JST CREST-PRESTO joint international symposium ― Structural Biological Dynamics: From Molecules to Life with 60 trillion Cells ― November 5-6, 2015 (Thursday & Friday) Ito Hall, Ito International Research Center, The University of Tokyo

Program organizers Plenary Speakers Tadashi Yamamoto Nahum Sonenberg Okinawa Institute of Science and Technology McGill University (OIST) Andrea Musacchio Soichi Wakatsuki Max-Planck Institute of Molecular Physiology SLAC National Accelerator Laboratory Stanford University Ian A. Wilson Scripps Research Institute Keiji Tanaka Tokyo Metropolitan Institute of Medical Science Plenary Speakers Hiroki Ueda Ursula Klingmüller The University of Tokyo/RIKEN QBiC German Cancer Research Center (DKFZ)

Yifan Cheng University of California, San Francisco

Feng Zhang Broad Institute of MIT and Harvard / Massachusetts Institute of Technology

JST CREST-PRESTO joint international symposium Welcome Note

It is our great pleasure to invite you to participate in Tadashi Yamamoto Professor, the Okinawa Institute of the international symposium, “Structural Biological Science and Technology Dynamics: From Molecules to Life with 60 trillion Program Officer at CREST Cells” . elcome“Biodynamics” WRecently, our CREST and PRESTO programs have been tackling the understanding of hierarchical dynamics (from molecules to life with 60 trillion cells) of biological phenomena via interdisciplinary analyses. The symposium focuses on recent Keiji Tanaka developments in systems biology, synthetic biology, Director, the Tokyo Metropolitan structural biology, and other relevant areas. It also Institute of Medical Science aims to increase the exchange of scientific knowledge Program Officer at CREST “Structural between local and international scientists in order to Life Science” provide an integrated approach on understanding the hierarchical biological dynamics, and to discuss various approaches to further the development of those areas. This will be a great opportunity for all of you to acquire the latest information and recent advances through networking and oral/poster Soichi Wakatsuki presentation. Professor, the SLAC National Accelerator Laboratory/Stanford University Program Officer at PRESTO “Structural Life Science”

Hiroki Ueda Professor, the University of Tokyo/RIKEN QBiC Program Officer at PRESTO “Design and Control of Cellular functions” Dear colleagues,

Yoshiko Shirokizawa On April 1, 2015, the government of Japan Executive Director, JST established the National Research and Development Agencies by transforming the former Independent Administrative Agencies for R&D, including JST elcome (Japan Scienceote and Technology Agency). The NNational Research and Development Agencies have greater flexibility in institutional management based on the characteristics of R&D agencies, and at the same time greater responsibility to maximize research achievements. On the occasion of institutional transformation, JST, as the core agency in the furtherance of Japan’ s STI, will further optimize its virtual network-based research institution structure and strive for higher achievements in contributing to the acceleration of Japan’ s growth strategies and realization of a sustainable society.

We are delighted to invite you to participate in the “JST CREST-PRESTO joint international symposium—Structural Biological Dynamics: From Molecules to Life with 60 trillion Cells” to be held on November 5–6, 2016, in Tokyo, Japan. The CREST and PRESTO are some of JST’ s main strategic basic research programs, striving to lead the world in research excellence. The symposium features hot topics within highly relevant areas of systems biology, synthetic biology, and structural biology. This time, we are inviting leading scientists from overseas, and from four CREST-PRESTO programs, who are exploring biological principles to address current and future developments. We expect this symposium to provide a platform for networking and for fostering research collaborations with participants or with researchers worldwide.

JST CREST-PRESTO joint international symposium Program

Thursday, November 5, 2015

12:00 - 13:00 Registration 13:00 - 13:10 Welcome Message Tadashi Yamamoto

Session 1 Biology and gene regulation Chair: Tadashi Yamamoto 13:10 - 13:40 Translational control of cancer and autism …………………………………………… 2 Nahum Sonenberg（McGill University） 13:40 - 14:00 Identifi cation of a sleep regulatory circuit and implications for the function and evolution of REM sleep ……………………………………………………………… 3 Yu Hayashi（University of Tsukuba） 14:00 - 14:25 Oscillatory control of neural stem cells ………………………………………………… 4 Ryoichiro Kageyama（Kyoto University） 14:25 - 14:35 Break

Session 2 Cellular module Chair: Masasuke Yoshida 14:35 - 15:05 The role of kinetochores in chromosome segregation and feedback control of cell division ………………………………………………………… 5 Andrea Musacchio（Max Planck Institute of Molecular Physiology） 15:05 - 15:25 Transport toward the center of the cell: structure & mechanism of cytoplasmic dynein ………………………………………………………………………… 6 Takahide Kon（Osaka University） 15:25 - 15:50 Proton pumping mechanism coupled with electron transfer of bovine cytochrome c oxidase …………………………………………………………… 7 Tomitake Tsukihara（University of Hyogo） 15:50 - 16:10 Molecular mechanism of V1 rotary motor ……………………………………………… 8 Takeshi Murata（Chiba University） 16:10 - 16:20 Break

Session 3 In vivo function and structure Chair: Keiji Tanaka 16:20 - 16:50 Broad Neutralization of Infl uenza Viruses and Implications for a Universal Vaccine and Therapy …………………………………………………… 9 Ian A. Wilson（Scripps Research Institute） 16:50 - 17:15 Structural study of TLR8 sensing single stranded RNA in innate immune system ………………………………………………………………………… 10 Toshiyuki Shimizu（The University of Tokyo） 17:15 - 17:35 Structural insight into multitasking molecular chaperone Trigger Factor ……… 11 Tomohide Saio（Hokkaido University） 17:35 - 18:00 Structural Basis of Bacterial Multidrug Effl ux Pumps and Development of Pump Inhibitors ………………………………………………………………………… 12 Akihito Yamaguchi（Osaka University） 18:00 - 18:10 Break 18:10 - 20:50 Poster Presentation & Reception Friday, November 6, 2015

9:00-10:00 Registration

Session 4 Biological dynamics Chair: Tetsu Akiyama 10:00-10:30 Systems medicine ‒ from molecules to patients …………………………………… 13 Ursula Klingmüller（The German Cancer Research Center ） 10:30-10:50 Generating diverse organ buds from stem cells …………………………………… 14 Takanori Takebe（Yokohama City University） 10:50-11:15 Temporal Coding and Transomic Analysis of Insulin Action ……………………… 15 Shinya Kuroda（The University of Tokyo） 11:15-13:00 On Own for Lunch & Poster Viewing

Session 5 State-of-the-art biological technologies Chair: Junichi Takagi 13:00-13:30 Structures of TRP ion channels by single particle cryo-EM ………………………… 16 Yifan Cheng（University of California, San Francisco） 13:30-13:55 Direct Visualization of Protein Molecules in Dynamic Action by High-speed Atomic Force Microscopy …………………………………………… 17 Toshio Ando（Kanazawa University） 13:55-14:15 Novel arrayed lipid bilayer chamber system for highly sensitive analysis of membrane transporters ………………………………………………………………… 18 Rikiya Watanabe（The University of Tokyo） 14:15-14:40 Probing the biological dynamics using Zero-Mode Waveguides ………………… 19 Sotaro Uemura（The University of Tokyo） 14:40-14:50 Break

Session 6 State-of-the-art biological technologies and biological dynamics Chair: Hiroki Ueda 14:50-15:20 Development and Applications of CRISPR-Cas9 for Genome Editing …………… 20 Feng Zhang（Broad Institute of MIT and Harvard / Massachusetts Institute of Technology） 15:20-15:40 Carrying skeletal elements by novel type of cells enables the self-organizing construction of 4D skeleton of sponges ……………………………………………… 21 Noriko Funayama（Kyoto University） 15:40-16:05 Roles of the tight junction (TJ)-apical complex in epithelial morphogenetic dynamics ………………………………………………………………………………… 22 Sachiko Tsukita（Osaka University） 16:05-16:25 A synthetic approach to understanding protein molecules ……………………… 23 Nobuyasu Koga（Institute for Molecular Science） 16:25-16:30 Closing remarks Yoshiko Shirokizawa（JST）

JST CREST-PRESTO joint international symposium General Information

Venue Ito International Research Center B2F

7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Entrance

Tel：+81-3-5841-0779 (9:00-17:30, Weekdays only)

Fax：+81-3-5841-0932

E-mail：[email protected]

Access Information：http://www.u-tokyo.ac.jp/ext01/iirc/en/access.html

From the Entrance

Poster Exhibition Area

Reception Desk Hall

B2F Please come to the Reception Desk

Hall Layout（Ito Hall B2F）

Notes 1. No power outlets are available nearby audience seats 2. Eating and drinking are prohibited in the Ito Hall 3. No smoking on the premises 4. Wireless LAN is available

Poster Presentation & Reception (18:10-20:50) Event Space (Ito International Research Center)

Please pay 5,000JPY for your participation in advance at the reception desk of the symposium. Structural Biological Dynamics: From Molecules to Life with 60 trillion Cells

Oral Presentation Session 1 Biological principles 13:10-13:40, November 5

Translational control of cancer and autism

Nahum Sonenberg ABSTRACT McGill University, Canada

Prof. Dr. Nahum Sonenberg joined the Translational control plays a critical role in the regulation of Roche Institute of Molecular Biology essential cellular processes including cell growth, proliferation, in Nutley after receiving his Ph.D. at development, and learning and memory. mRNA translation the Weizmann Institute of Science. He is dysregulated in many diseases, such as cancer and moved to McGill University in 1979, neurological disorders, via a combination of aberrant protein and is today a James McGill Professor expression and defects in the signaling pathways that converge in the Department of Biochemistry and on the translational machinery. Under most circumstances, the Rosalind and Morris Goodman Cancer Centre. Over the years, Prof. translational control is exerted at the initiation step where the Sonenberg has collected a number of eukaryotic translation initiation factor 4E (eIF4E) interacts prestigious prizes including the Robert with the mRNA 5’cap structure to facilitate the recruitment L. Noble Prize from the National of ribosomes and promote translation. Importantly, eIF4E Cancer Institute of Canada (2002), the preferentially stimulates a subset of mRNAs encoding Isaak-Walton-Killam Award for Health proliferation, survival, metabolic, and membrane proteins. The Sciences (2005) and the Gairdner activity of eIF4E is regulated at many levels, most profoundly by Foundation International Award two major signalling pathways: PI3K/Akt/mTOR and Ras/MAPK/ (2008); the Lewis S. Rosenstiel Award Mnk. mTOR directly phosphorylates the 4EBPs (eIF4E-binding (2011); the Royal Society of Canada’s proteins), which are inhibitors of eIF4E, to relieve translational McLaughlin Medal (2013): and the suppression; whereas Mnk phosphorylates eIF4E to stimulate Wolf Prize in Medicine (2014). Notably, he discovered many key factors such translation. Aberrations in these pathways result in dysregulated as mRNA 5' cap-binding protein eIF4E activity which contributes towards tumorigenesis and (eIF4E), internal ribosome entry site neurological disorders such as autism. (IRES) mode of translation and so on. His current research focuses on translational control in cancer, oncolytic viruses as anti-cancer drugs, microRNA control of translation, and translational control of plasticity, learning and memory.

2 Session 1 Biological principles 13:40-14:00, November 5 Identifi cation of a sleep regulatory circuit and implications for the function and evolution of REM sleep

Yu Hayashi ABSTRACT University of Tsukuba

Dr. Yu Hayashi is an assistant professor Mammalian sleep has evolved into a complex state composed and principal investigator at the of rapid eye movement (REM) sleep and non-REM (NREM) International Institute for Integrative sleep. NREM sleep produces cortical slow wave activity (SWA) Sleep Medicine (WPI-IIIS), University and contributes to memory consolidation, whereas the roles of of Tsukuba. Dr. Hayashi’s research REM sleep are less clear. The neural circuitry generating REM on sleep first began at RIKEN Brain sleep is also poorly understood. Here, using mouse genetics, Science Institute as a postdoctoral we established a method to manipulate neurons of a specific fellow, after receiving his PhD degree from the University of Tokyo in 2008. embryonic cell lineage. With this approach, we identified Since 2013, he has been appointed the brainstem neurons that either induce or inhibit REM sleep. current position. His current focus is These neurons shared a common developmental origin with on the function and evolution of sleep. neurons promoting wakefulness, all derived from a common He is a Japan Science and Technology pool of proneural hindbrain cells. Genetic manipulation of this Agency (JST) PRESTO researcher. sleep regulatory circuit allowed reduction or elongation of REM sleep, which in turn affected SWA during subsequent NREM sleep, implicating REM sleep in the regulation of NREM sleep quality.

JST CREST-PRESTO joint international symposium 3 Session 1 Biological principles 14:00-14:25, November 5

Oscillatory control of neural stem cells

Ryoichiro Kageyama ABSTRACT Kyoto University

Ryoichiro Kageyama received MD in Multipotent neural stem cells undergo self-renewal while 1982 and PhD in 1986 from Kyoto producing neurons, astrocytes, and oligodendrocytes. These University. After spending 3.5 years as processes are controlled by multiple basic helix-loop-helix a postdoctoral fellow at the National (bHLH) fate determination factors, such as Mash1/Ascl1, Hes1, Cancer Institute in USA, he returned to and Olig2, which regulate specifi cation of neurons, astrocytes, Kyoto University Faculty of Medicine as and oligodendrocytes, respectively. Time-lapse imaging analysis Assistant Professor in 1989 and was showed that these factors are expressed in an oscillatory later appointed Associate Professor in 1991. There, he began looking at manner by neural stem cells, whereas the expression of a basic helix-loop-helix (bHLH) genes, selected factor becomes dominant and sustained during cell 1,2 such as Hes1 and Mash1, and analyzed fate choice . We further showed by optogenetic approach their roles in neural development. that sustained expression of Mash1 promotes neuronal He then moved to Institute for Virus differentiation, whereas oscillatory expression of Mash1 Research, Kyoto University, to assume activates proliferation of neural stem cells, indicating that the a full professorship in 1997. He was expression dynamics is important for the function of Mash12,3. also appointed Deputy Director of These results suggest that the multipotency is a state controlled World Premier International Research by multiple oscillating fate-determination factors, and that Initiative-Institute for Integrated Cell- fate choice is a process of sustained expression of a selected Material Sciences (WPI-iCeMS), factor. We also found that the Notch ligand Deltalike1 (Dll1), a Kyoto University, in 2013. His current research involves studies on the downstream of Mash1 and Hes1, is expressed in an oscillatory dynamics of bHLH gene expression manner by neural stem cells. Dll1 oscillation seems to be during cellular proliferation and important for mutual activation of Notch signaling in neural stem differentiation. His group has cells. We will discuss the signifi cance of oscillatory expression developed time-lapse imaging and of these factors in neural development. light-controlled gene expression systems and is working to characterize 1. Shimojo et al. (2008) Neuron; 2. Imayoshi et al. (2013) Science; the significance of dynamic gene 3. Imayoshi and Kageyama (2014) Neuron expression in neural development.

4 Session 2 Cellular module 14:35-15:05, November 5 The role of kinetochores in chromosome segregation and feedback control of cell division

Andrea Musacchio ABSTRACT Max Planck Institute of Molecular Physiology Germany Andrea Musacchio is a Scientific When a mother cell divides, its daughters expect to receive Member of the Max Planck Society an exact copy of the genome. To meet this requirement, sister and Director of the Department chromatids must bi-orient at the metaphase plate of the spindle. of Mechanistic Cell Biology at the Subsequent dissolution of sister chromatid cohesion (anaphase) Max Planck Institute of Molecular allows the sisters to part in opposite directions. The timing of Physiology in Dortmund, Germany anaphase is regulated by a checkpoint control system named (since 2010). Musacchio is also spindle assembly checkpoint (SAC). Kinetochores, the sites of Honorary Professor at the University of Duisburg-Essen (since 2013). attachment of chromosomes to microtubules, recruit the SAC Musacchio carried out PhD studies with components to monitor the state of microtubule attachment the late Matti Saraste at the European and prevent mitotic exit prior to the bi-orientation of all sister Molecular Biology Laboratory (1990- chromatid pairs. My laboratory investigates the organization of 1995) and postdoctoral work with kinetochores and how it refl ects on microtubule binding and SAC Stephen C. Harrison at the Harvard signaling. Our main inroads into this question are emerging from Medical School (1995-1998). Before biochemical reconstitutions and structural analyses of SAC and moving to his current location in kinetochore components, coupled with functional investigations Germany, he directed a laboratory at in simple model systems, including HeLa cells and S. cerevisiae . the European Institute of Oncology in I will report on the reconstitution of large parts of the kinetochore Milan (1999-2010). Musacchio has and on the development of tools to measure the kinetics of been elected EMBO Member and is the recipient of grants of the European assembly of the SAC effector complex, the mitotic checkpoint Research Council. His current research complex (MCC). I will report how the combination of these tools focuses on structural and functional may provide a direct quantifi cation of the role of kinetochores as characterization of proteins involved catalysts for MCC production during SAC activation. in the control of cell division and of chromosome segregation. Klare K, Weir JR, Basilico F, Zimniak T, Massimiliano L, Ludwigs N, Herzog F & Musacchio A (2015) CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores. J Cell Biol. jcb.201412028. Overlack K, Primorac I, Vleugel M, Krenn V, Maffini S, Hoffmann I, Kops GJ & Musacchio A (2015) A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint. Elife 4:e05269. doi: 10.7554/eLife.05269. Basilico F, Maffi ni S, Weir JR, Prumbaum D, Rojas AM, Zimniak T, De Antoni A, Jeganathan S, Voss B, van Gerwen S, Krenn V, Massimiliano L, Valencia A, Vetter IR, Herzog F, Raunser S, Pasqualato S & Musacchio A (2014) The pseudo GTPase CENP-M drives human kinetochore assembly. Elife 8:e02978. doi: 10.7554/ eLife.02978

JST CREST-PRESTO joint international symposium 5 Session 2 Cellular module 15:05-15:25, November 5 Transport toward the center of the cell: structure & mechanism of cytoplasmic dynein

Takahide Kon ABSTRACT Osaka University

Takahide Kon is currently Professor Eukaryotic cells are equipped with an efficient intracellular of Biochemistry in the Department transport system that is critical for diverse cellular activities, of Biological Sciences, Graduate including cell motility, cell division, and intracellular traffi cking School of Science at Osaka University, and positioning of numerous cargoes such as protein Japan; a PRESTO researcher of Japan complexes, mRNA, organelles and viruses. Underscoring Science and Technology Agency in the the importance of the transport system, it is now clear that field of “structural life science”. He serious human and animal diseases are associated with joined Department of Life Sciences, Graduate School of Arts and Sciences, dysfunction of the transport machinery. The goal of our University of Tokyo, as an Assistant research is to understand the molecular mechanism underlying Professor, after receiving his PhD the intracellular transport system at near-atomic level. To degree in cell biology from University elucidate how the dynein motor, that is the heart of the transport of Tokyo in 2000. He subsequently machinery, generates force and movement, we have obtained served as an Associated Professor, crystal structures of dynein trapped in an intermediate state Institute for Protein Research at just after the force generation (1-2). To further elucidate the Osaka University (2010-2013) and as structural basis of the transport mechanism, we directly a Professor of Biochemistry at Hosei observed the structure of dynein molecules moving along a University (2013-2015), before he microtubule track in the presence of ATP at near-physiological was appointed as a Faculty Member at concentration by means of cryo-electron microscopy (3). These Osaka University in 2015. His research career has focused on cell motility findings provide a structural framework for understanding the at molecular level. His current focus mechanism of intracellular transport toward the center of the is on the structure and mechanism cells. of intracellular transport systems in eukaryotic cells, especially on a huge 1. Kon et al. (2012). The 2.8 Å crystal structure of the dynein motor domain. Nature 484: 345-350. 2. Kon et al. (2011). X-ray structure of a functional full-length dynein motor domain. Nature Struct. Mol. motor protein complex, dynein, that is Biol. 18: 638-642. 3. Imai et al. (2015). Direct observation shows superposition and large scale fl exibility within cytoplasmic the heart of the transport system. dynein motors moving along microtubules. Nature Commun. in press.

6 Session 2 Cellular module 15:25-15:50, November 5 Proton pumping mechanism coupled with electron transfer of bovine cytochrome c oxidase

Tomitake Tsukihara ABSTRACT University of Hyogo / Osaka University

Prof. Tomitake Tsukihara joined Cytochrome c oxidase (CcO) that is the terminal enzyme Department of Life Science, University of mitochondrial respiratory chain accepts electrons from of Hyogo in 2008, after he retired cytochrome c (Cyt.c) to reduce a dioxygen molecule and pumps Osaka University. He started research protons from the matrix space to the inter-membrane space of protein crystallography when he was across the inner membrane. Bovine CcO pumps 4 proton student. He has been collaborating with equivalents per catalytic cycle. We have proposed H-path Prof. Shinya Yoshikawa, University theory of proton pumping coupling with electron transfer and of Hyogo in structural and functional research of bovine cytochrome dioxygen reduction (1). Protons are pumped from the negative oxidase since middle of 1970s. They side to the positive side through H-pathway, which includes a determined crystal structure of bovine tandem hydrogen-bond network and water channel. The H-path cytochrome c oxidase in 1995. It theory was confirmed by site directed mutagenesis studies was the first structure of membrane and theoretical calculation, and reinforced by crystallographic protein complex of animals. He has studies of several reaction intermediate states. proposed H-path theory for proton The recent X-ray structures of oxidized/reduced CcO from at pumping mechanism of cytochrome 1.5/1.6 Å resolution reveal that a large water cluster, with 21 c oxidase that pumps four protons water molecules and a Mg2+ site, is linked to a short hydrogen coupling with dioxygen reduction. The bond network extending to the H-pathway. The cluster that H-path theory has been reinforced contains enough proton-acceptor groups to retain 4 proton with high-resolution X-ray structures of cytochrome c oxidase in several equivalents must be a crucial element of the proton-pumping reaction intermediates as well as site system of CcO. directed mutagenesis studies. He had served as President of Japanese Crystallographic Society of Japan (2006-2007) and President of Protein Society of Japan (2008-2009).

JST CREST-PRESTO joint international symposium 7 Session 2 Cellular module 15:50-16:10, November 5

Molecular mechanism of V1 rotary motor

Takeshi Murata ABSTRACT Chiba University

Dr. Takeshi Murata is a professor of V-ATPases function as proton pumps in many cellular processes Graduate School of Science at Chiba such as bone resorption and cancer metastasis, and are University. His research on V-ATPase thus attractive drug targets for osteoporosis and cancer. The first began at Tokyo University of hydrophilic V1 part is known as a rotary motor in which a central Science as a graduate research axis DF complex rotates inside a hexagonally arranged catalytic student in 1994. After receiving his A3B3 complex using ATP hydrolysis energy, although the PhD degree, he joined John Walker’s molecular mechanism was not well defi ned because of a lack of laboratory at MRC as a postdoctoral fellow in 2000. He focused on the high-resolution structural information. We have established the structural studies of the V-ATPase in vitro expression and purification systems of Enterococcus

and solved the crystal structure of the hirae V1-ATPase from the A3B3 and DF complexes (1), and

membrane rotor ring at 2005. He came obtained crystal structures for the V1 complex, corresponding to back to Japan at RIKEN as a researcher the catalytic dwell state (2). Recently, we also obtained crystal in 2005, and moved to Kyoto University structures corresponding to the ATP-binding dwell and ADP- as an assistant professor in 2007. release dwell states discovered by soaking ADP into nucleotide- During these periods, he developed free V1 crystals. In my talk, I would like to discuss about the experimental protocols to obtain molecular mechanism of the V-ATPase based on these structural monoclonal antibodies that recognize and biochemical, biophysical fi ndings. conformational epitopes of membrane proteins for co-crystallization, and (1) Saijo, S. et al. (2011) Proc. Natl Acad. Sci. USA 108, 19955-19960. solved crystal structures of membrane (2) Arai, S. et al. (2013) Nature 493, 703-707. protein-antibody complexes. He became a faculty of Chiba University in 2009. His current focus is still on structural studies of membrane proteins including V-ATPases, GPCRs, and transporters.

8 Session 3 In vivo function and structure 16:50-17:20, November 5 Broad Neutralization of Infl uenza Viruses and Implications for a Universal Vaccine and Therapy

Ian A. Wilson ABSTRACT Scripps Research Institute USA

Prof. Ian Andrew Wilson is Chair Until relatively recently, most antibodies to infl uenza virus were of the Department of Integrative thought to be strain-specific and protect only against highly Structural and Computational Biology related strains within the same subtype. Since 2008, a number at The Scripps Research Institute. of human antibodies have been isolated that are much broader Prof. Wilson’s research on influenza and neutralize across different subtypes and types of infl uenza virus hemagglutinin first began at viruses through binding to functionally conserved sites. The Harvard University as a postdoctoral major surface antigen, the hemagglutinin (HA), of influenza fellow, after receiving his PhD degree from Oxford University. He joined the virus is the main target of these neutralizing antibodies. We Scripps Research Institute in La Jolla in have determined crystal structures of many broadly neutralizing 1982. Since 2000, he has directed the antibodies in complex with variety of different HAs and show Joint Center for Structural Genomics they bind to highly conserved sites on the HA fusion domain (JCSG) that has pioneered innovative (stem) in both influenza A (1-3) as well as influenza B (4) new methods for high throughput viruses, as well as to the receptor binding site (e.g. 5-7). The structural studies, including x-ray and characterization of these broadly neutralizing antibodies along NMR. His research career has focused with their mode of binding and neutralization provide exciting on the recognition of microbial new opportunities for structure-assisted vaccine design and pathogens by the immune system. His for design of therapeutics that afford greater protection against current focus is on recognition of viral infl uenza viruses. pathogens, including HIV, influenza virus and HCV, by broadly neutralizing 1. Ekiert et al. (2009) Antibody recognition of a highly conserved infl uenza virus epitope. Science 324:246- antibodies to further vaccine design. 251. He is a Fellow of Royal Society of 2. Ekiert et al. (2011) A highly conserved neutralizing epitope on group 2 infl uenza A viruses. Science 333:843-850. London Fellow, a Fellow of the Royal 3. Friesen et al. (2014) A common solution to group 2 infl uenza virus neutralization. Proc. Natl. Acad. Sci. U.S.A. 111:445-450. Society of Edinburgh, and on the Board 4. Dreyfus et al. (2012) Highly conserved protective epitopes on Infl uenza B viruses. Science 337:1343- of Directors and Scientific Advisory 1348. 5. Ekiert et al. (2012) Cross-neutralization of influenza A viruses mediated by a single antibody loop. Board of Keystone Symposia. Nature 489:526-532 6. Xu et al. (2013) A recurring motif for antibody recognition of the receptor-binding site of infl uenza hemagglutinin. Nature Struct. Mol. Biol. 20:363-370 7. Lee et al. (2014) Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of infl uenza virus. Nature Commun. 5:3614.

JST CREST-PRESTO joint international symposium 9 Session 3 In vivo function and structure 17:20-17:45, November 5 Structural study of TLR8 sensing single stranded RNA in innate immune system

Toshiyuki Shimizu ABSTRACT The University of Tokyo

Prof. Toshiyuki Shimizu is a member Toll-like receptors (TLRs) constitute a family of innate immune of Graduate School of Pharmaceutical receptors that recognize pathogen-associated molecular Sciences, The University of Tokyo. He patterns (PAMPs)”. TLR7 and TLR8 recognize ssRNA and graduated from The University of Tokyo initiate innate immune responses. Moreover, several small at 1989. He got into Kirin Brewery molecule compounds have been identified as TLR7 and TLR8 Company and immediately joined activators. We determined the crystal structures of unliganded Protein Engineering Research Institute and ligand-induced activated human TLR8 dimers [1]. Ligand (PERI) in 1989. After receiving his PhD degree from The University of Tokyo binding induces reorganization of the TLR8 dimer, which in 1995, he worked at Nara Institute of enables downstream signaling processes. To elucidate how Science and Technology as a research TLR8 recognizes its natural ligand (ssRNA), as well as how associate. His research was focused the receptor can be activated by molecules as structurally on DNA recognition mechanism of and chemically different as ssRNA and the chemical ligands, transcription factor by determining we performed crystallographic studies of TLR8 in complex the protein-DNA complex by X-ray with ssRNAs [2]. The resultant structures revealed that TLR8 crystallography method. From 2001, recognizes, at distinct sites, uridine and small oligonucleotides he jointed Yokohama City University derived from degradation of ssRNA. Uridine bound the site on in 2001 as an associate professor. the dimerization interface where small chemical ligands are He focused on the structure function recognized, whereas short oligonucleotides bound a newly relationship of signaling molecule. He became a professor at The University identified site on the concave surface of the TLR8 horseshoe of Tokyo in 2010. His current research structure. Site-directed mutagenesis revealed that both binding is focused on structural biology of sites were essential for activation of TLR8 by ssRNA. These proteins in innate immunity and results demonstrate that TLR8 is a sensor for uridine and a nuclear proteins. short oligonucleotide derived from RNA.

[1] Tanji et al., Science (2013) [2] Tanji et al., Nature Struc Mol Biol (2015)

10 Session 3 In vivo function and structure 17:45-18:05, November 5 Structural insight into multitasking molecular chaperone Trigger Factor

Tomohide Saio ABSTRACT Hokkaido University

Dr. Tomohide Saio is Assistant Molecular chaperones prevent aggregation and misfolding of Professor of Department of Chemistry, proteins in the cellular environment and are thus central to Faculty of Science at Hokkaido maintaining protein homeostasis. However, scarcity of structural University, Japan, and JST PRESTO data has impeded an understanding of the recognition and anti- researcher. He focused on protein aggregation mechanisms of molecular chaperones. One of the NMR and paramagnetic lanthanide major chaperones in bacteria is Trigger Factor (TF) that forms probe during his Ph.D. study at a dimer of ~100 kDa in solution but binds to the ribosome or Hokkaido University. After receiving his Ph.D. in Life Science at Hokkaido a substrate protein as a monomer. Despite the large amount University in 2011, Dr. Saio joined of structural and biological knowledge for TF and TF-ribosome Rutgers University in Piscataway, NJ interaction, the molecular mechanism for the interaction as a postdoctoral fellow, to start his between TF and substrate protein remains unknown. research on molecular chaperones. He Here we report the NMR solution structure of three Trigger joined Hokkaido University in 2013. Factor (TF) chaperone molecules in complex with alkaline His main research interests are on phosphatase (PhoA) captured in the unfolded state. Our data molecular mechanisms of chaperones show that TF uses multiple sites, which extend over a distance in proteostasis controlling folding, of ~90 Å, to bind to several regions of the PhoA substrate translocation, and degradation of protein primarily through hydrophobic contacts. TF interacts proteins. with PhoA in a dynamic fashion, forming a complex with a lifetime of ~20 ms. Dynamic and transient interaction with the substrate is important for anti-aggregation activity and thus foldase activity. On the other hand, when TF is co-localized with a substrate, TF acts as strong holdase to prevent premature (mis) folding of the substrate on the ribosome.

Reference Saio, T.; Guan, X.; Rossi, P.; Economou, A.; Kalodimos, C.G. Science, 2014, 344, 1250494.

JST CREST-PRESTO joint international symposium 11 Session 3 In vivo function and structure 18:05-18:30, November 5 Structural Basis of Bacterial Multidrug Effl ux Pumps and Development of Pump Inhibitors

Akihito Yamaguchi ABSTRACT Osaka University

Prof. Akihito Yamaguchi is a former Multidrug efflux pumps are the major cause for multidrug director of the Institute of Scientific resistant Gram–negative pathogens, which are currently serious and Industrial Research (ISIR), Osaka problem in modern chemotherapy since there is no clinically- University. After the retirement of full useful drugs. Among them, RND-type efflux pumps including professorship of Osaka University AcrB and MexB have a most broad substrate spectrum and in 2013, prof. Yamaguchi is now act as a tripartite complex with outer membrane channel and specially-appointed professor of Osaka an adaptor protein. Multidrug recognition of effl ux pumps is a University and presides the Laboratory of Membrane Structural Biology in challenge for the concept of enzyme specifi city. We determined ISIR. Prof. Yamaguchi’s research the world’s fi rst crystal structure of RND-type effl ux pump and on bacterial drug efflux pumps has revealed the functional rotation mechanism of drug export. been started at Chiba University as an Multidrug recognition is based on the membrane vacuum assistant professor, after receiving his cleaner mechanism and the multisite drug binding in two PhD degree from University of Tokyo. voluminous drug binding pockets and multi entrances. A wide He was appointed to professor of range of drugs are recognized and exported from the multiple Osaka University in 1996 in ISIR. He entrances via two multisite drug binding pockets by a peristaltic succeeded to determine the world’s mechanism. Recently, we determined the world’s first crystal first crystal structure of a bacterial structure of inhibitor-binding effl ux pumps and thereby enables drug effl ux pump in 2002 and then he the structure-based drug design of pump inhibitors. continues to lead the world’s study of the structure-based molecular Murakami and Yamaguchi et al (2002) Crystal structure of bacterial multidrug effl ux transporter AcrB. mechanism of multidrug efflux. His Nature 419:587-593 current focus is to develop inhibitors of bacterial effl ux pumps to overcome multidrug resistant pathogens. He also performed pioneering work on the world’s first finding of specific secretion transporter of a signal transduction molecule, sphingoshine- 1-phosphate (S1P). He revealed that the transporter named SPNS2 plays an essential role leukocyte egress.

12 Session 4 Biological dynamics 10:00-10:30, November 6

Systems medicine – from molecules to patients

Ursula Klingmüller ABSTRACT The German Cancer Research Center, Germany

Prof. Dr. Ursula Klingmüller heads The hormone erythropoietin (Epo) is the key regulator of the division of “Systems Biology of erythropoiesis. Recombinant Epo is widely used for the Signal Transduction” at the German treatment of anemia. However, in the context of cancer related Cancer Research Center (DKFZ) in anemia the safety of the treatment is controversially discussed. Heidelberg. She started her career Clinical trials had to be terminated due to adverse effects on as a Senior Scientist at Harvard tumor progression and it was observed that the Epo receptor Medical School and the Whitehead (EpoR) can be present on lung cancer cells. As multiple effects Institute for Biomedical Research in Boston, USA, and afterwards headed might contribute to the adverse effects, we utilized a systems an independent junior group at the biology approach that combines quantitative data generation Max-Planck-Institute for Immunology with mathematical modeling to gain insights into underlying in Freiburg, Germany. She employs molecular mechanisms. We show that depending on the data-based mathematical modeling to binding properties of Erythropoiesis Stimulating Agents (ESAs) elucidate dynamic properties of EpoR these agents differentially activate signal transduction in lung signaling and the consequence of cancer cells. By our dynamic pathway model quantitatively cellular decisions. Her group focuses addressing the ligand receptor interaction we can predict on standardization of cellular systems ESA concentrations that could be used to preferentially and quantitative data generation. expand erythroid progenitor cells but not affect tumor cells. Prof. Dr. Ursula Klingmüller received We link our precisely calibrated dynamic pathway model FEBS Anniversary Prize and serves as an elected member of the to a pharmacokinetic and a pharmacodynamic model and German Research Foundation (DFG) quantitatively describe the time course of ESAs in patients. "Hinterzartner Kreis". Therefore, advancing a systems biology approach towards an application in systems medicine opens new possibilities to stratify patients and optimize treatment regimes for individual patients.

JST CREST-PRESTO joint international symposium 13 Session 4 Biological dynamics 10:30-10:50, November 6

Generating diverse organ buds from stem cells

Takanori Takebe ABSTRACT Yokohama City University

Takanori Takebe received his M.D. in In vitro organogenesis is now becoming a realistic goal of stem 2011 from Yokohama City University cell biology; however, one practical challenge is to develop School of Medicine, Japan after a few a four-dimensional (4-D) stem cell culture system whereby years training as a research associate multiple progenitors communicate in a spatiotemporal manner, at the Scripps Research Institute and as observed in organogenesis. During early hepatogenesis, the as an external medical student at multicellular communication that occurs among mesenchymal Columbia University Medical Center stem cells, undifferentiated vascular endothelial cells and in the US. After receiving his M.D., he worked as a research associate anterior visceral endodermal cells are required to initiate the at the same university with a joint budding of the rudimentary liver in the foregut. To recapitulate appointment as a researcher at Mirai early organogenesis, we recently showed that specifi ed hepatic Design Lab in Japan. He became a cells self-organized into 3-D iPSC-derived liver buds when project leader in 2012 at Yokohama co-cultivated on solidified Matrigel with multiple stromal cell City University Advanced Medical populations. By transplanting in vitro grown organ bud, we have Research and later in 2013 appointed demonstrated the vascularized and functional liver tissues in an associate professor position at an immunodeficient animal with therapeutic potential (Nature, Yokohama City University. He now has 2013). Furthermore, we also demonstrated the applicability of a joint position as a visiting associate this approach to other systems by delineating the mechanisms professor at Stanford University guiding organ bud formation. Specifically, mesenchymal in the US. His current focus is on recapitulating developmental 4-D progenitors initiated organ bud formation within these interactions between cells, tissues and heterotypic cell mixtures, which was dependent upon substrate organs; thereby, generating human matrix stiffness. Defining optimal mechanical properties of organ from stem cells towards therapy. the substrate promoted formation of 3D, transplantable organ buds from tissues including kidney, pancreas and cartilage (J Clin Invest, 2014 & Cell Stem Cell, 2015). In this talk, I will summarize the state-of-art of these organ bud based approaches, and discuss their future potential applications.

14 Session 4 Biological dynamics 10:50-11:15, November 6 Temporal Coding and Transomic Analysis of Insulin Action

Shinya Kuroda ABSTRACT University of Tokyo

Shinya Kuroda is a professor at Insulin selectively regulates many metabolic functions, such as Department of Biological Sciences, glycogenesis, gluconeogenesis and protein synthesis, through Graduate School of Science, University the AKT pathway depending on its temporal patterns, such as of Tokyo. Prof. Kuroda was originally additional secretion, which is a pulse-like secretion in response trained as a biochemist during to meals, and basal secretion, which is the low and constant his graduate school, when he has secretion during fasting. We developed a simple computational investigated the role of the Rho- model of the insulin-dependent AKT pathway, and found that small GTPase family. He moved into computational fi eld in Mitsuo Kawato’s the pathway uses “temporal patterns” for selective downstream lab at ATR where he learned basics of regulation via differences in their network structures and kinetics computation and started simulation (Kubota et al., Mol. Cell, 2012, Noguchi, Mol. Sys. Biol, 2013). study of cellular signaling. He became Our results demonstrate that the AKT pathway can multiplex an independent associate professor at distinct patterns of insulin and the downstream molecules Dept. Information Science, University selectively decode the temporal patterns of insulin. of Tokyo, where he started systems Insulin action involves dynamic molecular interactions biology work of cellular signaling between multiple layers including protein phosphorylation, including ERK-dependent cell fate and metabolites. We performed metabolomic and phospho- decision. During this work, he came proteomic analysis in insulin-stimulated Fao hepatoma cells, up with a concept of temporal coding and automatically reconstructed global molecular network of and began to analyze temporal coding of insulin action after he became insulin action by use of trans “omics” data together with several a professor at biological sciences, databases. We found a landscape of global network of insulin- University of Tokyo. He recently started dependent metabolic control that involves 13 protein kinases, 26 trans-omic analysis of insulin action. phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites.

References Kubota, H., Noguchi, R., Toyoshima, Y., Ozaki, Y., Uda, S., Watanabe, K., Ogawa, W., and Kuroda, S. (2012). Temporal coding of insulin action through multiplexing of the AKT pathway. Mol Cell 46, 820-832. Noguchi, R., Kubota, H., Yugi, K., Toyoshima, Y., Komori, Y., Soga, T., and Kuroda, S. (2013). The selective control of glycolysis, gluconeogenesis and glycogenesis by temporal insulin patterns. Mol Syst Biol 9, 664. Yugi, K., Kubota, H., Toyoshima, Y., Noguchi, R., Kawata, K., Komori, Y., . . . Kuroda, S. (2014) Reconstruction of insulin signal fl ow from phosphoproteome and metabolome data. Cell reports 8, 1171- 1183.

JST CREST-PRESTO joint international symposium 15 Session 5 State-of-the-art biological technologies 13:00-13:30, November 6 Structures of TRP ion channels by single particle cryo-EM

Yifan Cheng ABSTRACT University of California, San Francisco, USA

Dr. Yifan Cheng is currently an As a versatile tool in structural biology, single particle electron Associate Professor at Department cryo-microscopy (cryo-EM) has been used to determine of Biochemistry and Biophysics, the three-dimensional (3D) structures of proteins and University of California San Francisco macromolecular complexes without the need for crystals. (UCSF). He received his Ph.D. degree Recent technological breakthroughs in electron detector in 1991 from Institute of Physics, technologies have enabled developments of novel techniques Chinese Academy of Sciences in both data acquisition and image processing. Together, these (CAS). From 1991 to 1996, he was a postdoctoral fellow at Department novel technologies revolutionized single particle cryo-EM and of Physics, University of Oslo (NTNF enabled near atomic resolution structure determinations of a Fellow), and Max-Planck-Institut broad range of proteins complexes without the need of crystals. für Metallforschung (Alexander von Thus, it is now feasible to use single particle cryo-EM to Humboldt Fellow). In 1996, he changed determine structures of protein complexes that are challenging his research field from physics to for the more traditional X-ray crystallographic structure structural biology, and received further determination approach. This is particularly true for integral training in electron cryo-microscopy membrane proteins. The Transient Receptor Potential (TRP) ion (cryo-EM) from Professor Kenneth channel is a large and functionally diverse superfamily, second Taylor at Florida State University and only to potassium channels. Up to date, there is no crystal Professor Yoshinori Fujiyoshi at Kyoto structure of any member of TRP channel superfamily. Facilitated University. In 1999, he joined the laboratory of Professor Thomas Walz by novel single particle cryo-EM technology, we determined at Harvard Medical School. In 2006, he atomic structures of TRPV1 ion channels in three different became a faculty at UCSF and stayed conformations, as well as structure of TRPA1. These structures there ever since. His laboratory studies revealed the diverse structural architectures exist in TRP channel three-dimensional structures of family, providing a structural blueprint for understanding unique macromolecule by single particle cryo- aspects of TRP channel function. EM

16 Session 5 State-of-the-art biological technologies 13:30-13:55, November 6 Direct Visualization of Protein Molecules in Dynamic Action by High-speed Atomic Force Microscopy

Toshio Ando ABSTRACT Kanazawa University

Prof. Toshio ANDO is currently Directly observing protein molecules in dynamic action at studying at the Department of Physics high spatiotemporal resolution has long been a holy grail of Kanazawa University. After receiving for biological science. This is because proteins are dynamic his Dc. Sci. degree in physics from nature and because we long have had to infer how proteins Waseda University, he worked at the function from the static snapshots of their structures and the Cardiovascular Research Institute of dynamic behavior of optical makers attached to the molecules. UC San Francisco as a postdoctoral To materialize this long quested observation, I have been fellow and then an assistant research biophysicist from 1980 to 1986. developing high-speed atomic force microscopy (HS-AFM) since Then, he returned to Japan to 1993. Tremendous strides have recently been accomplished in establish a biophysics laboratory at its high-speed and low-invasive performances. Consequently, the Department of Physics, Kanazawa various dynamic molecular actions, including bipedal walking University. Prof. Ando’s has been of myosin V [1] and rotary propagation of structural changes studying the molecular mechanism of in F1-ATPase [2], have successfully been captured on video proteins, especially of myosin motors, (see Review [3]). The visualized dynamic images not only while developing new tools. In 1993, provided irrefutable evidence for speculated actions of the he embarked on the development of protein molecules but also brought new discoveries inaccessible high-speed atomic force microscopy with other approaches, thus giving great insights into how (HS-AFM) and fi nally materialized this the molecules function. For example, the molecular movies of new technique in 2008. Currently, his research focuses on the nano- myosin V directly demonstrated the swinging lever-arm motion visualization of protein molecules as powerstroke and more importantly brought a new discovery in dynamic action as well as on the on the chemomechanical coupling in this motor. HS-AFM is development of the second generation now transforming “classical” structural biology into dynamic of HS-AFM techniques. For his creation structural bioscience. of HS-AFM and new structural biology, he has been awarded a number of 1. Kodera et al. (2010) Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468:72-76. prizes including the Yamazaki-Tei’ichi 2. Uchihashi et al. (2011) High-speed atomic force microscopy reveals rotary catalysis of rotorless F1- Prize and Shimadzu Prize. ATPase, Science 333:755-758. 3. Ando et al. (2014) "Filming biomolecular processes by high-speed atomic force microscopy", Chem. Rev. 114:3120-3188.

JST CREST-PRESTO joint international symposium 17 Session 5 State-of-the-art biological technologies 13:55-14:15, November 6 Novel arrayed lipid bilayer chamber system for highly sensitive analysis of membrane transporters

Rikiya Watanabe ABSTRACT The University of Tokyo

Dr. Rikiya Watanabe is an Assistant Membrane transporters are responsible for the transport of Professor of Applied Chemistry at substrate molecules across biomembranes, and perform The University of Tokyo, and PRESTO physiological roles such as nutrient uptake, signal transduction, researcher at Japan Science and and energy synthesis. Transporters have frequently been Technology Agency. He received a B.S. targeted in pharmaceutical research owing to their physiological from Waseda University in 2004, an importance. Although extensive studies have attempted to M.S. from The University of Tokyo in elucidate the mechanism of transport, quantitatively and 2006, and a Ph.D. in single molecule biophysics from Osaka University reproducibly measuring the activity of transporters in a high in 2009. Thereafter, he worked as a throughput format has remained diffi cult due to the complexity postdoctoral fellow at ISIR, Osaka of processes involved in membrane formation. Here, we address University, before joining as an this issue by developing a novel microsystem that forms Assistant Professor. His research arrayed lipid bilayer chambers (ALBiC) in sub-million femtolitre interests are focused on elucidating reaction chamber format, each sealed with a stable lipid bilayer the mechanisms conserved among membrane with an efficiency of over 90 % (1-2). Due to the bio-molecular machines, mainly infinitesimal volume of these chambers, ALBiC can enhance through the development of novel the detection sensitivity by a factor of 106, demonstrating the single-molecule techniques, including single-molecule analysis of passive and active membrane microsystems fabricated at micron- transport in a high throughput manner. Moreover, we have or nano-scale. His current focus is on membrane transporters to extend recently demonstrated some physiological membrane aspects the versatility of single-molecule on ALBiC, such as asymmetric transbilayer phospholipid techniques for further analytical and distribution (3), and modulation of membrane potential across pharmacological applications, such lipid-bilayers. Thus, our new platform, ALBiC, holds promise for as drug screening. He has received understanding the mechanism of transporter proteins as well as The Young Scientists’ Prize from the for further analytical and pharmacological applications. Minister of Education, Culture, Sports, Science and Technology of Japan in 1. Watanabe, R. et al. (2014) Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity. Nat. Commun. 5: 4519. 2015. 2. Soga, R. et al. (2015) Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters. Sci. Rep. 5: 11025. 3. Watanabe, R. et al. (2014) High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition. Sci. Rep. 4: 7076.

18 Session 5 State-of-the-art biological technologies 14:15-14:40, November 6 Probing the biological dynamics using Zero- Mode Waveguides

Sotaro Uemura ABSTRACT The University of Tokyo

Prof. Sotaro Uemura is one of the Traditional total internal reflection fluorescence (TIRF) based youngest professors of the Department microscopy has revealed novel molecular mechanism in protein of Biophysics and Biochemistry, translation (1) but limits the concentration of fluorescently- Faculty of Science, University of Tokyo. labeled unbound components due to high background He specializes in single-molecule and fluorescence. Zero-Mode Waveguides (ZMWs) provides an single-cell biophysics. After attaining elegant solution to these problems. ZMWs are one such nano- his doctorate in 2004 at Waseda structure that can confine laser illumination to an extremely University, he joined the Steve Chu’s laboratory at the Stanford University small region above a bound fluorescent molecule. We had as 1st postdoctoral fellow. Since 2006 used the ZMW technology for direct tracking of translation he served as an assistant professor at under physiological conditions (2). We also could monitor the the University of Tokyo for 3 years, but processes of the initiation complex formation (3). This approach he decided back to Stanford University, provides a powerful tool that is a good match for measuring the joining the Joseph Puglisi’s laboratory dynamics of the broad biological processes. as 2nd postdoctoral fellow in 2009. In 2011 he served as a team leader 1. Uemura S et al. (2007) Nature 446, 454-457. 2. Uemura S et al. (2010) Nature 464, 1012-1017. at RIKEN before taking his current 3. Tsai A et al. (2012) Nature 487, 390-393. position in 2014. He has successfully developed visualized the world’s first protein translation mechanism through novel single-molecule technologies. His current focus is on visualization for wide varieties of biological processes, for example, RNA processing, membrane protein and genome editing.

JST CREST-PRESTO joint international symposium 19 Session 6 State-of-the-art biological technologies and biological dynamics 14:50-15:20, November 6 Development and Applications of CRISPR- Cas9 for Genome Editing

Feng Zhang ABSTRACT Broad Institute of MIT and Harvard / Massachusetts Institute of Technology, USA Feng Zhang is the Keck Career The Cas9 endonuclease from the microbial adaptive immune Development Professor of Biomedical system CRISPR can be easily programmed to bind or cleave Engineering at MIT, an Investigator of the specific DNA sequence using a short RNA guide. Cas9 is McGovern Institute for Brain Research, enabling the generation of more realistic disease models and a New York Stem Cell Foundation- is broadening the number of genetically-tractable organisms Robertson Investigator, and a Core that can be used to study a variety of biological processes. The Member of the Broad Institute of MIT Cas9 nuclease can also be modified to modulate transcription and Harvard. As a graduate student at Stanford University, Zhang worked and alter epigenetic states in living cells. In this presentation we with advisor Karl Deisseroth to invent will look at the latest developments including structure-guided a set of technologies for dissecting the engineering of Cas9 to achieve robust genome modulation functional organization of brain circuits. and inducible genome editing, and metagenomic prospecting His lab works on developing and applying to identify efficient Cas9 orthologs. We will also describe disruptive technologies including applications of the Cas9 nuclease for understanding the gene optogenetics and genome engineering functions in the nervous system and disease processes. (TALE and CRISPR) to understand The expanding Cas9 toolbox is enabling a broad range of nervous system function and disease. applications in basic research as well as medicine. Zhang’s long-term goal is to develop novel therapeutic strategies for disease treatment. He obtained a bachelor’s degree from Harvard University and a PhD in chemistry and bioengineering from Stanford University. Before joining the MIT faculty he was a junior fellow of the Harvard University Society of Fellows. He is widely recognized for the development of molecular technologies including optogenetics and CRISPR-Cas9, including receiving the Waterman Award from the National Science Foundation, the Perl/UNC Prize in Neuroscience, and the Gabbay Award in Biotechnology.

20 Session 6 State-of-the-art biological technologies and biological dynamics 15:20-15:40, November 6 Carrying skeletal elements by novel type of cells enables the self-organizing construction of 4D skeleton of sponges

Noriko Funayama ABSTRACT Kyoto University

Dr. Funayama is an Associate Professor How organisms develop complex, beautiful structures has long in the Department of Biophysics in the been an intriguing question. Despite advances in developmental Graduate School of Science of Kyoto biology, the mechanisms underlying the assembly of some University. Dr. Funayama worked biological structures remain virtually unexplored. The skeletons on molecular mechanisms of cell of sponges, seen, for example, in Haeckel’s famous drawings adhesion as PhD work in Dr. Shoichiro of glass sponges (hexactinellids), show great morphological Tsukita’s lab, found the nuclear diversity and beautiful macroscopic patterns. The question localization of β-catenin and its role in the signaling pathway that causes A-P naturally arises of how sponges – one of the evolutionarily axis formation in Xenopus embryo oldest extant animals – construct such elaborate skeletons, as a postdoctoral fellow in Dr. B.M. but it remains unanswered. We revealed that the skeleton Gumbiner’s lab, worked on the cell fate construction of sponges is fundamentally different from any specification and the mesenchymal other known skeleton construction. We succeeded in live to epithelial transition of lateral plate imaging by establishing fluorescent labeling of spicules and mesoderm in Dr. Y. Takahashi’s group, microscopic systems to obtain images from the side of sponges. and then joined Dr. Kiyokazu Agata’s We discovered that mature spicules are dynamically transported lab, where she started to work by from where they were produced by newly discovered transport focusing on the cellular and molecular cells, then pierce through outer epithelia, and their basal mechanisms of totipotent stem cells ends become fixed there to substrate or connected with of the evolutionarily oldest extant animal, sponge, and recently has done such fixed spicules. Our study revealed that division of labor landmark work elucidating the skeleton by manufacturer, transporter, and cementer cells, and that construction of sponges. iteration of the sequential mechanical reactions of “transport” “pierce” “raise up” and “cementation”, allows construction of the spiculous skeleton spicule by spicule as a self-organized biological structure, with the great plasticity in size and shape required for indeterminate growth, and generating the great morphological diversity of individual sponges.

JST CREST-PRESTO joint international symposium 21 Session 6 State-of-the-art biological technologies and biological dynamics 15:40-16:05, November 6 Roles of the tight junction (TJ)- apical complex in epithelial morphogenetic dynamics

Sachiko Tsukita ABSTRACT Osaka University

Prof. Sachiko Tsukita is a cell biologist at Epithelial cells adhere to each other by tight junctions (TJs) Osaka University, Japan. Prof. Tsukita’s to form cell sheets, which is a critical step in epithelial research on cell-cell adhesion and morphogenesis. We recently discovered that a network of cytoskeleton/signaling began when she was microtubules exists just below the apical membrane of the a graduate student at Tokyo University. After epithelial cell sheet. Because this apical microtubule network receiving her PhD degree, she continued her appears to be connected to TJs through TJ-associated proteins, research career at the Tokyo Metropolitan including cingulin and novel proteins that we have identified, Institute of Medical Science and the National Institute for Physiological Sciences. She we defined the TJ and its associated membrane and apical joined Kyoto University as a professor in structures as the “TJ-apical complex.” Here, we will fi rst present the College of Medical Technology in 1994 our recent studies on the role of the TJ-apical complex in the and then in the Faculty of Medicine in 2003. morphogenetic processes by which two-dimensional epithelial In 2007, she joined Osaka University in the cell sheets create three-dimensional tissue confi gurations. Next, Graduate School of Frontier Biosciences we will focus on the multi-ciliated tracheal epithelial cells as and the Graduate School of Medicine. As a typical example of highly differentiated epithelial cells. The part of her research on tight junctions(TJs) coordinated movement of cilia on these cells is required for the cytoskeleton and cell signaling, she mucociliary transport. We found that the TJ-apical complex established a method for isolating the cell- plays a specific role in orienting the basal bodies to enable cell junctions, including TJs, of epithelial-type this coordinated multi-ciliary movement. In collaboration with cells. Using these preparations, several tight junctional proteins, including ZO1, claudin, theoretical scientists, we propose a model showing how the self- occludin, and tricellulin, were identified. organization of the apical microtubules is critical for this ciliary Dr. Tsukita analyzed the function of these coordination. These studies explore the novel concept that TJs proteins at the molecular, cellular, and whole organize biological systems by integrating the TJ paracellular animal (mouse) level, with particular focus barrier and apical cortical systems. on paracellular barrier creation and biological systems. Her current research expands on this work, and explores the novel concept of the TJ-apical complex; that is, the critical roles of TJs and their associated apical cortical layer, which collectively form epithelial barriers in dynamic biological systems.

22 Session 6 State-of-the-art biological technologies and biological dynamics 16:05-16:25, November 6 A synthetic approach to understanding protein molecules

Nobuyasu Koga ABSTRACT Institute for Molecular Science

Dr. Nobuyasu Koga is an associate Protein molecules fold into unique tertiary structures specifi ed professor at the Research Center by their amino acid sequences from random coils, and then of Integrative Molecular Systems at exert functions based on the folded structures. Naturally the Institute for Molecular Sciences occurring proteins have complicated structural features such at the National Institute for Natural as kinked helices, bulged strands, strained loops, and buried Sciences. Dr. Koga studied on protein polar groups because of functional constraints and long folding and molecular motors using evolutional histories, which make it difficult to uncover the coarse-grained molecular dynamics simulations at Kobe University during fundamental principles for folding and functional mechanisms. his PhD course. After receiving his A synthetic approach, designing simple and ideal protein PhD degree from Kobe University in molecules completely from scratch, provides us opportunities 2006, he started to work on protein to explore the principles: hypothesis on folding or functions design in the Baker laboratory at can be evaluated by computationally designing proteins and the University of Washington as a experimentally assessing how they behave. We discovered a set postdoctoral researcher from 2007. of rules relating local structures to tertiary protein motifs, which Then, he joined the Institute for made possible the design of protein structures for various Molecular Science in Okazaki, Japan alpha-beta topologies. The results illuminate how proteins in 2014, and also joined the research fold into unique tertiary structures. Furthermore, based on our group for design and control of cellular developed design technologies, we are tackling the design of functions as a Sakigake (PRESTO) researcher. His research interest is functional proteins from scratch. to understand principles for protein Sarel J. Fleishman, Sagar D. Khare, Nobuyasu Koga, and David Baker, Protein Science, 20(4), 753-757, folding and functions by designing 2011 protein molecules from scratch Nobuyasu Koga, Rie Tatsumi-Koga, Gaohua Liu, Rong Xiao, Thomas B. Acton, Gaetano T. Montelione and David Baker, Nature, 491(7423), 222-227, 2012 and to develop the technologies for Yu-Ru Lin, Nobuyasu Koga, Rie Tatsumi-Koga, Gaohua Liu, Amanda F. Clouser, Gaetano T. Montelione, and engineering protein molecules toward David Baker, Proc. Natl. Acad. Sci, 112(40):E5478-85, 2015 the goal of regulating and designing cellular functions.

JST CREST-PRESTO joint international symposium 23

Structural Biological Dynamics: From Molecules to Life with 60 trillion Cells

Poster Presentation CREST “Biodynamics” No Title Authors Affi liation

Takayuki Teramoto1 2+ 2 Terumasa Tokunaga 1. Kyushu University Whole-brain Ca imaging of C. elegans 4 Osamu Hirose 2. Kyushu Institute of Technology reveals the multi-neuronal dynamics 5 CB01 Yu Toyoshima 3. The Institute of Statistical Mathematics that are coordinated by global synaptic Yuichi Iino5 4. Kanazawa University connectivity Ryo Yoshida3 5. The University of Tokyo Takeshi Ishihara1

Yuhki Nakatake1 Norio Goda1 Nana Chikazawa- Nohtomi1 Miyako Murakami1 Miki Sakota1, Shunichi Wakabayashi1 Mak Siu San1 Martin Jakt1 Tomoo Ueno1 Misako Matsushita1 Mayumi Oda1 Utsumi Noriko2 1. Keio University 2. Kazusa DNA Research Institute Madoka Ishikawa- Large-scaled transgene activation on 3. DNA Chip Research Inc CB02 3 Hirayama 4. National Center for Child Health and Development human embryonic stem cells 3 Noriko Itoh 5. Xcoo, Inc. Motohiko Tanino3 Yukari Ikeda3 Hiroshi Iijima3 Takumi Miura4 Masakazu Machida4 Kahori Minami4 Shigeru B.H.Ko1 Hiroyuki Nishimura5 Ryo Matoba3 Hidenori Akutsu4 Osamu Ohara2 Minoru S.H. Ko1

Junpei Higashi1 Formation of the 3D structure of Atsushi Kawamoto2 1. Osaka University CB03 vertebrate bones by topology Tsuyoshi Nomura2 2 2. Toyota Central R&D Labos optimization Tadayoshi Matsumori Shigeru Kondo1

Neurogenesis suggests a novel algorithm Anthony J. DeCostanzo CB04 RIKEN for pattern classifi cation in sparse code Tomoki Fukai

Hiroki Kurihara1 Experimental and mathematical analysis 1 1. The University of Tokyo and Tetsuji Tokihiro 2. Tokyo Medical and Dental University CB05 of collective cell movement during Youichiro Wada1 angiogenesis Kenji Yasuda2

Masahiko Kumagai Understanding 3D Chromatin Dynamics Yuichi Motai CB06 Ryohei Nakamura The University of Tokyo in the medaka genome Shinichi Morishita Hiroyuki Takeda

Kyosuke Shinohara1 2 Duanduan Chen 1. Tokyo University of Agriculture and Technology Absence of Radial Spokes in Mouse Node 2 Tomoki Nishida 2. Osaka University CB07 Cilia Is required for Rotational Movement Kazuyo Misaki3 3. RIKEN but Confers Ultrastructural Instability Shigenobu Yonemura3 Hiroshi Hamada2

Structural approaches for dynamics of Atsushi Mochizuki CB08 RIKEN complex network systems. Takashi Okada

Understanding synapse dynamics Shigeo Okabe CB09 Shinji Tanaka The University of Tokyo through nanoscale structural analyses Hirohide Iwasaki

Identifi cation of a new class of GPCR Genzui Setsu CB10 Masao Doi Kyoto University signaling that tunes the central clock Hitoshi Okamura

Modeling spontaneous pattern formation CB11 Takashi Miura Kyushu University of capillary meshwork in vitro

26 PRESTO ”Design and Control of Cellular functions” No Title Authors Affi liation Substrate and curvature dependence of PD01 Satoshi Sawai The University of Tokyo ventral F-actin waves in Dictyostelium Enzymatic propagation of large DNA Masayuki Suetsugu PD02 Rikkyo University circles Hiroko Tsujimoto Spatiotemporal dynamics of non- PD03 Masahiro Takinoue Tokyo Institute of Technology equilibrium artifi cial cell model Kazuhito Tabata Reconstitution of a cell in femto litter Yoshiki Moriizumi PD04 The University of Tokyo sized microchamber array Rikiya Watanabe Hiroyuki Noji Protein phosphatase makes a mitotic PD05 Satoru Mochida Kumamoto University switch together with kinase. Analysis of gene expression regulation PD06 through precise reconstitution of ‘epi- Takashi Umehara RIKEN nucleosome’ Imaging and manipulation of PD07 Kohki Okabe The University of Tokyo temperature in single living cells Phytohormone-mediated spatiotemporal PD08 Shigeyuki Betsuyaku JST PRESTO regulation of plant immune responses Kaoru Sugimura1 Mechanical control of epithelial 1 1. Kyoto University PD09 Keisuke Ikawa 2. Meiji University morphogenesis Shuji Ishihara2 Assembly of the olfactory neural circuit in PD10 Haruki Takeuchi JST PRESTO mice Electrically-induced bubble injector for PD11 Yoko Yamanishi Shibaura Institute of Technology biomedical applications

CREST “Structural Life Science” No Title Authors Affi liation Yasunori Watanabe1 Structural basis of phospholipid transfer 1 Shin Kawano 1. Kyoto Sangyo University CS01 between the ER and mitochondria and Yasushi Tamura2 2. Yamagata University within mitochondria Toshiya Endo1 Toshiya Senda1 Hironobu Suzuki1 2 Structural biology of carcinogenesis by Takeru Hayashi 1. High Energy Accelerator Research Organization, KEK CS02 1 Helicobacter pylori Miki Senda 2. The University of Tokyo Lisa Nagase1 Masanori Hatakeyama2 Yoshiki Higuchi X-ray structure analysis of NAD+- Midori Taketa CS03 University of Hyogo reducing [NiFe]-hydrogenase Hanae Nakagawa Yasuhito Shomura Shuya Fukai1 Atsushi Yamagata1 Tomoyuki Yoshida2 1 Yusuke Sato 1. The University of Tokyo Mechanisms of splicing-dependent trans- 1 Sakurako Goto-Ito 2. University of Toyama CS04 synaptic adhesion for inducing synaptic Takeshi Uemura3 3. Shinshu University diff erentiation Asami Maeda1 4. Ritsumeikan University Tomoko Shiroshima1 Hisashi Mori2 Masayoshi Mishina4 Toshiaki Isobe1 Hiroshi Nakayama2 Masato Taoka1 Yoshio Yamauchi1 Molecular studies of RNA-dysmetabolic 1 1. Tokyo Metropolitan University Yuko Nobe 2. RIKEN CS05 syndrome ‒ From ribonucleoproteomics 3 Toshinori Kozaki 3. Tokyo University of Agriculture and Technology to structural life science - Kazuo Ishii3 Keiichi Izumikawa3 Hideaki. Ishikawa3 Nobuhiro Takahashi3

JST CREST-PRESTO joint international symposium 27 Yutaka Ito1 “Motion capture” of proteins inside living 2 1. Tokyo Metropolitan University CS06 Takanori Kigawa 2. RIKEN cells Yuji Sugita2 Risa Mutoh1 Yuko Misumi1 2 Hisako Kubota-Kawai 1. Osaka University NMR study of the interaction sites on the 2 Ryutaro Tokutsu 2. National Institute for Basic Biology CS07 ferredoxin isoforms for photosynthetic Takahisa Ikegami3 3 Yokohama City University protein complexes Michael Hippler4 4. University of Münster Jun Minagawa2 Genji Kurisu1 Maintenance for ER homeostasis through Ryo Ushioda CS08 Kyoto Sangyo University disulfi de reductase, ERdj5 Kazuhiro Nagata Nobuo N. Noda1 Yuko Fujioka1 Hironori Suzuki1 2 1. Institute of Microbial Chemistry, Tokyo Takeshi Kaizuka Structure and function of the autophagy 2. The University of Tokyo CS09 3 initiation complex Sho W. Suzuki 3. Tokyo Institute of Technology Hayashi Yamamoto3 Noboru Mizushima2 Yoshinori Ohsumi3 Toshiyuki Oda A molecular ruler determines the repeat Haruaki Yanagisawa CS10 The University of Tokyo length in eukaryotic cilia and fl agella. Ritsu Kamiya Masahide Kikkawa Yasunori Shintani Osaka University CS11 Positive Regulator of OXPHOS Seiji Takashima Atsushi Nakagawa1 Hirotaka Narita1 Yasushi Okamura1 1 Akira Kawanabe 1. Osaka University 1 Yuichiro Fujiwara 2. Japan Advanced Institute of Science and Technology Structural studies of the novel protein 2 Hidekazu Tsutsui 3. Nagoya Institute of Technology CS12 family working with cell membrane Hideki Kandori3 4. Hiroshima City University potential signal Masayo Iwaki3 5. Kinki University 6. Tohoku University Yu Takano4 Hiroko Kondo4 Yasushige Yonezawa5 Kengo Kinoshita6 Miyo Terao Morita1 Yoshinori Hirano2 Mechanism of gravitropic signaling in Masatoshi Taniguchi1 1. Nagoya University CS13 gravity sensing cells ～ From molecular Takeshi Nishimura1 2 2. Nara Institute of Science and Technology structure to plant organ response ～ Megumi Kagiyama Kohei Kawashima2 Toshio Hakoshima2

PRESTO ”Structural Life Science” No Title Authors Affi liation Yoshiki Tanaka1 Yasunori Sugano1 Mizuki Takemoto2 Takaharu Mori3 Arata Furukawa1 Cytoplasmic insights into the protein- 2 1. Nara Institute of Science and Technology Tsukasa Kusakizako 2. The University of Tokyo PS01 conducting channel implied by crystal 2 Kaoru Kumazaki 3. RIKEN structures of SecYEG Ayako Kashima1 Ryuichiro Ishitani2 Yuji Sugita3 Osamu Nureki2 Tomoya Tsukazaki1

CryoEM structure of muscle thin fi lament 1 Takashi Fujii 1. JST PRESTO PS02 with the tropomyosin and troponin Yurika Yamada2 2. Osaka University complex Structure-Based Design of Chemical PS03 Probes for Imaging and Functional Yuichiro Hori Osaka University Regulation of Proteins Conformational dynamics of GPCRs under PS04 Takumi Ueda The University of Tokyo lipid bilayer condition revealed by NMR

28 ATP-free unidirectional walking of Noriyuki Kodera PS05 Takayuki Uchihashi Kanazawa University myosin V Toshio Ando PS06 Crystal structures of CRISPR-Cas9 Hiroshi Nishimasu The University of Tokyo Ultrastructural analysis of infl uenza virus PS07 Takeshi Noda Kyoto University genome transcription Correlative studies on autophagy PS08 proteins using 3D electron microscopy Maho Hamasaki Osaka University with precise spatial information Structural basis for higher order structure PS09 Kyohei Arita Yokohama City University formation of UHRF1 Structural and Biochemical analysis of PS10 Narinobu Juge Okayama University Vesicular glutamate transporter X-ray Crystal Structure of Voltage-Gated PS11 Kohei Takeshita Osaka University Proton Channel, VSOP Dissection of the circadian clock PS12 machinery by integrating chemical Tsuyoshi Hirota Nagoya University biology and structural biology

Poster Layout

PD09～PD11 PS01～PS04 P S 8 0 0 5 D ～ P P ～ S 1 1 0 2 D P

CB04 CB11 ～ Poster

3 Exhibition 0 B

C Area ～ 1 0 B C

CS01～CS13

JST CREST-PRESTO joint international symposium 29 CB01 CB02

Whole-brain Ca2+ imaging of C. elegans Large-scaled transgene activation reveals the multi-neuronal on human embryonic stem cells dynamics that are coordinated by Yuhki Nakatake1, Norio Goda1, Nana Chikazawa- global synaptic connectivity. Nohtomi1, Miyako Murakami1, Miki Sakota1, Shunichi Wakabayashi1, Mak Siu San1, Martin Jakt1, Tomoo Takayuki Teramoto1, Terumasa Tokunaga2, Osamu Ueno1, Misako Matsushita1, Mayumi Oda1, Utsumi Hirose4, Yu Toyoshima5 Yuichi Iino5, Ryo Yoshida3 and Noriko2, Madoka Ishikawa-Hirayama3, Noriko Itoh3, Takeshi Ishihara1 Motohiko Tanino3, Yukari Ikeda3, Hiroshi Iijima3, 1. Kyushu University Takumi Miura4, Masakazu Machida4, Kahori Minami4, 2. Kyushu Institute of Technology Shigeru B.H.Ko1, Hiroyuki Nishimura5, Ryo Matoba3, 3. The Institute of Statistical Mathematics Hidenori Akutsu4, Osamu Ohara2 and Minoru S.H. Ko1 4. Kanazawa University 5. University of Tokyo 1. Keio University 2. Kazusa DNA Research Institute 3. DNA Chip Research Inc One of the fundamental challenges in neurosciences 4. National Center for Child Health and Development has been visualization of brain network dynamics 5. Xcoo, Inc. at cellular resolution. To accomplish this challenge by using a simple nervous system, we devised a 4D Large scaled-gene perturbation is one of the most imaging microscope system and developed an image powerful tools for uncovering gene regulatory network. processing software. This system, combining with a Such comprehensive approaches are well studied in 2+ Ca probe and mCherry as a cellular marker, enable simple model animals, but not in higher eukaryotes. us to analyze the neuronal activities of the whole Previously, we have reported comprehensive studies on central nervous system in C. elegans at the individual mouse embryonic stem (ES) cells, and demonstrated cellular levels. In wild-type animals, multiple neurons key regulatory gene networks that govern cell fates (1-3). exhibited synchronized rhythmic activity under the Here, we report an establishment of a set of transgenic non-stimulus condition, whereas the number of human ES cell lines that carry tetracycline inducible the synchronized neurons was decreased in unc-7 expression gene cassette for more than 500 single animals, which is defective in locomotive behavior transcription factors. Transcriptome data was obtained because of a mutation in a gap-junction component, 48h after transgene induction by RNA-seq. This data expressed in many neurons. This result suggests set has multiple characteristic properties such as 1) the that the electrical synaptic couplings are required same platform across data set, 2) analyzed with dynamic for the synchronized rhythmic activity in the central cell status, 3) pre-determined initiation point on gene nervous system. In addition, our 4D imaging system perturbation, 4)inducible in different cell status. Massive will provide a new insight for exploring brain network and specifi c cell differentiation was found after transgene dynamics in C. elegans . induction that correlate with gene expression patterns of mature organs. These observations contribute to understand transcriptional hierarchy and species difference between human and mouse.

1. Nishiyama A, et al. (2009). Uncovering early response of gene regulatory networks in ES cells by systematic induction of transcription factors. Cell Stem Cell. 2009 Oct 2;5(4):420-33.

30 CB03 CB04

Formation of the 3D structure of Neurogenesis suggests a novel vertebrate bones by topology algorithm for pattern classifi cation optimization in sparse code Junpei Higashi1, Atsushi Kawamoto2, Tsuyoshi Anthony J. DeCostanzo and Tomoki Fukai Nomura2, Tadayoshi Matsumori2 and Shigeru Kondo1 RIKEN 1. Osaka University 2. Toyota Central R&D Labos Hippocampal dentate gyrus (DG) is thought to play an active role in the contextual discrimination of Topology optimization is a mathematical approach that memory episodes. DG is also known for its extreme optimizes material layout within a given design space, sparse neural activity and adult neurogenesis, which for a given set of loads and boundary conditions such continuously produces new neurons for functional that the resulting layout meets a prescribed set of [1] integration into existing networks . The relationship performance targets. Using topology optimization, between adult neurogenesis and sparse activity in engineers can fi nd the best concept design that meets cognition is unknown. Here, we propose a novel the design requirements. We utilized this technique to algorithm of adult neurogenesis for pattern separation construct the 3D structure of vertebrae of zebrafi sh in in a three-layer neural network, in which entorhinal the computer. cortical inputs projecting to DG with feed-forward Zebrafi sh vertebra in juvenile fi sh is a simple cylinder excitation to CA3. We found that local network structure, then the ends of the cylinder expand to integration of newborn cells into DG requires sparse form the corn structure. At the lateral sides of the corn activity that, in turn, enables adult neurogenesis to structure, libs are added to enforce the rigidity. Then cluster related memories together thereby reducing the neural arch and hemal arch connect to the edge errors in pattern separation. Our results demonstrate of the corns. We have investigated the position of the how neurogenesis enhances memory encoding in tendons that would apply the force to the vertebrae cooperation with sparse neural activity. and with the assumption that the new bone is added in response to the local deformation. The calculation [1] Inokuchi K. Adult neurogenesis and modulation of neural circuit function. Curr Opin Neurobiol 21:360-364 (2011). result with COMSOL successfully reproduced some of the specific characteristics of the vertebrae fine structure.

JST CREST-PRESTO joint international symposium 31 CB05 CB06

Experimental and mathematical Understanding 3D Chromatin analysis of collective cell movement Dynamics in the medaka genome during angiogenesis Masahiko Kumagai, Yuichi Motai, Ryohei Nakamura, Hiroki Kurihara1, Tetsuji Tokihiro1, Youichiro Wada1 Shinichi Morishita and Hiroyuki Takeda 2 and Kenji Yasuda The University of Tokyo

1. The University of Tokyo 2. Tokyo Medical and Dental University The 3D structure of chromosomes affects gene expression and has been implicated in the maintenance Angiogenesis is a morphogenetic process that of pluripotency, lineage-specific cell differentiation produces branching vascular structures during and pathological transformation including cancer and embryogenesis and various (patho-)physiological neurological disease. To elucidate the mechanisms conditions. We have identified characteristic cellular that determine the 3D structure, we perform a behaviors in angiogenic processes, including dynamic genome-wide study to collect 1D epigenetic codes changes in forward-backward movement, tip cell and observe 2D chromatin interactions from the overtaking and resultant cell mixing. Although the genomes of medaka to build a 3D model of chromatin cellular behaviors appear complex and arbitrary, architecture. We recently succeeded in Hi-C analysis, different types of mathematical modeling and collecting genome interaction data, of cultured cells, experimental verification indicated that some native tissues as well as blastula-stage embryos. deterministic cell-cell interactions are critical For 3D modeling, we are implementing a program for vascular elongation and possibly branching. which estimates consensus 3D models based on Recently, we found differences in branch-forming multidimensional scaling. This method enables us to capacity among cell types and some regularities in calculate 3D structure from vast amount of interaction directional cell movement using in vitro angiogenesis data in a realistic time. We will discuss the logic and experiments using mouse vascular explants and an model that can serve as a reliable tool to understand endothelial cell line by refined cell-tracking system. the differentiation state and potency of specific-cell Together with single-cell analyses of cell movement lineages. and gene expression, novel mathematical modeling and experimental verification using constitutional approaches will be discussed to elucidate the possible cellular mechanisms underlying branch formation in angiogenesis.

32 CB07 CB08

Absence of Radial Spokes in Structural approaches for dynamics Mouse Node Cilia Is required for of complex network systems Rotational Movement but Confers Atsushi Mochizuki and Takashi Okada Ultrastructural Instability RIKEN Kyosuke Shinohara1, Duanduan Chen2, Tomoki 2 3 3 Nishida , Kazuyo Misaki , Shigenobu Yonemura and By the success of modern biology we have many 2 Hiroshi Hamada examples of large networks which describe 1. Tokyo University of Agriculture and Technology interactions between a large number of species of 2. Osaka University bio-molecules. On the other hand, we have a limited 3. RIKEN understanding for quantitative details of biological systems, like the regulatory functions, parameter Determination of left-right asymmetry in mouse values of reaction rates. To overcome this problem, embryos is established by a leftward fluid flow that we have developed structural theories for dynamics is generated by clockwise rotation of node cilia. How of network systems. By our theories, important node cilia achieve stable unidirectional rotation has aspects of the dynamical properties of the system remained unknown, however. Here we show that can be derived from information on the network brief exposure to the microtubule-stabilizing drug structure, only, without assuming other quantitative paclitaxel (Taxol) induces randomly-directed rotation details. In this presentation, we will introduce a new and changes the ultrastructure of node cilia. In vivo theory for chemical reaction networks. We present a observations and a computer simulation revealed that mathematical method, named structural sensitivity a regular 9+0 arrangement of doublet microtubules analysis, to determine the sensitivity of reaction is essential for stable unidirectional rotation of node systems from information on the network alone. We cilia. The 9+2 motile cilia of the airway, which manifest establish and prove a general law which connects planar beating, are resistant to Taxol treatment. the network topology and the sensitivity patterns of However, airway cilia of mice lacking the radial spoke metabolite responses directly. head protein Rsph4a undergo rotational movement instead of planar beating, are prone to microtubule rearrangement, and are sensitive to Taxol. Our results suggest that the absence of radial spokes allows node cilia to rotate unidirectionally but renders them ultrastructurally fragile as a trade-off

JST CREST-PRESTO joint international symposium 33 CB09 CB10

Understanding synapse dynamics Identifi cation of a new class of GPCR through nanoscale structural signaling that tunes the central analyses clock Shigeo Okabe, Shinji Tanaka and Hirohide Iwasaki Genzui Setsu, Masao Doi and Hitoshi Okamura

The University of Tokyo Kyoto University

Synaptic connectivity is thought to be highly The circadian pacemaker that governs daily rhythms in stabilized, but synaptic molecules show continuous behavior and physiology resides in the hypothalamic replacement locally. How synapses are generated and suprachiasmatic nucleus (SCN). Malfunction of this stabilized using specific molecular machinery is one brain clock has been linked to the pathogenesis of of the central questions in the field of neurobiology a variety of diseases, but the development of drugs (1-3). We are developing a new quantitative imaging targeting this brain locus still remains uncovered. method that enables us to efficiently characterize G protein-coupled receptors (GPCRs) are important nanoscale features of synaptic structures in vitro. drug targets, and there are still more than 140 orphan Multiple parameters related to nanoscale synaptic GPCRs waiting for decipherment of their function. In features are analyzed and components important order to identify a new GPCR that tunes the central for synapse stability are extracted. These in vitro clock, here we launched the SCN orphan GPCR experiments are combined with in vivo two-photon project, in which we (i) surveyed all known orphan imaging of synapses within the mouse neocortex and GPCRs expressed in the SCN, (ii) generated knockout the nanoscale features extracted from in vitro study mice of candidate GPCR genes of interest, and (iii) are correlated with properties of stable synapses in asked whether there is a defect in their circadian vivo. We will also describe the model of molecular rhythms in behavior. Based on this screening strategy, dynamics within synapse cytoplasm, which can we have been able to show that the SCN possesses a explain the behavior of single molecules within new class of GPCR that controls circadian behavior. synapses measured by microscopic techniques, such as fl uorescence correlation spectroscopy.

1. Ishida et al. (2012) Neuron 76, 549-564. 2. Shin et al. (2013) Nat. Commun 4, 1440. 3. Isshiki et al. (2014) Nat. Commun 5, 4742.

34 CB11 PD01

Modeling spontaneous pattern Substrate and curvature formation of capillary meshwork in dependence of ventral F-actin waves vitro in Dictyostelium Takashi Miura Satoshi Sawai

Kyushu University The University of Tokyo

Pattern formation by endothelial cells has been In many cell types ranging from keratocytes to extensively studied as an example of spontaneous fibroblasts, traveling wave dynamics of F-actin pattern formation. When HUVECs are cultivated are observed. In Dictyostelium, F-actin waves are in fibrin gel, they spontaneously form meshwork observed at the ventral side of the plasma membrane structure within 24 hours just as HUVEC-Matrigel whose roles are unclear but appear to be related to system. Then they start to form lumen and finally macropinocytosis and phagocytosis. Here, by live- perfusable capillary meshwork is formed. This cell TIRF and IRM imaging of Dictyostelium cells system is very useful in tissue engineering point of together with the use of micropatterned substrates, view because it can be used as a synthetic capillary we show that the wave front where Arp2/3 complex network to cultivate 3D tissue structure in vitro (1). is enriched consist of member fold decorated with We try to understand the phenomenon by dividing members of F-BAR protein. When wave fronts the phenomenon into early and late phase. At fi rst we escaped to the lateral side of the plasma member, they assume that in the early phase, randomly distributing transformed to membrane ruffles indicating that the cell can connect by a certain probability depending waves and ruffl es are inter-convertible. Furthermore, on the distance. Then, to model the later phase of we show that the occurrence of waves is dependent pattern formation, we simply assume that the tubes on substrate adhesiveness and the direction of "grow" at constant velocity V with surface tension. propagation can be manipulated by micrometer size By this model, we can generate the experimentally indentation on the substrate. Based on the findings, observed pattern. These models are simple enough we propose that the wave dynamics are the result to analyze mathematically (percolation theory can be of a core feedback property that couples dendritic applicable), and experimental verifi cation is underway actin fi laments, membrane curvature and membrane to quantitate model parameters. tension.

1. S. Kim, H. Lee, M. Chung, N. L. Jeon, Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip. 13, 1489–1500 (2013).

JST CREST-PRESTO joint international symposium 35 PD02 PD03

Enzymatic propagation of large DNA Spatiotemporal dynamics of non- circles equilibrium artifi cial cell model Masayuki Suetsugu and Hiroko Tsujimoto Masahiro Takinoue

Rikkyo University Tokyo Institute of Technology

We have reconstituted an entire process of replication Living systems are achieved by complex chemical cycle of the Escherichia coli mini-chromosome reaction dynamics far from equilibrium. The design, using purified enzymes that catalyze initiation at the construction, and control of bio-inspired self- chromosomal origin, bidirectional fork progression, organized phenomena are challenges in a wide Okazaki fragment maturation, and decatenation range of fields such as synthetic biology and bio- of the interlinked sister mini-chromosomes. The inspired nano/microtechnology. In general, chemically decatenation produces the supercoiled monomer open systems with well-controlled chemical fluxes molecules that are competent for the next round into/out of the systems are essential for complex of replication initiation. Thus, the replication cycle chemical reactions far from equilibrium. Therefore, repeats autonomously in isothermal condition. This the construction of microreactors with chemical system termed RCR (Replication Cycle Reaction) fluxes is necessary. Here, we report a droplet-based allows exponential propagation of circular DNA microfluidic method that can control time-variable molecules. Large DNA circles up to several hundred chemical fluxes into/out of a microreactor. Our method kilobases can be propagated. Combining with a 1-step is inspired by the universal molecular transportation DNA assembly reaction, we develop a cell-free cloning systems in cells based on vesicular fusion and method to create synthetic circular genomes, in which fission, such as endo- and exo-cytotic processes. RCR supersedes biological hosts to propagate circular This method allowed precise control of time-variable DNA products. In addition to such a practical aspect, chemical fluxes, resulting in successful control of RCR could provide a valuable platform for a bottom- chemical oscillation far from equilibrium. We believe up approach to constructing self-replicative biological that this system brings innovations in chemical and systems. biomedical studies in terms of dynamical control of self-organized phenomena far from equilibrium.

36 PD04 PD05

Reconstitution of a cell in femto Protein phosphatase makes a litter sized microchamber array mitotic switch together with kinase. Kazuhito Tabata, Yoshiki Moriizumi, Rikiya Watanabe Satoru Mochida and Hiroyuki Noji Kumamoto University The University of Tokyo Switch-like behavior is often observed in many Hybrid chamber cells are devices of femtoliter (fL) biological phenomena, behind which complex size that house membrane and cellular components molecular network should exist. In this project, we of the cell and allow for the fusion of animate and aim to reconstitute in vitro the switch-like behavior inanimate materials. It is considered as an ideal in the cell cycle by which division phase (mitosis/ M platform for cell reproduction and reconstitution. To phase) is temporary separated from interphase to explore the feasibility of cell reconstitution with fL- secure faithful segregation of our genetic materials, chambers, we attempted to integrate a living bacterial chromosomes. Biochemical basis of mitosis is cell with fL-chamber. Protoplasts from E. coli cells protein phosphorylation and cyclin-dependent kinase were placed on fL-chambers, the top of which was (CDK) is the major enzyme that catalyzes mitotic sealed with lipid bilayer. Some of protoplasts showed phosphorylation. CDK activity appears and gradually membrane fusion that released the intracellular increases during interphase and peaks in mitosis, components into a chamber and the membrane however, phosphorylation of CDK substrates is components into the artifi cial bilayer membrane. We induced all in sudden on entering into mitosis. How are currently conducting viability assessments of the can this be possible? I hypothesized that interaction hybrid system. between two antagonizing enzymes, CDK and PP2A phosphatase, could make such abrupt changes of equilibrium possible on their substrates (ref.). With only 3 polypeptides in addition to CDK and PP2A, we succeeded in making a switch-like behavior on substrate phosphorylation, which is similar to what we observe on mitotic entry.

Reference: Mochida et al. (2010) Science, 330 (6011), p1670.

JST CREST-PRESTO joint international symposium 37 PD06 PD07

Analysis of gene expression Imaging and manipulation of regulation through precise temperature in single living cells reconstitution of ‘epi-nucleosome’ Kohki Okabe

Takashi Umehara The University of Tokyo

RIKEN Temperature, a key regulator of biochemical reactions, Human gene expression is not only regulated by infl uences many physiological functions of organisms. their DNA sequence information but also by their We previously demonstrated monitoring and imaging epigenetic information. However, reconstitution of of intracellular temperature based on a fluorescent a genomic DNA containing epigenetic information polymeric thermometer and quantitative fl uorescence of interest (i.e. designer epigenome) has been imaging techniques, showing temporal and spatial technologically difficult thus far. This study aims to variation of temperature in single living cells (1) precisely reconstitute designed ‘epi-nucleosome’ associated with cellular functions , shedding light on containing a variety of epigenetic information in vitro an intriguing hypothesis: temperature change inside of such as lysine acetylation and methylation, through a cell is involved in cell biology. Conceiving this idea, technological innovation of biochemistry and genetic we have been investigating the intrinsic connection code engineering (1-2). Through the reconstitution of between intracellular temperature change and cell ‘epi-nucleosomes’, I aim to quantitatively understand functions. By observing intracellular temperature the nature of an epigenome and its effect on and the dynamic mRNA behavior in stressed cells, eukaryotic gene expression. Recent proceedings on we have revealed that stress-responsive mRNA the chromatin transcription analysis using precisely granule formation essentially depends on intracellular acetylated ‘epi-nucleosomes’ will be presented. local thermogenesis. Furthermore, we manipulated intracellular temperature by irradiating infrared laser 1) Wakamori et al. Anal. Biochem. 423, 184 (2012). onto a cell to observe the thermodynamics and cell 2) Yanagisawa et al. Chembiochem 15, 1830 (2014). response. These techniques are indispensable for the advancement of thermal biology, which explores the commitment of temperature to life.

1. Okabe et al., (2012) Intracellular temperature mapping with a fl uorescent polymeric thermometer and fl uorescence lifetime imaging microscopy Nat. Commun. 3:705.

38 PD08 PD09

Phytohormone-mediated Mechanical control of epithelial spatiotemporal regulation of plant morphogenesis immune responses Kaoru Sugimura1, Keisuke Ikawa1 and Shuji Ishihara2

Shigeyuki Betsuyaku 1. Kyoto University 2. Meiji University JST PRESTO

Plant immune responses, such as hypersensitive How do cells push and pull each other to trigger response (HR), are regulated spatially and temporally. precise deformations of a tissue when shaping In the case of HR, programmed cell death was the body? The answer to this central question is observed at the site of pathogen infection and the essential for understanding the development of surrounding cells are thought to exert immune animal forms including our body. We have developed responses promoting accumulation of antimicrobial key techniques to approach this question, including 1 compounds. Formation of a concentration gradient of Bayesian force inference , whole-tissue imaging, and salicylic acid (SA), a key molecule in plant immunity, image processing tools. By using these techniques, upon pathogen infection has been proposed to we previously reported that the extrinsic stretching regulate such a spatiotemporal pattern of plant force provides directional information for assignment immune responses in HR. This “French-flag” of the orientation of cell rearrangement and promotes 2 hypothesis for SA action was originally raised by hexagonal cell packing in the Drosophila wing . We are studies of Tobacco immunity nearly two decades ago currently addressing the mechanism by which force- and was further formulated by a number of studies in generating and force-sensing properties of F-actin other species. However, the hypothesis has not been trigger extrinsic force-driven cell rearrangements. In clearly examined. In order to examine this “French- addition, we investigate how the rate and directionality flag” hypothesis for SA action in spatiotemporal of cell rearrangements are determined with respect aspects, I have developed a live-imaging system to the physical properties of cells and molecular of plant immune responses. The system enabled regulation of the cytoskeleton and cell adhesion. us to detect dynamic immune responses in non- 1. Ishihara and Sugimura. J. Theor. Biol. (2012) detached living Arabidopsis leaves. Our cell biology- 2. Sugimura and Ishihara. Development (2013) based approach revealed that mutually antagonistic interaction of two phytohormones, SA and jasmonic acid (JA), shapes the HR pattern, rather than the simple French-fl ag model.

JST CREST-PRESTO joint international symposium 39 PD10 PD11

Assembly of the olfactory neural Electrically-induced bubble injector circuit in mice for biomedical applications Haruki Takeuchi Yoko Yamanishi

JST PRESTO Shibaura Institute of Technology

In the mouse olfactory system, odorants are Minimally-intrusive ablation of cell has been detected by ~1,000 different odorant receptors (ORs) successfully carried out by using the breakdown of expressed in olfactory sensory neurons (OSNs). electrically-induced bubble. This novel method of Each OSN expresses only one functional OR species, fi ne ablation method provide non-thermal damage of which is referred to as the "one neuron-one receptor" the surface of biological cell as well as the ablation rule. Furthermore, OSN axons bearing the same OR region can be downsized because this resolution is converge to a specifi c projection site in the olfactory depend on the width of capillary bubble. Cavitation bulb (OB) forming a glomerular structure, i.e., the of a single bubble occurs at the tip of inner electrode "one glomerulus-one receptor" rule. Based on these and perforates biological tissue immediately when the basic rules, binding signals of odorants detected voltage is applied to the electrode. Subsequently, the by OSNs are converted to spatial information of bubble is launched at high speed from the electrode activated glomeruli in the OB. The glomerular map transporting reagent into the biological tissue. This is formed by two different genetic processes: one is device can be used under liquid as well as under air OR-independent projection along the dorsal-ventral environment. This novel device is applicable to various axis, and the other is OR-dependent projection along typs of cells such as animal cell or plant cell with the anterior-posterior axis. The map is further refi ned minimally invasiveness. This method will contribute in an activity-dependent manner during the neonatal to wide range of biological applications such as gene period. Recent progress in elucidating molecular therapy and gene transformations mechanisms of the glomerular map formation will be presented.

40 CS01 CS02

Structural basis of phospholipid Structural biology of carcinogenesis transfer between the ER and by Helicobacter pylori mitochondria and within Toshiya Senda1, Hironobu Suzuki1, Takeru Hayashi2, mitochondria Miki Senda1, Lisa Nagase1 and Masanori Hatakeyama2

1 1 2 Yasunori Watanabe , Shin Kawano , Yasushi Tamura , 1. High Energy Accelerator Research Organization, KEK and Toshiya Endo1 2. The University of Tokyo

1. Kyoto Sangyo University 2. Yamagata University Helicobacter pylori , a gram-negative bacterium that colonizes the human gastric mucosa, has Mitochondria consist of the four distinct sub- been recognized as a major risk factor for gastro- compartments, the outer membrane (OM), duodenal diseases, such as peptic ulcers and gastric intermembrane space, inner membrane (IM), and the carcinomas. H. pylori delivers an effector protein CagA matrix, and their biogenesis requires synthesis and into gastric epithelial cells and the EPIYA and CM transport of its constituent proteins and phospholipids. segments in the C-terminal region of CagA (CagA-C) Mitochondrial phospholipids are synthesized by promiscuously interact with cellular signaling multiple phospholipidsynthetic enzymes located molecules, such as SHP2 and PAR1, to deregulate in different organelle membranes including the ER these target proteins. To uncover the structure and membrane and mitochondrial IM. However, identity functional details of a large protein complex formed and functions of the phospholipid transfer systems by CagA, SHP2, and PAR1, we initiated a structural among the ER membrane and mitochondrial OM and biology study of CagA. Since our group determined IM, which are required for correct distribution and the crystal structure of the N-terminal region of CagA compositions of phospholipids such as cardiolipin (1), we are working on structure and function of the in mitochondria, are largely unknown. Here we CagA-C, which has been known as an intrinsically determined the crystal structures of Ups1-Mdm35, disordered region. We have revealed a lariat-loop like which is responsible for phosphatidic acid transfer structure of CagA-C and obtained crystal structures between the OM and IM, and Mdm12, which of the SH2 domain of SHP2 in complex with various constitutes the mitochondria-ER tethering structure EPIYA segments from CagA-C. and is responsible for phospholipid transfer between 1. Hayashi, T. et al. (2012) Tertiary structure and functional analysis of the Helicobacter the ER membrane and OM, with and without substrate pylori CagA oncoprotein. Cell Host Microbe, 12, 20-33. phospholipids. These structures as well as in vitro phospholipid transfer assays allowed us to reveal the detailed mechanisms of phospholipid transfer between the ER and mitochondria and within mitochondria.

Watanabe et al. (2015) Structural and mechanistic insights into phospholipid transfer by Ups1-Mdm35 in mitochondria. Nature Commun. in press.

JST CREST-PRESTO joint international symposium 41 CS03 CS04

X-ray structure analysis of NAD+- Mechanisms of splicing-dependent reducing [NiFe]-hydrogenase trans-synaptic adhesion for inducing Yoshiki Higuchi, Midori Taketa, Hanae Nakagawa and synaptic differentiation Yasuhito Shomura Shuya Fukai1, Atsushi Yamagata1, Tomoyuki Yoshida2, 1 1 3 University of Hyogo Yusuke Sato , Sakurako Goto-Ito , Takeshi Uemura , Asami Maeda1, Tomoko Shiroshima1,Hisashi Mori2 and Masayoshi Mishina4 NAD+-reducing [NiFe]-hydrogenases (NAD-h2ases) catalyze the reversible electron transfer from 1. The University of Tokyo hydrogen to NAD+1. They generally consist of two 2. University of Toyama 3. Shinshu University catalytic heterodimeric subcomplexes: HoxFU NADH 4. Ritsumeikan University oxidoreductase (diaphorase) and HoxYH hydrogenase units. The four subunits are phylogenetically related Synapse formation is induced by trans-synaptic 2 to NADH:quinone oxidoreductase (Complex I) . We interactions between membrane receptor-like have solved the x-ray crystal structure of the NAD- adhesion molecules known as ‘synapse organizers’. h2ase in the as-isolated form. The two active sites of Selection of synapse targets depends on selective hydrogenase and diaphorase are separated by ~60 pairing between pre- and postsynaptic organizers, Å connected via 5 Fe–S clusters. While the relative which is regulated by short splice inserts called mini- disposition of the Fe–S clusters in each subunit exon peptides. Presynaptic type IIa receptor protein 3 closely resemble Complex I , the NAD-h2ase is tyrosine phosphatases (RPTPs) can induce synaptic equipped with a minimum set of clusters for electron differentiation through mini-exon peptide-dependent transfer. Furthermore, the orientation between HoxFU interactions with interleukin-1 receptor accessory and HoxYH subcomplexes is different from that of protein (IL-1RAcP), IL-1RAcP-like 1 (IL1RAPL1) Complex I, supporting the hypothesis that the two or Slit- and Trk-like (Slitrk) family proteins. Based 3 subcomplexes have evolved as prebuilt modules . The on their complex structures and structure-based Ni–Fe active site of the hydrogenase in the oxidized mutational analyses at the molecular and cellular form includes the carboxy group of a glutamic acid levels, we reveal a structural basis for mini-exon residue as the bidentate ligand. peptide-dependent trans-synaptic adhesion mediated by the type IIa RPTP complexes for inducing synaptic 1. Horch, M., Lauterbach, L., Lenz, O., Hildebrandt, P. & Zebger, I., FEBS Lett. 586, 545–56 (2012). differentiation. 2. Sazanov, L. A & Hinchliffe, P., Science 311, 1430–6 (2006) 3.Efremov, R. G. & Sazanov, L. A., Biochim. Biophys. Acta 1817, 1785–95 (2012). 1. Yamagata et al. (2015) Mechanisms of splicing-dependent trans-synaptic adhesion by PTPδ–IL1RAPL1/IL-1RAcP for synaptic differentiation. Nat. Commun., 6, 6926 2. Yamagata et al. (2015) Structure of Slitrk2–PTPδ complex reveals mechanisms for splicing-dependent trans-synaptic adhesion. Sci. Rep., 5, 9686

42 CS05 CS06

Molecular studies of RNA- “Motion capture” of proteins inside dysmetabolic syndrome – From living cells ribonucleoproteomics to structural Yutaka Ito1, Takanori Kigawa2 and Yuji Sugita2 life science - 1. Tokyo Metropolitan University Toshiaki Isobe1, Hiroshi Nakayama2, Masato Taoka1, 2. RIKEN Yoshio Yamauchi1, Yuko Nobe1, Toshinori Kozaki3, 3 3 3 Kazuo Ishii , Keiichi Izumikawa , Hideaki. Ishikawa In living cells, a variety of macromolecules exist in 3 and Nobuhiro Takahashi a very crowded environment, thus influencing the 1. Tokyo Metropolitan University kinetics and thermodynamics of protein folding and 2. RIKEN various binding events. We have reported the first 3. Tokyo University of Agriculture and Technology 3D protein structure calculated exclusively on the basis of information obtained in living E.coli cells This study aims to maximize the performance of [1-2]. We have also introduced a novel approach our state-of-the-art mass spectrometry (MS)-based for observing in-cell NMR spectra using the sf9/ “ribonucleoproteomics” technology and its application baculovirus system [3]. However, the low intracellular to the structure-function studies of ribonucleoprotein concentration of target proteins in eukaryotic cells complexes responsible for human refractory RNA- makes it diffi cult to perform detailed NMR analyses. dysmetabolic syndrome. To date, we have succeeded In our CREST project, by developing a new approach to increase the sensitivity of our RNA MS system integrating NMR and computational sciences, we to the extent that allows for the direct analysis of focus on investigating 3D structures, dynamics, and cellular microRNAs1 and developed the method interactions of proteins "at work" inside eukaryotic for comprehensive quantitative determination of cells, aiming at establishing a new "in situ" structural posttranscriptional RNA modification2. We also applied biology, which is supposed to be one of the major our method to the molecular studies of a number of fields in the novel structural life science. We will RNA-binding proteins responsible for human RNA- report our recent methodological developments for dysmetabolic syndrome such as ALS and SMN: We “motion capture” of proteins inside HeLa cells by found a novel metabolic pathway that ensures the analysing long-range distance/angle constraints from quality of U snRNAs important for pre-mRNA splicing NMR pseudocontact shifts. and isoform expression3. We expect that our study will provide an innovative tool for the global analysis of 1. Sakakibara et al. (2009) Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458: 102-105. cellular RNA-protein interaction networks and expand our 2. Ikeya et al. (2010) NMR protein structure determination in living E. coli cells using understanding of the pathogenesis of RNA-dysmetabolic nonlinear sampling. Nat. Protoc. 5:1051-1060. 3. Hamatsu et al. (2013) High-resolution heteronuclear multidimensional NMR of proteins syndrome for the early diagnosis and new drug discovery. in living insect cells using a baculovirus protein expression system J. Am. Chem. Soc. 135: 1688-1691.

1. Nakayama H et al, (2015) Direct identifi cation of human cellular microRNAs by nanofl ow liquid chromatography-high resolution mass spectrometry and database searching. Anal. Chem. 87, 2884-91. 2. Taoka M et al, (2015) A mass spectrometry-based method for comprehensive quantitative determination of posttranscriptional RNA modifications: the complete chemical structure of Shizosaccaromyses pombe ribosomal RNAs. Nucleic Acids Res. pii: gkv560. [Epub ahead of print] PMID: 26013808. 3. Ishikawa H et al, (2014) Identifi cation of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis. Nucleic Acids Res. 42, 2708-24.

JST CREST-PRESTO joint international symposium 43 CS07 CS08

NMR study of the interaction sites Maintenance for ER homeostasis on the ferredoxin isoforms for through disulfi de reductase, ERdj5 photosynthetic protein complexes Ryo Ushioda and Kazuhiro Nagata 1 1 2 Risa Mutoh , Yuko Misumi , Hisako Kubota-Kawai , Kyoto Sangyo University Ryutaro Tokutsu2, Takahisa Ikegami3, Michael Hippler4, Jun Minagawa2 and Genji Kurisu1 Secretory and membrane proteins are folded in the 1. Osaka University ER and only correctly folded proteins are secreted 2. National Institute for Basic Biology through the Golgi. Although the misfolded proteins 3. Yokohama City University are retained in the ER for the further attempt of re- 4. University of Münster folding by the aid of molecular chaperones, terminally In chloroplast, Ferredoxin (Fd) is reduced by misfolded proteins are discarded by a process known Photosystem I (PSI) and oxidized by Fd-NADP+ as ER-associated degradation (ERAD). We found and reductase (FNR). Green algae possess six Fd isoforms reported two novel ER-resident proteins involved in (Fd1 – Fd6), implying isoform-specific involvement this process; EDEM and disulfide reductase ERdj5, in the formation of NADPH and ATP, respectively. both of which facilitated ERAD in association with BiP However, the structural basis for the isoform-specifi c (Ushioda et al., Science 2008, Hagiwara et al., Mol. electron transfer and complex formation is still Cell 2011). elusive. In this study, we analyzed them using NMR Here we report the new role of ERdj5 as disulfide 2+ to determine the interaction sites on Fd1 and Fd2 reductase in ER homeostasis. ERdj5 regulates Ca with FNR and PSI for NADPH production, and those homeostasis in the ER through the redox activity. with cyclic electron fl ow (CEF) supercomplex for ATP Thus, we propose that protein homeostasis, redox homeostasis and calcium homeostasis in the ER have synthesis, composed of PSI, Cyt b6f, light-harvesting complexes, FNR and other membrane proteins. close cross-talks each other and ERdj5 has a key role Although application of NMR to a huge membrane in regulating these major homeostasis in the ER. protein complex is a challenge, 1H-15N HSQC spectra of [15N]-Fds with and without these target proteins have been successfully recorded. We will show that the interaction sites on Fd1 for single FNR, PSI and CEF supercomplex are at lease partly distinct from each other.

44 CS09 CS10

Structure and function of the A molecular ruler determines the autophagy initiation complex repeat length in eukaryotic cilia and Nobuo N. Noda1, Yuko Fujioka1, Hironori Suzuki1, fl agella. 2 3 3 Takeshi Kaizuka , Sho W. Suzuki , Hayashi Yamamoto , Toshiyuki Oda, Haruaki Yanagisawa, Ritsu Kamiya 2 3 Noboru Mizushima and Yoshinori Ohsumi and Masahide Kikkawa

1. Institute of Microbial Chemistry, Tokyo The University of Tokyo 2. The University of Tokyo 3. Tokyo Institute of Technology Existence of cellular structures with specific size raises a fundamental question in biology: How do Autophagy, an intracellular degradation system cells measure length? One conceptual answer to this conserved among most eukaryotes, is mediated by question is by a molecular ruler, but examples of such concerted actions of autophagy-related (Atg) proteins. rulers in eukaryotes are lacking. Under autophagy-inducing conditions, Atg proteins target to the site of autophagosome formation in a Here, we studied the FAP59/172 complex by using hierarchical manner, in which the autophagy initiation genetics and cryo-electron tomography (Cryo- complex functions as the most upstream factor (1). ET). The two proteins form a complex, and their The autophagy initiation complex consists of Atg1, absence disrupts 96-nm repeats in axonemes. Cryo- Atg13, Atg17, Atg29 and Atg31 in the case of budding ET revealed that the FAP59/172 complex takes a yeast. We determined the crystal structures of Atg1- 96-nm-long extended conformation along axonemal Atg13 and Atg13-Atg17-Atg29-Atg31 complexes and microtubules. Elongation of the complex resulted in established the architecture and phospho-regulation extension of the repeats and duplication of specific of the autophagy initiation complex (2). axonemal components. We conclude that the In the case of higher eukaryotes such as mammals, FAP59/172 complex is the molecular ruler that defi nes the autophagy initiation complex possesses Atg101 96-nm repeats in cilia/fl agella. instead of Atg29 and Atg31. We determined the crystal structure of the Atg101-Atg13 complex and revealed two important roles of Atg101 in mammalian autophagy initiation: one is stabilization of Atg13 and the other is recruiting other Atg proteins to the autophagosome formation site (3).

1. Noda & Inagaki. (2015) Mechanisms of autophagy. Annu Rev Biophys 44: 101-122. 2. Fujioka et al. (2014) Structural basis of starvation-induced assembly of the autophagy initiation complex. Nat Struct Mol Biol 21:513-521. 3. Suzuki et al. (2015) Structure of the Atg101-Atg13 complex reveals essential roles of Atg101 in autophagy initiation. Nat Struct Mol Biol 22: 572-580

JST CREST-PRESTO joint international symposium 45 CS11 CS12

Positive Regulator of OXPHOS Structural studies of the novel Yasunori Shintani and Seiji Takashima protein family working with cell membrane potential signal Osaka University Atsushi Nakagawa1, Hirotaka Narita1, Yasushi 1 1 1 OXPHOS is a mitochondrial inner membrane protein- Okamura , Akira Kawanabe , Yuichiro Fujiwara , 2 3 3 complex where the most endogenous ATP is Hidekazu Tsutsui , Hideki Kandori , Masayo Iwaki , Yu Takano4, Hiroko Kondo4, Yasushige Yonezawa5 and produced. Energy transfer efficiency of OXPHOS is Kengo Kinoshita6 very high, however, we recently discovered the novel positive regulators of OXPHOS activity1,2. HigD1 and 1. Osaka University 2. Japan Advanced Institute of Science and Technology G0s2 bind directly to the Complex IV and Complex V 3. Nagoya Institute of Technology of OXPHOS respectively and both increase the ATP 4. Hiroshima City University production rate. We revealed the structural changes 5. Kinki University of Complex IV by binding HigD1 analyzed by Raman 6. Tohoku University spectroscopy. The results indicate that HigD1 binding Electric signal is important in many living processes, alters the structure of Complex IV around heme a such as nerve signal and heart beat. Recently novel including H-proton pathway and Complex IV has a voltage-sensing proteins have been identified, and structural background which was activated by direct these molecules transduce electrical signals into binding of small ligand. These structural analysis intracellular signals in a different way from the might lead to the development of new therapeutic classical voltage-gated ion channels. Our project aims tools for various disease by upregulating the OXPHOS to reveal dynamic mechanism of signal transduction activity. by the new voltage-sensing proteins, such as a

1. Hayashi T et al, Higd1a is a positive regulator of cytochrome c oxidase. Proc Natl Acad voltage-gated proton channel (VSOP or Hv1) and a Sci U S A. 2015 Feb 3;112(5):1553-8.(2015) 2. Kioka H. et al, Evaluation of intramitochondrial ATP levels identifi es G0/G1 switch gene voltage-sensing phosphatase (VSP), based on their 2 as a positive regulator of oxidative phosphorylation. Proc Natl Acad Sci U S A. 111(1): atomic structure. These results will open a new 273-8. (2014) paradigm of life science and our results will give valuable information for strategic development of new tools for visualization of electrical signal and will be extended to the studies for medical application.

46 CS13 PS01

Mechanism of gravitropic signaling Cytoplasmic insights into the in gravity sensing cells ～ From protein-conducting channel implied molecular structure to plant organ by crystal structures of SecYEG response ～ Yoshiki Tanaka1, Yasunori Sugano1, Mizuki 2 3 1 Miyo Terao Morita1, Yoshinori Hirano2, Masatoshi Takemoto , Takaharu Mori , Arata Furukawa , Tsukasa 2 2 1 Taniguchi1, Takeshi Nishimura1, Megumi Kagiyama2, Kusakizako , Kaoru Kumazaki , Ayako Kashima , 2 3 2 Kohei Kawashima2 and Toshio Hakoshima2 Ryuichiro Ishitani , Yuji Sugita , Osamu Nureki and Tomoya Tsukazaki1 1. Nagoya University 2. Nara Institute of Science and Technology 1. Nara Institute of Science and Technology 2. The University of Tokyo 3. RIKEN In general, plant shoots grow upward (negative gravitropism), whereas roots grow downward Sec proteins drive protein translocation across the (positive gravitropism) so as to be in a suitable membrane, which is one of the essential processes. position for survival. The goal of our research is to The bacterial SecYEG translocon functions as an understand the molecular mechanisms by analyzing evolutionally conserved protein-conducting channel. structures and functions of proteins involved in the Conformational transitions of SecYEG allow protein gravity signaling.We have studied a plant-specific translocation without perturbation of membrane protein family DLLs (DGE1/LAZY1 like proteins) permeability. Previously we determined and discussed found by transcriptomic analyses focusing on gravity crystal structures of SecYE complex and SecDF (1)- sensing cells. We demonstrated that DLLs are key (2). Here, we report the two new crystal structures factors not only for the gravity signaling process but of SecYEG. One of the structures suggested that a also for growth angle control of lateral organs both in cytoplasmic loop covering the hourglass-shaped shoots and roots. Recently, we found that RLD (RCC1- channel may be involved in protein translocation. like domain containing proteins) 1-4, interacting In addition, another crystal structure implies that factors of DLLs, have function in root gravitropism. interactions in the cytoplasmic side of SecY is related In addition, rld multiple mutants showed auxin- to lateral gate opening at the first step of protein related phenotypes, suggesting that gene function translocation. These SecYEG structures provide a of RLDs is not limited to gravity signaling. Analyses number of structural insights into the Sec machinery for the crystal structure of a complex of DLLs-RLD for further studies. interacting domains are on going.

1. Tsukazaki et al. (2008) Conformational transition of Sec machinery inferred from bacterial SecYE structures. Nature 455: 988-991. 2. Tsukazaki et al. (2011) Structure and function of a membrane component SecDF that enhances protein export. Nature 474: 235-238.

JST CREST-PRESTO joint international symposium 47 PS02 PS03

CryoEM structure of muscle thin Structure-Based Design of Chemical fi lament with the tropomyosin and Probes for Imaging and Functional troponin complex Regulation of Proteins Takashi Fujii1 and Yurika Yamada2 Yuichiro Hori

1. JST PRESTO Osaka University 2. Osaka University Protein labeling with synthetic probes is a powerful Muscle contraction is driven by cyclic interactions method for verifying protein localization and function of myosin in the thick filament with thin filament in living cells. In this method, a protein tag is fused composed of actin, tropomyosin (Tm) and troponin to a protein of interest (POI) and is labeled with its 2+ (TnC, TnI, TnT). It is thought that the binding of Ca specifi c probe, which typically contains a fl uorophore. released from sarcoplasmic reticulum to TnC causes Its advantage is that POIs can be conditionally a conformational change of Tm on the actin fi lament visualized at particular time points, allowing precise to allow actin-myosin interaction. To understand this spatiotemporal analyses of proteins. In addition, POIs regulatory mechanism, it is indispensable to elucidate can be modified with various synthetic molecules the structure of thin filament at high resolution. We that give a new function to proteins. So far, we have 2+ established a method to purify intact, Ca -free and developed a protein labeling system based on a 2+ Ca -bound thin filaments from skeletal muscle of novel protein tag, PYP-tag, and its synthetic probes a crab, Portunus trituberculatus, at high yield. We including fluorogen and inhibitor molecules (1-3). developed a novel image analysis approach for 3D By using this system, various biological phenomena reconstruction of thin filament. A cryoEM density including protein trafficking, protein degradation, 2+ map of thin filament in the absence of Ca shows signal transduction, and epigenetic phenomena have interesting features of actin-Tm-Tn interactions never been investigated. In this symposium, the detailed seen before. strategy of molecular design and some biological applications of this method will be presented.

1. Y. Hori, H. Ueno, S. Mizukami, K. Kikuchi, J. Am. Chem. Soc., 2009, 131, 16610. 2. Y. Hori, K. Nakaki, M. Sato, S. Mizukami, K. Kikuchi, Angew. Chem. Int. Ed., 2012, 51, 5611. 3. Y. Hori, T. Norinobu, M. Sato, K. Arita, M. Shirakawa, K. Kikuchi, J. Am. Chem. Soc., 2013, 135, 12360.

48 PS04 PS05

Conformational dynamics of GPCRs ATP-free unidirectional walking of under lipid bilayer condition myosin V revealed by NMR Noriyuki Kodera, Takayuki Uchihashi, and Toshio Takumi Ueda Ando

The University of Tokyo Kanazawa University

G-protein-coupled receptors (GPCRs) exist Our previous high-speed AFM (HS-AFM) observations in conformational equilibrium between active of myosin V (M5) suggest that M5 does not require and inactive states, and the former population the ADP.Pi bound state to rebind to actin and that determines the efficacy of signaling (1). However, the powerstroke performed by the leading head the conformational equilibrium of GPCRs in lipid spontaneously occurs and does not require the Pi bilayers is unknown owing to the low sensitivities of release process, which is challenging the widely their NMR signals. To increase the signal intensities, accepted chemomechanical coupling model of a deuteration method was developed for GPCRs myosin motor. Here, we performed an experiment to expressed in an insect cell/baculovirus expression further evidence our suggestion, in which under the system. The NMR sensitivities of the methionine nucleotide-free conditions as well as in the presence of ADP we applied a controlled strong tapping- methyl resonances from the β2-adrenergic receptor force onto either head of a two-headed bound M5 (β2AR) in lipid bilayers of reconstituted high-density lipoprotein (rHDL) increased by approximately 5-fold to dissociate the head from actin using a newly upon deuteration (2). NMR analyses revealed that developed scanning mode of HS-AFM. When the the exchange rates for the conformational equilibrium trailing head is detached in this mechanical way, M5 almost always steps forwards by ~36 nm, whereas of β2 AR in rHDLs were remarkably different from those measured in detergents (2). The timescales of it moves neither backwards nor forwards when GPCR signaling, calculated from the exchange rates, the leading head is detached. This result strongly are faster than those of receptor tyrosine kinases and supports our suggestion and requires reconsideration thus enable rapid neurotransmission and sensory of the chemomechanical coupling model of myosin perception. motor.

1. Ando et al. (2001) A high-speed atomic force microscope for studying biological 1. Kofuku Y. et al. (2012) Efficacy of the β2-adrenergic receptor is determined by macromolecules. PNAS 98:12468-12472. conformational equilibrium in the transmembrane region. Nature Commun. 3: 1045 2. Kodera et al (2010) Video imaging of walking myosin V by high-speed atomic force 2. Kofuku Y. et al. (2014) Functional dynamics of deuterated β2-adrenergic receptor in microscopy. Nature 468: 72-76. lipid bilayers revealed by NMR spectroscopy. Angew. Chem. Int. Ed. 53: 13376-9 3. Kodera & Ando (2014) The path to visualization of walking myosin V by high-speed atomic force microscopy. Biophys. Rev. 6: 237-260.

JST CREST-PRESTO joint international symposium 49 PS06 PS07

Crystal structures of CRISPR-Cas9 Ultrastructural analysis of infl uenza Hiroshi Nishimasu virus genome transcription

The University of Tokyo Takeshi Noda Kyoto University The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer Influenza A virus genome consists of eight- adjacent motif (PAM) and complementarity to segmented, negative-sense, single-stranded RNAs the guide RNA. Recently, Staphylococcus aureus (vRNAs). Each vRNA is associated with multiple Cas9 (SaCas9), which is significantly smaller copies of viral nucleoprotein (NP) and a viral RNA than Streptococcus pyogenes Cas9 (SpCas9) (1, polymerase complex (PB1, PB2, and PA), together 2), was harnessed for in vivo genome editing. forming a helical ribonucleoprotein complex (RNP). Here, we present the crystal structures of SaCas9 The RNP functions as a molecular machine for in complex with a guide RNA and its double- transcription and replication of virus genome. , stranded DNA target containing the 5 -TTGAAT- Although the processes involved in vRNA , , , 3 PAM and the 5 -TTGGGT-3 PAM at 2.6 and 2.7 transcription are largely revealed by biochemical Å resolutions, respectively (3). The structures and molecular biological studies, no ultrastructural revealed the mechanism of the relaxed recognition information on the RNPs during transcription exists. , , of the 5 -NNGRRT-3 PAM by SaCas9. A structural It remains unclear how the RNP changes in terms comparison of SaCas9 with SpCas9 highlighted both of its helical structure during mRNA synthesis as structural conservation and divergence, explaining well as how mRNA is produced from the RNP. To their distinct PAM specifi cities and orthologous guide understand the mechanism of infl uenza virus genome RNA recognition. These structural information about transcription from an ultrastructural point of view, this minimal Cas9 will further expand the CRISPR- in vitro-transcribed RNPs were investigated on an Cas9 genome editing toolbox. ultrastructural level by atomic force microscopy and cryo-electron microscopy. This study discusses 1. Nishimasu et al. (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935-949. the possible mechanism of influenza virus genome 2. Anders et al. (2014) Structural basis of PAM-dependent target DNA recognition by the transcription. Cas9 endonuclease. Nature 513:569-573. 3. Nishimasu et al. (2015) Crystal structure of Staphylococcus aureus Cas9. Cell, in press.

50 PS08 PS09

Correlative studies on autophagy Structural basis for higher order proteins using 3D electron structure formation of UHRF1 microscopy with precise spatial Kyohei Arita information Yokohama City University Maho Hamasaki

Osaka University DNA methylation and histone posttranslational modifications are major epigenetic traits, which are Autophagy is a tightly regulated intracellular inherited through mitosis. Ubiquitin-like containing degradation system that plays fundamental roles PHD and RING finger domains, 1 (UHRF1), an in cellular homeostasis. Autophagy is induced by essential factor for inheritance of DNA methylation starvation, bacterial infection, accumulation of pattern in somatic cells, contains histone reader aggregate proteins, organelle damages and so on. modules and hemi-methylated DNA binding domain. Autophagosomes are quite unique among organelles, While the binding property of each domain to their as they are not preexisting in the cytosol and only substrates is well studied, mechanism by which form when they are in need. The site of this organelle UHRF1 cooperative recognize the epigenetic traits formation was determined to be at ER-mitochondria remains elusive. Here we show the structural basis contact sites1. To see more precise relations among for cooperative recognition of epigenetic marks by ER, mitochondria and autophagy, proteins involved UHRF1. The reader modules folded into compact in the early step of autophagosome formation were structure without the substrates, which inhibits correlated and observed with light and electron binding of the reader modules to their substrates. microscopy (CLEM) combined with 3D electron Synergic binding of the epigenetic marks by the tomography. This technique could also be applied on reader modules leads to structural alteration of other protein involved in autophagy to link between the domains from closed to open. We also show the membrane dynamism and protein localization to that the cooperative recognition of the epigenetic solve the function of protein. marks by UHRF1 is required for chromatin loading of a maintenance DNA methyltransferase (DNMT1) 1. Hamasaki M, Furuta N et al. (2013) Nature, 495(7441), 389-93. and ubiquitylation of histone H3. Thus, cooperative binding of epigenetic marks by UHRF1 is essential for maintenance of DNA methylation.

JST CREST-PRESTO joint international symposium 51 PS10 PS11

Structural and Biochemical analysis X-ray Crystal Structure of Voltage- of Vesicular glutamate transporter Gated Proton Channel, VSOP Narinobu Juge Kohei Takeshita

Okayama University Osaka University

Vesicular glutamate transporters (VGLUTs) are VSOP consists of four transmembrane helices as responsible for the vesicular storage of L-glutamate a voltage-sensor domain (VSD). VSOP belongs to and play an essential role in glutamatergic signal superfamily of voltage-gated ion channels but VSOP transmission in the central nervous system. We have lacks an authentic pore domain. In most popular already developed a procedure to assess the activity biological function, VSOP involves a high level of VGLUT with purified and reconstitution system. production of the superoxide in phagocytes through We have already found following three things that (1) its tight functional coupling with NADPH oxidase Essential three amino acids residues are responsible to eliminate pathogens, and also the activities of for VGLUT2 activity. (2) VGLUT has two different VSOP relates several pathological or physiological transport mechanisms. (3) VGLUT activity is regulated implications such as exacerbation of ischemic brain by Chloride ion. However, molecular mechanism of damage, progression of cancer or human sperm this transporter is still unknown because the structural motility. In this study, we determined the crystal and biochemical analysis is still missing ling. On this structure of mouse VSOP chimeric channel. This meeting, I would like to talk about the recent fi ndings structure showed the first resting state structure in of this protein. reported VSD structures by binding zinc ion, which reported to inhibit VSOP activation, to extracellular Juge et al. (2010) metabolic control of vesicular glutamate transport and release. Neuron, 68: 99-112 region of VSOP. Interestingly, this structure showed unique features that is double hydrophobic layers in the protomer. These layers may prevent the penetration of water molecules as a proton transfer carrier. These layers may play distinct roles in proton pathway.

52 PS12

Dissection of the circadian clock machinery by integrating chemical biology and structural biology Tsuyoshi Hirota

Nagoya University

The circadian clock is an intrinsic time-keeping mechanism that coordinates the daily rhythms of physiology and behavior. Circadian rhythms are generated in a cell-autonomous manner through transcriptional regulatory networks of the clock genes. To search for novel clock modifiers, we have developed a high-throughput circadian assay in human cells and conducted chemical screens. From hundreds of thousands of small molecules, we identified two compounds named longdaysin and LH846 that potently lengthen the period of the circadian clock through inhibition of casein kinase I family proteins (1, 2). We further discovered a new class of period lengthening compound named KL001 that specifi cally interacts with the core clock protein CRY to inhibit its degradation (3). We are characterizing other clock- modifying compounds to reveal their molecular targets. Structural analysis of the complex between these compounds and target proteins will provide unique opportunities to understand and control the circadian clock system.

1. Hirota et al. (2010) High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase. PLoS Biol. 8: e1000559. 2. Lee, Hirota et al. (2011) A small molecule modulates circadian rhythms through phosphorylation of the period protein. Angew. Chem. Int. Ed. 50: 10608-10611. 3. Hirota et al. (2012) Identifi cation of small molecule activators of cryptochrome. Science 337: 1094-1097.

JST CREST-PRESTO joint international symposium 53