of NPCs, termed collectively nucleoporin, have been identified, how nucleoporins arrange in NPC, however, is still an enigma. NPC is a highly symmetric protein complex, which contains an eight-fold rotational symmetry across the center of the pore and a two-fold symmetry in the plane of the nuclear envelope. In addition, according to electron microscopic reconstruction model, NPC can also be considered schematically as a series of concentric cylinders. A peripheral cylinder coating the nuclear pore membrane contains a heptameric Nup84 complex, which harbors Nup145C/Sec13 complex in the center. Hence, X-ray crystallographic approach was employed to determine the Nup145C/Sec13 structure. Notably, in the two crystal forms I obtained, Nup145C/Sec13 oligmerized to form a slightly bent hetero-octamer rod. In the meantime, the oligomerization state could be observed in solution as well. Furthermore, as indicated by structural analyses, the interacting interfaces not only are evolutionarily conserved but also cover huge area. Taken together, the Nup145C/Sec13 hetero-octamer rod could be physiologically relevant without much doubt. In concordance with the stoichiometric considerations of NPC, then we confidently propose a model with respect to a coat for the nuclear pore membrane. In the model, the coat is constructed of four rings and each ring is composed of eight heptamers. Moreover, due to the three axes on the Nup145C/Sec13 rod, the hetero-octamer is also places vertically in order to connect these four anti-parallel rings. Remarkably, the Nup85/Seh1p crystal structures that were determined recently indicated that the assembly of this pair bears reseblance in its shape and dimension to that of another pair, Nup145C/Sec13. Thus, Nup85/Seh1 does form another hetero-octamer pole as we predicted in the model. Surprisingly, in contrast to the rigidity of the Nup145C/Sec13 hetero-octamer, the Nup85/Seh1 structures reveal a hinge motion that may facilitate the conformational change in the NPC for importing the integral membrane proteins

Architecture of a Coat for the Nuclear Pore Membrane

The symmetric core of the nuclear pore complex can be considered schematically as a series of concentric cylinders. A peripheral cylinder coating the pore membrane contains the previously characterized, elongated heptamer that harbors Sec13-Nup145C in its middle section. Strikingly, Sec13-Nup145C crystallizes as a hetero-octamer in two space groups. Oligomerization of Sec13-Nup145C was confirmed biochemically. Importantly, the numerous interacting surfaces in the hetero-octamer are evolutionarily highly conserved, further underlining the physiological relevance of the oligomerization. The hetero-octamer forms a slightly curved, yet rigid rod of sufficient length to span the entire height of the proposed membrane-adjacent cylinder. In concordance with the dimensions and symmetry of the nuclear pore complex core, we suggest that the cylinder is constructed of four antiparallel rings, each ring being composed of eight heptamers arranged in a head-to-tail fashion. Our model proposes that the hetero-octamer would vertically traverse and connect the four stacked rings

DNA Binding and Degradation by the HNH Protein ColE7

The bacterial toxin ColE7 bears an HNH motif which has been identified in hundreds of prokaryotic and eukaryotic endonucleases, involved in DNA homing, restriction, repair, or chromosome degradation. The crystal structure of the nuclease domain of ColE7 in complex with a duplex DNA has been determined at 2.5 Å resolution. The HNH motif is bound at the minor groove primarily to DNA phosphate groups at and beyond the 3′ side of the scissile phosphate, with little interaction with ribose groups and bases. This result provides a structural basis for sugar- and sequence-independent DNA recognition and the inhibition mechanism by inhibitor Im7, which blocks the substrate binding site but not the active site. Structural comparison shows that two families of endonucleases bind and bend DNA in a similar way to that of the HNH ColE7, indicating that endonucleases containing a “ββα-metal” fold of active site possess a universal mode for protein-DNA interactions

Landslide Evolution and Analysis of Failure Mechanism in LanTai Area

臺灣地區由於山坡地面積佔全區70%以上，且常受颱風豪雨及地震之侵襲，大型坡地崩塌時常發生並造成災害。97 年辛樂克颱風之廬山地滑及98 年莫拉克颱風之高屏溪流域，均造成嚴重災害，故大型坡地崩塌災害潛勢及影響範圍分析實為重要研究課題。 宜專一線為宜蘭縣通往太平山森林遊樂區之重要觀光道路，其中蘭台苗圃往白嶺地區部分路段曾受到坡地崩塌之影響而損壞。根據前人文獻及既有資料，該區域之道路及擋土結構，皆經過多次修補，且現地調查時道路可觀察多處裂縫、植生傾斜及崩崖陷落等崩塌潛勢特徵。 本研究透過航照及衛星影像蒐集來判釋崩塌發生，並由現地調查之地表特徵可研判蘭台地區潛在崩塌及可能崩塌機制，同時配合多種監測系統及多時段影像輔助判釋，利用質點影像測速法 (PIV)來分析蘭台地區之地形演化，並藉由其他調查項目以及岩心箱取樣，研判研究區的地層位態以及進行材料性質試驗，利用PIV分析所得點資料的位移量以及位移方向，透過ArcGIS計算以及玫瑰圖統計來劃設研究區於各期影像中的可能滑動範圍以及主要滑動方向來決定分析剖面方向，配合鑽孔資料與孔內攝影成果建立潛勢區域之力學模型，在確認所分析剖面為最具代表性的分析剖面後，結合現地雨量及地下水位監測資料，以模擬方式分析崩塌潛勢區域之破壞機制以及滑動面。 研究結果發現，透過多時段影像可觀察到研究區在各期影像分析中主要崩塌推移位置、範圍以及主要滑動方向，而經過力學分析所得滑動弧位置與地表變遷分析有相對應趨勢，說明利用地表變位量所求得之可能發生破壞位置和力學分析所得滑動面於坡頂與坡趾位置相接近。Due to slope land in Taiwan accounting for more than 70% of the region, the slope land often suffers typhoons and earthquakes induced landslides. For example, Typhoon Sinlaku caused the Lusan landslide in 2008, and landslides in Kaoping River Basin during Typhoon Morakot in 2009. Both caused severe damage, thus the potential and evolutions of large landslide become very important research topics. The Yilan Special Route No.1 is the main road between Yilan City and Taipin Mountain Forest Recreation Area. According to previous study, the road pavement and retaining walls both went through many rounds of repairement, especially in Lantai area. In the field investigation, we could observe that there were many cracks on the road and trees inclined toward the dowm slope direction. In this research, multi-stage aerial photos and the satellite images to determine landslide crown, and the results from field investigation were used to decide the potential scar and identified the failure criteria. The Particle Image Velocimetry (PIV) analysis was conducted for the terrain evolution in Lantai area, and results were referred to geological investigations and sampled the specimens from borehole cores, to determine the statue of ground formation and the laboratory tests were conducted. After transferring the point data from PIV analysis using ArcGIS and rosette statistics, the potential landslide area and the main sliding direction can be decided. Based on these results and borehole inclinometer results and drill reports, the model of potential landslide area could be established. This study combined the model with rainfall monitoring data in the field to back analyze the failure surface in each cases. The results showed that by using multi-stage images we could observe the potential landslide scar’s position, range, main sliding direction and the evolution of landslide in each cases. The scar location matched the failure surface location well. Key: Lantai area, Large-scale landslide, PIV analysis, Evolution of landslide, Stability of slop

Generation of FHL2 homozygous knockout lines from human embryonic stem cells by CRISPR/Cas9-mediated ablation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Mutations in the FHL2 (Four and a half LIM domains protein 2) gene are associated with cardiomyopathy in patients. Here, we generated two homozygous knockout lines using CRISPR/Cas9-mediated ablation in a human embryonic stem cell (hESC) WA09 line. These knockout lines exhibit a normal karyotype without expressing FHL2 protein, while maintaining pluripotency and differentiation properties. These isogenic mutation lines will be provided as a disease model for cardiomyopathy studies and drug screening

MZT Proteins Form Multi-Faceted Structural Modules in the γ-Tubulin Ring Complex

Microtubule organization depends on the g-tubulin ring complex (g-TuRC), a 2.3-MDa nucleation factor comprising an asymmetric assembly of g-tubulin and GCP2-GCP6. However, it is currently unclear how the g-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex. Here, we report cryo-EM structures of MZT1 and MZT2 in the context of the native human g-TuRC. MZT1 forms two subcomplexes with the N-terminal a-helical domains of GCP3 or GCP6 (GCP-NHDs) within the g-TuRC ‘‘lumenal bridge.’’ We determine the X-ray structure of recombinant MZT1/GCP6-NHD and find it is similar to that within the native g-TuRC. We identify two additional MZT/GCP-NHD-like subcomplexes, one of which is located on the outer face of the g-TuRC and comprises MZT2 and GCP2-NHD in complex with a centrosomin motif 1 (CM1)-containing peptide. Our data reveal how MZT1 and MZT2 establish multi-faceted, structurally mimetic ‘‘modules’’ that can expand structural and regulatory interfaces in the g-TuRC.ISSN:2666-3864ISSN:2211-124