bims-nucpor 2022-01-16 papers

Nat Cell Biol. 2022 Jan;24(1): 112-122

Super-resolved 3D tracking of cargo transport through nuclear pore complexes.

Rajdeep Chowdhury, Abhishek Sau, Siegfried M Musser.

Nuclear pore complexes (NPCs) embedded within the nuclear envelope mediate rapid, selective and bidirectional traffic between the cytoplasm and the nucleoplasm. Deciphering the mechanism and dynamics of this process is challenged by the need for high spatial and temporal resolution. We report here a multicolour imaging approach that enables direct three-dimensional visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring structure of the NPC scaffold. The success of this approach is enabled by the high positional stability of NPCs within permeabilized cells, as verified by a combined experimental and simulation analysis. Hourglass-shaped translocation conduits for two cargo complexes representing different nuclear transport receptor pathways indicate rapid migration through the permeability barrier on or near the NPC scaffold. Binding sites for cargo complexes extend more than 100 nm from the pore openings, which is consistent with a wide distribution of the phenylalanine-glycine polypeptides that bind nuclear transport receptors.

DOI: https://doi.org/10.1038/s41556-021-00815-6

Protein Cell. 2022 Jan 11.

8 Å structure of the outer rings of the Xenopus laevis nuclear pore complex obtained by cryo-EM and AI.

Linhua Tai, Yun Zhu, He Ren, Xiaojun Huang, Chuanmao Zhang, Fei Sun.

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.

Keywords: AlphaFold2; Xenopus laevis; cryo-EM; cytoplasmic ring; nuclear pore complex; nuclear ring

DOI: https://doi.org/10.1007/s13238-021-00895-y

Sci Rep. 2022 Jan 10. 12(1): 315

Structural characterization of human importin alpha 7 in its cargo-free form at 2.5 Å resolution.

S Tsimbalyuk, C M Donnelly, J K Forwood.

Shuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure-function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

DOI: https://doi.org/10.1038/s41598-021-03729-3

Mol Biomed. 2020 Dec 30. 1(1): 18

GEF-independent Ran activation shifts a fraction of the protein to the cytoplasm and promotes cell proliferation.

Jinhan Zhou, Yuping Tan, Yuqing Zhang, Aiping Tong, Xiaofei Shen, Xiaodong Sun, Da Jia, Qingxiang Sun.

Ran (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other functions in the cytoplasm, as well as in cellular transformation when switched on. Unlike other members of the GTPase superfamily, Ran binds more tightly to GDP than to GTP due to the presence of an auto-inhibitory C-terminal tail. Multiple missense mutations in the C-terminus of Ran occur in cancers, but their biological significance remains unclear. Here, the quantitative GDP/GTP binding preference of four engineered mutations with unstable C-termini was analyzed using a devised mant-GDP dissociation assay. The results showed that the impact of different C-terminal mutations depends on multiple factors. Although these mutants were more GTP-loaded in human cells, they were shown to be more cytoplasmic, and to support nuclear transport with minimally or partially reduced efficiency. Further, several Ran cancer mutants were compromised in autoinhibition, slightly more GTP-bound, more cytoplasmic, and enhanced the proliferation of A549 and HeLa cells in vitro. Thus, our work reveals a new route of Ran activation independent of guanine nucleotide exchange factor (GEF), which may account for the hyper-proliferation induced by Ran cancer mutations.

Keywords: Activation; Cancer mutations; GTP bias; Nuclear transport; Small GTPases

DOI: https://doi.org/10.1186/s43556-020-00011-2

J Assist Reprod Genet. 2022 Jan 13.

The function of Nucleoporin 37 on mouse oocyte maturation and preimplantation embryo development.

Qianying Guo, Qiang Liu, Nan Wang, Jing Wang, Andi Sun, Jie Qiao, Liying Yan.

PURPOSE: Nucleoporin 37 (NUP37) has been reported to activate the YAP-TEAD signaling, which is crucial for early embryo development. However, whether NUP37 is involved in oocyte meiosis and embryo development remains largely unknown. The study aimed to clarify the function of Nup37 in oocyte maturation and early embryo development, and to explore the mechanism.
METHODS: The expression level and subcellular localization of NUP37 were explored. After knocking down of Nup37 by microinjecting interfering RNA (siRNA), the oocyte maturation rate, aberrant PB1 extrusion rate, and blastocyst formation rate were evaluated. In addition, the effect of the downregulation of Nup37 on YAP-TEAD signaling was confirmed by immunofluorescence staining and real-time quantitative PCR.
RESULTS: NUP37 was highly expressed in oocytes and early embryos; it mainly localized to the nuclear periphery at mice GV stage oocytes and early embryos. Nup37 depletion led to aberrant PB1 extrusion at the MII stage oocyte and a decreased blastocyst formation rate. The reduction of NUP37 caused YAP1 mislocalization and decreased the expression of Tead1, Tead2, and Tead4 during mice embryo development, thus affecting the YAP-TEAD activity and embryo developmental competence.
CONCLUSIONS: In summary, NUP37 played an important role in mice oocyte maturation and preimplantation embryo development.

Keywords: Blastocyst formation; Nuclear pore complexes; Nucleoporin 37; Oocyte maturation; YAP-TEAD

DOI: https://doi.org/10.1007/s10815-021-02330-x