Takako Koujin

Spectral imaging fluorescence microscopy

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint

Abstract. The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

Molecular communication through gap junction channels: System design, experiments and modeling

Molecular communication is engineered biological communication that allows nanomachines to communicate through chemical signals. Nanomachines are small scale biological devices that either exist in nature or are artificially engineered from biological materials, and that perform simple functions such as sensing, processing, and actuation. As nanomachines are too small and simple to communicate through a traditional communication means (e.g. electromagnetic waves), molecular communication provides a mechanism for nanomachines to communicate by propagating molecules that represent information. In this paper, we propose to explore biological cells for engineering a molecular communication system. Its system characteristics and key networking services are first discussed, and then our current status of experimental and modeling studies is briefly reported.

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to ∼80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.

Molecular Communication through Gap Junction Channels

Molecular communication is engineered biological communication that allows biological devices to communicate through chemical signals. Since biological devices are made of biological materials and are not amenable to traditional communication means (e.g., electromagnetic waves), molecular communication provides a mechanism for biological devices to communicate by transmitting, propagating, and receiving molecules that represent information. In this paper, we explore biological cells and their communication mechanisms for designing and engineering synthetic molecular communication systems. The paper first discusses the characteristics and potential design of communication mechanisms, and then reports our experimental and modeling studies to address physical layer issues of molecular communication.

Microplatform for intercellular communication

A microplatform was designed, fabricated, and tested for demonstrating the propagation of molecular signals through a line of patterned HeLa cells expressing gap junction channels (HeLa Cx43 cells). The microplatform was capable of patterning cells onto a predefined design with lithography and surface chemical treatment. Lucifer Yellow was first used as a fluorescent marker to demonstrate the formation of functional gap junction channels between patterned HeLa Cx43 cells. The cells at one end of the cell line were next chemically stimulated to induce the propagation of intercellular calcium waves along the cell line, which was successfully monitored with Fluo4. The designed microplatform allowed intercellular communication over an arbitrary network topology of cells, which may provide new insight into mechanisms of intercellular communication.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live‐cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior‐posterior polarized nuclei: MAC‐specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC‐specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light‐electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC‐specific NPCs in the division‐failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.

A locally-induced increase in intracellular Ca2+ propagates cell-to-cell in the presence of plasma membrane Ca2+ ATPase inhibitors in non-excitable cells

Intercellular Ca2+ waves are commonly observed in many cell types. In non‐excitable cells, intercellular Ca2+ waves are mediated by gap junctional diffusion of a Ca2+ mobilizing messenger such as IP3. Since Ca2+ is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca2+ to intercellular Ca2+ waves is limited. Here, we report that in the presence of plasma membrane Ca2+ ATPase inhibitors, locally‐released Ca2+ from the flash‐photolysis of caged‐Ca2+ appeared to induce further Ca2+ release and were propagated from one cell to another, indicating that Ca2+ was self‐amplified to mediate intercellular Ca2+ waves. Our findings support the notion that non‐excitable cells can establish a highly excitable medium to communicate local responses with distant cells.

Biological excitable media based on non-excitable cells and calcium signaling

Abstract In this paper, we investigate a design of biological excitable media based on non-excitable cells and intercellular calcium signaling mechanisms. The calcium induced calcium release mechanism in non-excitable cells is exploited to transform the non-excitable cells into excitable media that propagate calcium signals cell-to-cell. The biological excitable media investigated in this paper represent versatile media for controlling biological systems owing to the nature and function of calcium signals as the universal second messenger for the cell. The enhanced calcium excitability of non-excitable cells is experimentally demonstrated and a mathematical model is developed to investigate the condition for non-excitable cells to increase the calcium excitability.

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.