A recipe for scientific synergy -Series 4- “Advancing biotechnology through multidisciplinary approaches”

The recent advances in Raman microscopy have enabled molecular-level analysis of living cells by detecting molecular vibrations, allowing the examination of chemical compositions and states in living organisms. Raman microscopy offers visualization of the biological environment and has been utilized for differentiating cell types, states, and activities. Raman microscopy can also detect tiny functional groups that can be used to visualize small molecules, which is unattainable through traditional labeling methods. Utilizing the sharp emission peak of Raman tags, simultaneous visualization of multiple targets is possible, which can be used for ultra-fast profiling with rich information content. These advancements allow for the identification of complex biological activities and states and can contribute to deepening our understanding of biological systems and functions.

Neuroscience has always been a multidisciplinary field, with cross-disciplinary innovations being a key aspect of advancing our understanding of the brain. I will describe some approaches we take in our investigations of the mammalian olfactory system (sense of smell in rodents), including psychophysics for quantitative characterization of complex, sensory-guided behaviours, efforts to decipher the meaning of neural activities through large-scale physiological recordings and analyses, as well as associated instrumentation to start a conversation about potential areas of collaboration.

The CCR4-NOT deadenylase complex is pivotal for life through its unique roles in gene regulation

Our life develops from fertilized eggs and is maintained according to a program engraved in the genome. Genomic information is converted into RNA by transcription, and information from the environment affects production and degradation of RNA. Recent studies from various laboratories show that the CCR4-NOT complex is an important factor that regulates RNA metabolism. To elaborate the role of this complex of eight proteins, we generated mice deficient in each of them, and found that this complex plays an important role in determining whether cells live or die. For example, we have shown that the CCR4-NOT complex is essential for insulin production and immune cell differentiation, and that it is important for gene regulation during early development. Our research on CCR4-NOT adds new knowledge to the fundamental principles of expression of information encoded in genomic DNA, and is expected to have a major impact on the medical field.

Statistical genetics is a research field that evaluates causality of human genetic variations on diseases. Recent developments of sequencing technologies have successfully identified comprehensive catalogues of genetic susceptible loci. However, little is known regarding how to develop methodology to integrate large-scale human genome data with diverse biological resources. We have developed such methods and applied to a pioneering example of large-scale genetic association studies on a variety of human complex traits. Our results should empirically show the value of statistical genetics to dissect disease biology, novel drug discovery, and personalized medicine. Further, we would like to introduce our activity on young researcher developments (“Summer school of statistical genetics” in Osaka Univeristy).

Conventional scientific approaches generally focus on the major components that constitute the observed object and exclude outliers from the analysis, which are far from other data in the measured values. Therefore, little knowledge has been accumulated on rare components with outliers. In order to focus on such rare components (especially cells) that have been overlooked until now, we developed trans-scale-scope called AMATERAS, an imaging device that enables simultaneous observation of more than 1 million cells with micrometer-level spatial resolution. In this symposium, I will introduce the outline of the device, examples of observation and analysis, and new focus on life science research.

Enzyme-catalysed reactions happen every second in the complex crowded environment of cellular cytosol. Here, we introduce a model system to mimic the high macromolecular crowding found in the cellular cytosol. Our system takes advantage of the tendency of proteins to phase-separate in membrane-less compartments, the lack of membrane barrier allows substrates and products to freely diffuse in the system. In general, this system can be used to study enzyme behavior in a macromolecular crowded and controlled environment. Further we demonstrate how this system can self-generate in situ a pH gradient. We discovered that this pH gradient controls droplets direction and speed, ultimately inducing migration.  Our system recapitulates putative biological and pre-biotic conditions.

Mitotic duration is tightly constrained, with extended mitotic duration being a characteristic of potentially problematic cells prone to chromosome missegregation and genomic instability. We show that memories of mitotic duration are integrated by a p53-based mitotic stopwatch pathway to exert tight control over proliferation. The stopwatch halts proliferation of the products of a single significantly extended mitosis or of successive modestly extended mitoses. Time in mitosis is monitored via mitotic kinase-regulated assembly of stopwatch complexes that are transmitted to daughter cells. The stopwatch is inactivated in p53-mutant cancers, as well as in a significant proportion of p53-wildtype cancers, consistent with classification of stopwatch complex subunits as tumor suppressors. Stopwatch status additionally influences efficacy of anti-mitotic agents currently used or in development for cancer therapy.

The germ cell lineage is the sole lineage that transmits genetic and epigenetic information across the next generation, thus perpetuating species. A series of germ cell differentiation processes, such as epigenetic reprogramming and meiosis, must be robust, as it has been evolved through the numerous germline cycles. For understanding mechanisms underlying the germline cycle, reconstitution of germ cell development in culture using pluripotent stem cells will provide a useful platform to analyze the differentiation process. More practically, gametes made in culture would be an infinite source for reproduction of animals, perhaps including human. In the joint symposium, I will introduce technologies for the reconstitution of germ cell development using pluripotent stem cells.

Skin aging reduces the barrier function, leading to skin dysfunction. Therefore, reducing the rate of skin aging is desirable to maintain skin homeostasis. Here, we aimed to control skin aging by clarifying the upstream regulators through omics analysis and by constructing a mathematical model of the regulatory network. Analysis of multi-omics data using senescent dermal fibroblasts identified TGFβ1 as a key regulator. Thrombospondin-1 (THBS1) and Fibromodulin (FMOD) were found to be highly correlated with gene expression levels and skin aging. An ordinary differential equation model was developed to show that THBS1 expression is sensitive to changes in TGFβ1 while FMOD expression is associated with a robust regulatory mechanism. These results suggest THBS1 is a promising drug target for skin aging.

The inositol phospholipid PI(4,5)P2 is well known signaling molecules, and regulate a wide variety of membrane proteins. In particular, a kind of the voltage-gated potassium channel, KCNQ2/3, has long been investigated for its regulation by PI(4,5)P2. In highly polarized neurons, KCNQ2/3 asymmetrically localized to axon initial segment (AIS), and the characteristic spatial distribution of KCNQ2/3 is directly related to the neuronal excitability. In this study, to elucidate the role of PI(4,5)P2 in the regulation of channel trafficking, the spatial dynamics of KCNQ3 were quantitatively analyzed in living neurons at multiple- and single-molecule level. As a result, it was revealed that the trafficking efficiency of mutant KCNQ3, which lacks PI(4,5)P2 binding ability, was decreased depending on its channel activity.

Staphylococcus aureus frequently causes infection outbreaks in a hospital, causes life-threatening infections in immunocompromised patients, and becomes multi-drug resistant. However, how bacteria adapt to environments remains unknown. We collected S. aureus isolates during an outbreak in a hospital. We found that a specific S. aureus lineage evolved from a clone expressing the accessory gene regulator (Agr) system—enabling various toxin productions in response to population—to subclones reversibly silencing Agr. We found that S. aureus with flexible Agr regulation showed an increased ability to acquire antibiotic-resistant plasmids, tolerate antibiotics, escape host immunity, and persistently colonize mice. Thus, S. aureus gained flexible regulation of Agr virulence, which led to antibiotic resistance and persistent colonization in the hospital environment.

Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. Herein I will present how the key remote loop leads to the emergence of function and how these loop additions/removals effect enzyme’s properties.

CCR4-NOT is a major cellular complex, which role and functions are broadly conserved from yeast to humans. One of its main functions is mRNA metabolism through the deadenylation of the poly(A) tail, which in turns destabilizes the mRNA and causes its degradation. Given this central role in mRNA homeostasis, we hypothesized that viruses have, over time, evolved different ways to evade this deadenylation-mediated degradation of their mRNAs. In this study, we propose to use evolutionary analyses to look for the presence of hallmarks of genetic conflicts on CCR4-NOT genes and associated genes, which might potentially have arisen from selective pressure by viruses.  We will here highlight the results obtained from looking at 22 genes encoding the CCR4-NOT complex and adjacent proteins. We hope that these results will provide new avenues for studying the intersection between mRNA metabolism and viral infection, by providing insight in the evolutionary history of those mechanisms.

Centrosomes are the major microtubule organizing centers essential for chromosome segregation during cell division. Centrosomes consist of centrioles that duplicate once per cell cycle and recruit pericentriolar material (PCM). During cell division, PCM increases in size and microtubule nucleating capacity to catalyze spindle assembly. By combining biochemical reconstitution and live-cell imaging in Caenorhabditis elegans embryos, we found that a mitotic kinase PLK1 phosphorylates and converts the PCM into a mitotically active microtubule nucleation site. Despite the essentiality for cell proliferation, centrosomes are eliminated during oogenesis in many organisms. This elimination is believed to be important for maintaining the centrosome number in a zygote, however, underlying mechanisms are unclear. We observed this process in the C. elegans germline and found that several centrosome components are eliminated in late pachytene when the MAP kinase is activated. We currently investigate the underlying mechanisms of centriole elimination in the germline.