Shouhei Kobayashi

Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

After bacterial invasion, ubiquitin is conjugated to host endosomal proteins and recognized by the autophagic machinery independent of LC3.

Artificial induction of autophagy around polystyrene beads in nonphagocytic cells

Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.

Externally Controllable Molecular Communication

In molecular communication, a group of biological nanomachines communicates through exchanging molecules and collectively performs application dependent tasks. An open research issue in molecular communication is to establish interfaces to interconnect the molecular communication environment (e.g., inside the human body) and its external environment (e.g., outside the human body). Such interfaces allow conventional devices in the external environment to control the location and timing of molecular communication processes in the molecular communication environment and expand the capability of molecular communication. In this paper, we first describe an architecture of externally controllable molecular communication and introduce two types of interfaces for biological nanomachines; bio-nanomachine to bio-nanomachine interfaces (BNIs) for bio-nanomachines to interact with other biological nanomachines in the molecular communication environment, and inmessaging and outmessaging interfaces (IMIs and OMIs) for bio-nanomachines to interact with devices in the external environment. We then describe a proof-of- concept design and wet laboratory implementation of the IMI and OMI, using biological cells. We further demonstrate, through mathematical modeling and numerical experiments, how an architecture of externally controllable molecular communication with BNIs and IMIs/OMIs may apply to pattern formation, a promising nanomedical application of molecular communication.

The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates

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 1O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of 1O2 was confirmed using 1O2 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 1O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

Significance Rapid detection of invasion of exogenous materials and subsequent responses are important for living organisms to survive hazards, such as pathogen infection. Understanding cellular responses against exogenous DNA provides clues not only for controlling pathogen infections that bring exogenous DNA into host cells, but also for designing efficient DNA delivery vectors for transgene expression. Here, by monitoring the invasion of exogenous DNA-coated polystyrene beads into living cells, we show that barrier-to-autointegration factor detects exogenous DNA immediately after its appearance at endosome breakdown and plays a role in DNA avoiding autophagy. These findings provide new insights into the mechanisms by which a cell detects and responds to exogenous double-stranded DNA. Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell’s response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF+ DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

Performance Evaluation of Leader–Follower-Based Mobile Molecular Communication Networks for Target Detection Applications

This paper proposes a leader–follower-based model of mobile molecular communication networks for target detection applications. The proposed model divides the 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. Furthermore, insight into the application-level performance of molecular communication networks will be provided based on the proposed model.

Live correlative light-electron microscopy to observe molecular dynamics in high resolution.

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.

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells

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.

Externally Controllable Molecular Communication Systems for Pattern Formation

A key open research issue in the area of molecular communication is establishing interfaces between a molecular communication environment where molecular communication takes place and its external environment. In this paper, we describe an architecture of externally controllable molecular communication systems and introduce two types of interfaces: one type for bio-nanomachines to interact with each other in a molecular communication environment, and the other type for bio-nanomachines to interact with conventional device in the external environment. The architecture of externally controllable molecular communication systems is then applied to control the spatio-temporal dynamics of molecular concentrations in a molecular communication environment, demonstrating the applicability of the architecture to pattern formation for medical applications such as tissue regeneration.

Early entry and deformation of macropinosomes correlates with high efficiency of decaarginine-polyethylene glycol-lipid-mediated gene delivery.

Decaarginine‐polyethylene glycol‐conjugated 3,5‐bis(dodecyloxy)benzamide/plasmid DNA [Arg10‐polyethylene glycol (PEG)‐lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated.