Shouhei Kobayashi’s research while affiliated with National Institute of Information and Communications Technology and other places

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

DNA transfection is an important technology in the life sciences, wherein nuclear entry of DNA is necessary to achieve expression of the exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe tools for transfection. However, they have no effects for transfection of non-proliferating cells, the reason for which remains unknown. This study aimed to elucidate the mechanism by which transfected DNA enters the nucleus for gene expression. To monitor the intracellular behavior of transfected DNA, we introduced a plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observed plasmid behavior and RFP expression. RFP expression appeared only after mitosis. Electron microscopy showed that plasmids were wrapped with nuclear envelope (NE)-like membranes or associated with chromosomes at telophase. Depletion of BAF, which is involved in NE reformation, delayed plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus via NE reformation at telophase.

This paper provides a comprehensive review of the emerging research area of mobile molecular communication. In mobile molecular communication, sender and receiver bionanomachines as well as associated nodes in the environment exhibit dynamic behavior in the sense that they are mobile and communicate while they move. This paper presents a model of mobile bionanomachines and uses the model to discuss how groups of such bionanomachines working in unison can provide useful functionalities. This paper illustrates several functionalities by applying mobile molecular communication to the concept of cooperative drug delivery. Unsolved research challenges in this area are outlined and discussed.

Reformation of a functional nucleus at the end of mitosis is crucial for normal cellular activity. Reconstitution approaches using artificial beads in frog egg extracts have clarified the molecules required for nuclear formation in vitro. However, the spatiotemporal regulation of these components, which is required for the formation of a functional nucleus in living embryos, remains unknown. Here we demonstrate that exogenous DNA introduced in the form of DNA-conjugated beads induces the assembly of an artificial nucleus in living mouse cleavage-stage embryos. Live-cell imaging and immunofluorescence studies revealed that core histones and regulator of chromosome condensation 1 (RCC1) assembled on the DNA, suggesting that nucleosomes were formed. Electron microscopy showed that double-membrane structures, partly extended from annulate lamellae, formed around the beads. Nuclear pore complex-like structures indistinguishable from those of native nuclei were also formed, suggesting that this membranous structure resembled the normal nuclear envelope (NE). However, the reconstituted NE had no nuclear import activity, probably because of the absence of Ras-related nuclear protein (Ran). Thus, DNA is necessary for NE reassembly in mouse embryos but is insufficient to form a functional nucleus. This approach provides a new tool to examine factors of interest and their spatiotemporal regulation in nuclear formation.

Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by “Live CLEM” imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in post‐mitotic NPC formation, we used artificial beads conjugated with anti‐GFP antibody, which captures GFP‐fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133‐coated beads or Nup153‐coated beads demonstrated that Nup133 and Nup153, as the sole effector molecules, assembled the NPC‐like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133‐coated beads showed that Nup133 effectively assembled Nup107 and ELYS, while minimal assembly of Nup98 and Nup62 was observed; the Nup153‐coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations. This article is protected by copyright. All rights reserved.

Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time-lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 minutes after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62-knockout murine embryonic fibroblast (MEF) cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation-mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not.. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

This paper proposes a leader-follower-based model of mobile molecular communication networks for target detection applications. The proposed model divides application functionalities of molecular communication networks into two types of mobile bio-nanomachine: leader and follower bio-nanomachines. Leader bio-nanomachines distribute in the environment to detect a target and create an attractant gradient around the target. Follower bio-nanomachines move according to the attractant gradient established by leader bio-nanomachines; they approach the target and perform necessary functionalities such as releasing drug molecules. This paper develops mathematical expressions for the proposed model, describes wet laboratory experiments designed to estimate model parameters, and performs biologically realistic computer simulation experiments to evaluate the performance of the proposed model. The main contributions of this paper are to demonstrate the functional division of molecular communication networks, which will facilitate the design and development of molecular communication networks. Further, insight into the application-level performance of molecular communication networks will be provided based on the proposed model.

Fluorescence microscopy (FM) is a powerful tool for observing specific molecular components in living cells, but its spatial resolution is relatively low. In contrast, electron microscopy (EM) provides high-resolution information about cellular structures, but it cannot provide temporal information in living cells. To achieve molecular selectivity in imaging at high resolution, a method combining EM imaging with live-cell fluorescence imaging, known as live correlative light-EM (CLEM), has been developed. In this method, living cells are first observed by FM, fixed in situ during the live observation and then subjected to EM observation. Various fluorescence techniques and tools can be applied for FM, resulting in the generation of various modified methods that are useful for understanding cellular structure in high resolution. Here, we review the methods of CLEM and live-cell imaging associated with CLEM (live CLEM). Such methods can greatly advance the understanding of the function of cellular structures on a molecular level, and thus are useful for medical fields as well as for basic biology.

Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. This article is protected by copyright. All rights reserved.

In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated 1 O 2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of 1 O 2 was confirmed using 1 O 2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than 1 O 2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.