Biology subjects

Fujita, J.

Publications and source records attributed to Fujita, J..

4 recordsLinked to original sources

bioRxiv2022

High-resolution structure of a microtubule-like tube composed of FtsZ-monobody complexes

FtsZ, a bacterial tubulin homologue, forms protofilaments and the Z-ring, which acts as a scaffold for accessory proteins during cell division. Although various studies have revealed its molecular mechanisms, the lack of high-resolution solution structures has hindered the understanding of the detailed mechanisms. Here, we developed a monobody (Mb) that binds FtsZs from Escherichia coli and Klebsiella pneumoniae (KpFtsZ) without affecting their GTPase activities. When expressed in E. coli cells, the Mb did not inhibit Z-ring formation but did inhibit cell division. The crystal structures of the KpFtsZ-Mb complexes revealed the epitope, and the cryoEM structure at 2.67 [A] resolution showed a double helical tube consisting of two KpFtsZ protofilaments stabilized by the Mb filling interfilament gaps. Our structural analyses highlight the similarity between the microtubule and the FtsZ tube and the importance of the plasticity of FtsZ protofilaments.

molecular biology↗

bioRxiv2022

Onecut1 partially contributes to liver progenitor cell transition and acquisition of metastatic potential in hepatocellular carcinoma.

Metastasis-initiating cells are considered to originate from stem cell-like cancer cells. In hepatocellular carcinoma, liver progenitor-like cells are reported to be derived from hepatocytes, indicating the possible acquisition of metastatic potential during hepatocyte-to-cholangiocyte transdifferentiation. Consistent with the expression pattern observed during ductal plate formation, we revealed an LPC transition with Onecut1 accumulation both during hepatocyte-to-cholangiocyte transdifferentiation and in a cell model. This event may be associated with transient acquisition of metastatic potential.

cancer biology↗

bioRxiv2021

Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2* and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 [A] resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 [A] resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, {beta}-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 [A] resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.

molecular biology↗

bioRxiv2021

Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination1-3--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

molecular biology↗