20-21 MAY 2017 | SINGAPOREJOINTSYMPOSIUM ON
BIOIMAGINGBETWEEN
SINGAPORE
AND JAPAN

MAY 20-21, 2017

Venue:NATIONAL UNIVERSITY OF SINGAPORE

Organizers
Paul MATSUDAIRA, National University of Singapore
Kazuo SUZUKI, Bio-imaging Society of Japan
Bioimaging Society, Japan,
Cooperation in Singapore: WABIOSDear All,
Welcome to Singapore! It is our pleasure to host the Joint Symposium on BioImaging,the 5th Annual Meeting of the Bioimaging Society of Japan, and the Women in Science
Symposium. The MechanoBiology Institute and the Centre for BioImaging Scienceswere established on the NUS campus only eight years ago but in this short period bothresearch centers have developed novel light and electron microscopy methods andapplied them to problems in structural biology, cell and developmental biology, andbiophysics. The opportunity to present our research to our colleagues from Japan will
open new collaborations.

Many thanks go to Prof. Thorsten Wohland, (DBS/CBIS), Prof. Linda Kenney (MBI),
Ms Latha.K.S (MBI), and Ms Nurzanna (CBIS) for their hard work in planning andorganizing this symposium. Finally, I would like to express my sincere gratitude to
JEOL for sponsoring the conference dinner. Please take time to enjoy our wonderful
campus and city before returning to Japan.

Sincerely,
Paul Matsudaira

Joint Symposium on Bioimaging between Singapore & Japan
National University of Singapore
May 20-21, Singapore
Saturday, May 20, 2017
Venue: University Hall Auditorium, 2nd Storey, Lee Kong Chian Wing
Time0830-0900am

0900-0910am

Speaker
Registration
University Hall Auditorium
Opening remarks
Paul MATSUDAIRA
Centre for Bioimaging Sciences & Department of Biological Sciences, National Universityof Singapore

Symposium 1
Min WU
0910-0935am

National University of Singapore

Information Content of Intracellular Patterns
Lu GAN
0935-1000am

Centre for Bioimaging Sciences & Department of Biological Sciences, National Universityof Singapore

Exploration of Eukaryotic Nuclei at Molecular Resolution
Pakorn (Tony) KANCHANAWONG
1000-1025am

Department of Biomedical Engineering & Mechanobiology Institute, National University ofSingapore

Nanoscale Architecture of Cadherin-based Cell Adhesions
Yusuke TOYAMA
1025-1050am

Mechanobiology Institute, Department of Biological Sciences, National University ofSingapore & Temasek Lifesciences Laboratory, Singapore

Tension-Dependent Vinculin Dynamics at Adherens Junction During Cell
Boundary Oscillation
1050-1120am

Coffee BreakPlenary Lecture 1
Shin’ichi ISHIWATA

1120-1220am

Department of Physics, Faculty of Science &Engineering, Waseda University, Tokyo,Japan

Bio-motile Functions Studied by Microscopic Imaging of PhysicalParameters
1220-0210pm

Lunch at University Hall, CBIS visit (starting at 13.10)

WOMEN IN SCIENCE SYMPOSIUM
Organizers: Linda J Kenney & Naoko Iida -Tanaka, Kahoko Hashimoto
Opening Address: Organizer, Japan
0210-0215pm
Naoko Iida-TANAKA, Otsuma Women’s University, Japan
Kahoko HASHIMOTO, Chiba Institute of Technology, Japan
Reina IKAGA
0215-0230pm
Department of Nutritional Science, National Institute of Health and Nutrition, JapanThe Role of Aquaporin-8 on Mitochondria
Tomoko MASAIKE
0230-0255pm

Department of Applied Biological Science, Faculty of Science & Technology, Tokyo
University of Science, Japan

Imaging Movements and Reactions of Biomolecules

Linda J KENNEY
0255-0320pm

0320-0345pm

0345-0350pm

Mechanobiology Institute, National University of Singapore

Super-resolution Imaging of Salmonella Virulence Regulation and
Secretion
Carol TANG
National Neuroscience Institute, Duke-NUS Medical School, National Cancer Centre,
Singapore

Targeting Brain Tumors: Improving Lives through Precision Medicine
Closing Remarks: Organizer, Singapore
Linda J KENNEY
Mechnobiology Institute, National University of Singapore

0350-0400pm

Presentation by Sponsor - JEOL Ltd.

0400-0600pm

Coffee break & Poster presentations

0630-0830pm

Dinner at Kent Ridge Guild House

Sunday, May 21, 2017
Venue: Mechanobiology Institute, Level 5, T-Lab, NUS
Time

Speaker

Symposium 2: Organizer: Takeharu NAGAI
Hiroki R UEDA
0900-0925am

Systems Pharmacology, Graduate School of Medicine, University of Tokyo, Laboratory ofSynthetic Biology, Quantitative Biology Centre, RIKEN, Japan

Whole-body and Whole-organ Clearing and Imaging with Single-cell
Resolution Toward Organism-Level Systems Biology in Mammals
Satoshi NISHIMURA
0925-0950am

0950-1015am

Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan

Minor Changes but Major Future Impacts, can be Revealed by Extensive
and High-Through-Put In Vivo Visualization Using 4K/8K CMOS Sensors
Katsumasa FUJITA
Department of Applied Physics, Osaka University, Japan

Raman Microscopy for Molecular Imaging of Living Cells
Takeharu NAGAI
1015-1040am
1040-1110am
1110-1210am

The Institute of Scientific and Industrial Research, Osaka University, Japan

Development of Techniques for Imaging Physiological Functions Toward
Visualization of Minority Cells
Coffee BreakPlenary Lecture 2
Michael SHEETZ
Mechanobiology Institute, National University of Singapore

Integrin Clusters: A Robust Way to Organize Adhesions for Cell Mechanics

1210-1220pm

Closing Remarks
Kazuo SUZUKI
Asia International Institute of Infectious Diseases Control, Teikyo University
Department of Immunology, National Institute of Infectious Diseases, Japan

1220-0200pm

Lunch at MBI, Level 10; MBI visit starting at 0100 pm

Information Content of Intracellular Patterns
Wu MIN
National University of Singapore
Periodic wave patterns are widely observed in oscillatory or excitable chemical systems and in multicellular
systems such as cardiac tissue and slime moulds. More recently, waves of cortical activity, linked to actin
dynamics in many cases, have been documented in a variety of single-cell systems, including various immune
cell types (1, 2). The appearance of order in these patterns raises the intriguing possibility that molecular
networks favouring their generation are evolved due to specific functional advantages they confer. One
particular appealing hypothesis is that wave propagation in space generates location-dependent phase
information that could be harnessed to encode spatial information. However, little is known about whether
or how they are employed by biological systems, especially higher eukaryotes. We will discuss our recent
results characterizing cortical waves of active Cdc42, which suggest that mitotic cortical waves might
potentially integrate positional and cell size information in modulating final sites of cell division.
1. Wu M, Wu X, De Camilli P. Calcium oscillations-coupled conversion of actin travelling waves to standing
oscillations. Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):1339-44.
2. Xiong D, Xiao S, Guo S, Lin Q, Nakatsu F, Wu M. Frequency and amplitude control of cortical oscillations byphosphoinositide waves. Nat Chem Biol. 2016 Mar;12(3):159-66.

Exploration of Eukaryotic Nuclei At Molecular Resolution
Lu GAN
Centre for BioImaging Sciences and Department of Biological Sciences, National University ofSingapore
The nucleus is the information warehouse of the eukaryotic cell. Essential functions like transcription,
replication, and chromosome segregation happen in the nucleus. Many fundamental questions remainunanswered because we lack a molecular understanding of the nucleus. To address this shortfall, my grouphas used electron cryotomography (cryo-ET) to obtain 3-D nanometer-level atlases of plankton and yeast
nuclei. We found that the chromosomes in vivo do not form highly ordered 30-nm fibers and that they do notcondense very much either. In contrast, we found that the chromatin readily forms compact 30-nm fibers
when released from the nucleus. These observations suggest that while one of the lowest energy states ofnatural chromatin is the 30-nm fiber, the crowded and metabolically active interior of nuclei keeps chromatin
in a more liquid-like state. Such disordered chromatin would enable the high levels of transcription detectedin yeast and might explain how other eukaryotes remodel their chromatin in response to transcriptional
upregulation.

Nanoscale Architecture of Cadherin-based Cell Adhesions
Pakorn (Tony) KANCHANAWONG
Department of Biomedical Engineering and Mechanobiology InstituteNational University of Singapore
Cadherin-mediated cell adhesions are supramolecular complexes that play essential roles in ligating andmechanically integrating neighboring cells, supporting dynamic coupling between cell-cell adhesions and thecontractile actin cytoskeletons. Despite well-documented functions in major aspects of tissue morphogenesis
and multicellularity, the ultrastructural organization within cadherin-based adhesions remains unknown, thus
obscuring insights into the underlying molecular mechanisms. We mapped the nanoscale organization of key
cell-cell junction proteins within cadherin-based adhesions formed on planarized biomimetic cadherin
substrate. The enhanced optical accessibility of the planar substrate together with interference-based
nanoscopy methods enabled high precision (~10-nm) axial (z) position measurement using common
fluorescent proteins. We observed a surprisingly well-organized molecular architecture that stratified along
the z-axis, with the cadherin-catenin layer and the actin compartment separated by ~30 nm, interposed by avinculin-containing interface zone. Our results indicated that vinculin can undergo a conformational
activation to span between the cadherin-catenin layer and the actin compartment. The nanoscale positioningof vinculin is determined by alpha-catenin, while vinculin conformational state is controlled by contractility
and Abl kinase phosphorylation on the residue Y822 of vinculin. Vinculin activation, in turn, modulates the
positioning of VASP and zyxin, inducing VASP-mediated actin polymerization, that likely results in apositive feedback loop that regulates junction strengthening. In conclusion, our measurements reveal a
modular nanoscale architecture of cadherin-based adhesions, suggesting a control principle whereby vinculin
serves as a molecular clutch that integrates mechanical and biochemical signals to differentially engage thecadherin-catenin complexes to the actomyosin contraction machinery under different contexts such as
developmental processes or diseases states.

Tension-Dependent Vinculin Dynamics At Adherens Junction During Cell Boundary
Oscillation
Yusuke HARA1, Murat SHAGIROV1, Issac LIM1, Yusuke TOYAMA1,2,3
1) Mechanobiology Institute, Singapore, 2) Department of Biological Sciences, National University ofSingapore, 3) Temasek Lifesciences Laboratory, Singapore

Throughout development, cell boundaries undergo contraction and elongation to change tissue morphology.
In comparison to cell boundary contraction, which is mainly driven by Myosin II dependent contraction, themechanisms of cell boundary elongation are still elusive. We used amnioserosa cells, which exhibit cell
oscillation during Drosophila dorsal closure, as a model system to uncover the mechanics underling cellboundary elongation, and to elucidate how the junctional tension evolves during cell oscillation. Here weshow that the cell boundary elongation is predominantly driven by non-autonomous active process, which is
associated with the transient flow of Myosin II in the neighboring cells pulling the vertices. Moreover,measurements of the junctional tensions at the specific phases of boundary oscillation by laser ablation,
showed that the elongating boundaries exhibited lower tension in compare to the contracting boundaries. We
extended our individual tension measurements to non-invasively estimate a tension map across the tissue.
We further found that the vinculin, which is known to interact with adherens junction component a-catenin
in tension dependent manner, accumulates to or dissociates from oscillating boundary, and the dynamics
correlated with the estimated junctional tension. We propose that the level of vinculin at the cell boundary
could be used to approximate junctional tension

Bio-Motile Functions
Studied by Microscopic Imaging of Physical Parameters
Shin’ichi ISHIWATA
Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, JapanThe functions of bio-motile systems depend on physical parameters such as force and temperature. Here I
will present the optical-microscopic imaging showing the dynamic properties of bio-motile systems (motor
proteins such as myosin and kinesin, cytoskeletons such as actin filaments and microtubules, a meiotic
spindle, muscle and nerve cells etc.), where the effects of mechanical perturbation and heat pulse are focused.
First, I will introduce single-molecular studies: that is, unbinding force of myosin II, V and VI with an actin
filament [1,2], that of kinesin with a microtubule [3,4], the polymerization dynamics of individual actin
filaments [5], distortion of protomers in an actin filament by external force [6], supercoiling and rotational
movement of sliding actin filaments [7,8], helical and robust processive movement of myosin V [9,10], andthe force-generation of a microtubule depolymerizer named MCAK [11]. Next, I will report the viscoelastic
properties of spindles self-organized in a Xenopus egg extract [12,13], and how the external mechanicalimpulse regulates the size and shape of spindles [11,14], and the timing of chromosome segregation within
a HeLa cell at metaphase [15]. Then, I will introduce how environmental temperature and local heat pulse
regulate the protein and cellular functions [16,17] such as the activities of molecular motors in vitro [18] andin a cell [19], the contractile properties of striated muscle [20-22], Ca-burst in a HeLa cell [23-26] and anerve cell [27]. Finally, I will introduce spontaneous formation of a ring-shaped contractile actin bundle [28]and rotational cytoplasmic flow [29] within an artificial cell system composed of protein solutions in a cellsized water-in-oil droplet surrounded by a monolayer phospholipid membrane.
References: 1) Nishizaka, T. et al. (1995) Nature 377, 251-4. 2) Oguchi, Y. et al. (2008) PNAS 105, 77149. 3) Kawaguchi, K. & Ishiwata, S. (2001) Science 291, 667-9. 4) Uemura, S. & Ishiwata, S. (2003) Nat.
Struct. Biol. 4, 308-11. 5) Fujiwara, I. et al. (2002) Nat. Cell Biol. 4, 666-73. 6) Shimozawa, T. & Ishiwata,
S. (2009) Biophys. J. 96, 1036-44. 7) Nishizaka, T. et al. (1993) Nature 361, 269-71. 8) Sase, I. et al. (1997).
PNAS 94, 5646-50. 9) Ali, M.Y. et al. (2002) Nat. Struct. Biol. 9, 464-7. 10) Oguchi, Y. et al. (2010) Nat.
Chem. Biol. 6, 300-5. 11) Oguchi, Y. et al. (2011) Nat. Cell Biol. 13, 846-53. 12) Itabashi, T. et al. (2009)
Nat. Meth. 6, 167-72. 13) Shimamoto, Y. et al. (2011) Cell 145, 1062-74. 14) Takagi, J. et al. (2013) Cell
Rep. 5, 44-50. & (2014) Biophys. J. 106, 735-40. 15) Itabashi, T. et al. (2012) PNAS 109, 7320-5. 16) Takei,
Y. et al. (2013) ACS Nano 8, 198-206. 17) Suzuki, M. et al. (2015) Nat. Meth. 10, 802-3. 18) Kato, H. et al.
(1999) PNAS 96, 9602-6. 19) Oyama, K. et al. (2012) Lab Chip 12, 1591-3. 20) Oyama, K. et al. (2012)
BBRC. 417, 607-12. 21) Shintani, S.A. et al. (2015) BBRC. 457, 165-70. 22) Kobirumaki-Shimozawa, F. etal. (2016) J. Gen. Physiol. 147, 53-62. 23) Suzuki, M. et al. (2007) Biophys. J. (Lett.) 92, L46-L48. 24)
Tseeb, V. et al. (2009) HFSP J. 3, 117-23. 25) Oyama, K. et al. (2015) Biophys. J. 109, 355-64. 26) Itoh, H.
et al. (2016) Chem. Comm. 52, 4458-61. 27) Oyama, K. et al. (2015) Sci. Rep. 5, 16611. 28) Miyazaki, M.
et al. (2015) Nat. Cell Biol. 17, 480-9. 29) Suzuki, K. et al. (2017) PNAS 114, 2922-7.

The Role of Aquaporin-8 on Mitochondria
Reina IKAGA1,2
1)Department of Nutritional Science, National Institutes of Biomedical Innovation, Health and Nutrition,2)Department of Food Science, Otsuma Women’s University
Aquaporins (AQPs) are a family of membrane transport proteinswhich play a role in water transport acrossbiological membranes.At least thirteen AQPs (AQP0-12) have been identified in mammals.Most ofthem are
localized in the plasma membrane and are involved in water transport into and/or out of cells. AQP8 is also
expressed in various tissue and cells, including liver, testis, and pancreas.It is interesting that AQP8 appears
to have functions on theplasma membrane and/or on the mitochondrial inner membrane. The major functionof mitochondria is the production of ATP by oxidative phosphorylation. Through this process, a large amountof water is also formed. However, the mechanisms of mitochondrial water handling remain largely unknown.
We found that, in mouse adipose tissues and 3T3-L1 preadipocytes, the mitochondrial type AQP8 isdominantly expressed. We therefore created an AQP8 knock-down cell line in which we have investigated
the role of AQP8 onmitochondrial function.The rate of oxygen consumption was decreased and the cellular
ATP level wassignificantly reduced in the AQP8-knockdowncells.In the AQP8-knockdown cells, marked
swelling of the mitochondria was observed by electron microscopy(Figure). These observations lead us tothe hypothesis that the reduced AQP8 expression disrupts normal mitochondrial water flux, and lowers
electron transport activity and ATP synthesis through inhibition of the water-generating processes. We also
found that knockdown of AQP8 had multiple effects on the cellular metabolism including reduction of fat
accumulation and changes in glycolysis and lipolysis. Thus, mitochondrial AQP8 functionally supports
mitochondrial respiration probably via water excretionand cellular metabolism through maintenance of
mitochondrial function.

Figure.
Electronmicrographsofmitochondria.Typicaltransmissionelectronmicroscopyimagesofmitochondriainacontrolcell(A)an
dinanAQP8-knockdown cell (B).Scalebaris 200nm.

Imaging Movements and Reactions of Biomolecules
Tomoko MASAIKE
Department of Applied Biological Science,
Faculty of Science and Technology, Tokyo University of Science
The aim of our bio imaging is to clarify how unitary steps of motions and chemical reactions lead to biological
functions.
Our main target is F1-ATPase, the world’s smallest rotary molecular motor. Its rotational behaviors havebeen studied in detail by the single-molecule observation method. Nevertheless, how chemical steps in ATPhydrolysis reaction drive rotation of the central shaft still remains unclear. Our aim for this target is todescribe the relationship between ATP hydrolysis reaction and rotational motion by conformational mappingof the catalytic subunit. We monitored domain motions in several local regions of the catalytic subunit bydetecting orientation of single-molecule fluorescent probes under TIRF microscopy, while simultaneously
observing rotation of the bead attached to central shaft by dark-field microscopy.
We recently detected changes in orientation of a fluorophore attached to helices that are close to the catalytic
site of the ATPase reaction. In addition, manipulation of the central shaft through magnetic beads undermagnetic field seems to induce conformational changes of the catalytic subunit. These results suggest how
rotation of the central shaft enhances conformational changes of the catalytic subunit. Taken together, theysuggest concerted intra-subunit conformational changes of the secondary structures in the catalytic subunit.
We try to discuss how conformational changes of each domain are relayed to the C-terminal helix in order toassist rotation of the central shaft.
Further studies include trials for visualization of chemical reactions catalyzed by single molecules. There has
been studies that utilized fluorescent analogs of substrate ATP for observation of binding and release fromsingle F1-ATPase molecules. Nevertheless, these analogs could not report timing of Pi release fromtheenzyme. Although we have not succeeded in detection of single inorganic phosphate (Pi) molecules sofar, we are developing a system to detect Pi released from a single-molecule F1-ATPase.
Finally, our target is expanding to higher levels of biological hierarchy such as organelles. They require othertechniques of microscopy such as 3-D tracking microscopy and subsequent interpretation of multiple
molecules that work cooperatively. Therefore, future studies should be directed at single-molecule studies
within an assembly of molecules.
We hope that our observation studies will open novel strategies of bio imaging and contribute tounderstanding of biological systems.

Super-resolution Imaging of Salmonella Virulence Regulation and Secretion

Yunfeng GAO1, Christopher SPAHN2, Yong Hwee FOO1, Andrew LIEW1,
Mike HEILEMANN2, and Linda J. KENNEY1,3,4
1)Mechanobiology Institute, National University of Singapore, Singapore; 2)Goethe University, Frankfurt,
Germany; 3)Jesse Brown VAMC; 4)University of Illinois-Chicago, Chicago, IL
In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of asensor kinase and a response regulator. In Salmonella, the EnvZ/OmpR system responds to osmotic stressand also positively regulates the Salmonella Pathogenicity Island 2 (SPI-2)-encoded type III secretion system
required for its survival in the macrophage vacuole. EnvZ/OmpR acidifies the Salmonella cytoplasm in
response to the acidified vacuole, driving virulence factor secretion into the host cytosol.
Acidification is required for the secretion of virulence factors, blocking acidification results in a neutralized
cytoplasm that is defective for SPI-2 secretion. Using super-resolution microscopy, we visualized theemergence of Salmonella-secreted effectors into the host cytoplasm and followed the resulting endosomal
tubulation. Our results suggest a mechanical role in needle elongation of the type three secretory apparatus
and subsequent gating of effector secretion. Comparison of our laboratory strain with a recently emerging
invasive strain of S. Typhimurium provides evidence for its invasive properties. Our work challenges existing
views that bacteria regulate their pH to maintain neutrality, and provides a new model for Salmonella
virulence factor secretion and infection. Supported by the Research Center of Excellence in Mechanobiology
from the Ministry of Education, Singapore, VA 5I01BX000372 and NIH AI123640 to LJK.

Targeting Brain Tumors: Improving Lives through Precision Medicine
Carol TANG1,2,3, Christopher Beng Ti ANG1,2,4,5
1) National Neuroscience Institute, 2) Duke-NUS Medical School, 3)National Cancer Centre, 4)Singapore
Institute for Clinical Sciences, A*STAR, 5)Department of Physiology, NUS
In mid-2016, the National Neuroscience Institute was awarded the NMRC Translational and ClinicalResearch (TCR) Flagship Program grant for neuro-oncology. This 5-year endeavour seeks to addresssignificant questions of unmet medical need, particularly in the most devastating form of adult malignant
gliomas. The multi-disciplinary team comprises scientific and clinical partners from several institutes andindustry.
The theme of Patient Stratification towards Precision Medicine serves to define specific biomarkers which
can stratify patients with malignant glioma into therapeutic classes so that specific chemotherapeutics can beadministered. The theme of Deciphering Molecular Heterogeneity focuses on elucidating the molecularprofile of grade IV glioblastoma multiform (GBM) at the time of recurrence and progression. This is thestage of disease where at present, management protocols are most arbitrary and require refinement. The
knowledge obtained by creation of a genomic database for recurrent GBM will be both novel and of great
clinical significance. The third theme of Novel Imaging Technologies and Predictive Biomarker Profiles aims
to develop new platforms such as imaging probes which are not only specific to malignant glioma but arealso closely correlated with the molecular stratification of the tumor1,2. In addition, we also describe thedevelopment of a “fluid biopsy” exosome-based microfluidic biomarker platform which allows for clinicalmonitoring of the patient at diagnosis and along the course of the disease3-5. In this theme, we also address
the issue of optimization of surgical resection of malignant gliomas. In this disease, the interface between
tumor tissue and normal brain is indistinct, making aggressive resection hazardous. As such, we describe theclinical development and trial of a Raman spectroscopic probe fashioned for use in neurosurgery that has inaddition, the capability to distinguish molecular subtype profiles. We capitalize on the fact that our clinicalcenter has an intraoperative MR imaging facility which allows the surgeon to ascertain extent of surgicalresection during surgery. This modality will be integrated into the clinical effort to determine the utility of
the Raman probe in maximizing tumor resection. In summary, this TCR proposal addresses the key
considerations in the management of a patient with malignant glioma from time of diagnosis onwards. It is
composed of efforts which will refine diagnosis, classification of disease, surgical resection technique andselection of chemotherapeutic protocol.
References

1 Atlas, T. C. G. Comprehensive genomic characterization defines human glioblastoma genes and core pathways.
Nature 455, 1061-1068, (2008).
2 Verhaak, R. G., Hoadley, K. A., Purdom, E., Wang, V., Qi, Y., Wilkerson, M. D., Miller, C. R., Ding, L., Golub,T., Mesirov, J. P., Alexe, G., Lawrence, M., O'Kelly, M., Tamayo, P., Weir, B. A., Gabriel, S., Winckler, W.,
Gupta, S., Jakkula, L., Feiler, H. S., Hodgson, J. G., James, C. D., Sarkaria, J. N., Brennan, C., Kahn, A., Spellman,
P. T., Wilson, R. K., Speed, T. P., Gray, J. W., Meyerson, M., Getz, G., Perou, C. M. & Hayes, D. N. Integrated
genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in
PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98-110, (2010).
3 Im, H., Shao, H., Park, Y. I., Peterson, V. M., Castro, C. M., Weissleder, R. & Lee, H. Label-free detection andmolecular profiling of exosomes with a nano-plasmonic sensor. Nat Biotechnol 32, 490-495, (2014).
4 Shao, H., Chung, J., Balaj, L., Charest, A., Bigner, D. D., Carter, B. S., Hochberg, F. H., Breakefield, X. O.,
Weissleder, R. & Lee, H. Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma
therapy. Nat Med 18, 1835-1840, (2012).
5 Shao, H., Chung, J., Lee, K., Balaj, L., Min, C., Carter, B. S., Hochberg, F. H., Breakefield, X. O., Lee, H. &Weissleder, R. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat Commun
6, 6999, (2015).

Whole-body and Whole-organ Clearing and Imaging with Single-cell Resolution
Toward Organism-Level Systems Biology in Mammals
Hiroki R. Ueda
Systems Pharmacology, Graduate School of Medicine, University of Tokyo
Laboratory for Synthetic Biology, Quantitative Biology Centre, RIKEN
Organism-level systems biology aims to identify and analyse cellular circuits in organisms. To this end, oneof the most powerful methods is optical imaging in combination with fluorescent labelling. However, thelong-standing obstacle has been tissue opacity. Recently, the solutions to this problem have started to emerge
by whole-body/organ clearing techniques that employ new tissue-clearing chemistry. In this talk, I introduce
these advancements and discuss how to combine new clearing techniques with efficient production of
genome-engineered animals, rapid volume imaging and efficient image informatics in order to obtain
quantitative organ-wide single-cell-resolution data. These technologies start to bring us closer to systemlevel understanding of physiology and diseases of complex mammalian systems.
References
1. Ueda, H.R. et al, Nature 418, 534-539 (2002).
2. Ueda, H.R. et al, Nat. Genet. 37, 187-92 (2005).
3. Sato T. K. et al, Nat Genet. 38, 312-9 (2006).
4. Ukai H. et al, Nat Cell Biol. 9, 1327-34 (2007).
5. Ukai-Tadenuma M. et al, Nat Cell Biol. 10, 1154-63 (2008).
6. Minami Y. et al PNAS 106, 9890-5 (2009).
7. Isojima Y. et al, PNAS 106, 15744-49 (2009).
8. Masumoto KH. et al, Curr Biol.20(24):2199-206.(2010).
9. Ukai-Tadenuma M et al. Cell 144(2):268-81 (2011).
10. Hogenesch JB, Ueda HR. Nature Rev. Genet. 12(6):407-16 (2011).
11. Jolley Cc, Ode KL, Ueda H.R. Cell Reports 2(4):938-50 (2012).
12. Sasagawa et al. Genome Biol. 14(4):R31 (2013).
13. Susaki et al. Cell, 157(3): 726–39, (2014).
14. Tainaka et al. Cell, 159(6):911-24(2014).
15. Susaki et al. Nature Protocols, 10(11):1709-27(2015).
16. Sunagawa et al, Cell Reports, 14(3):662-77 (2016).
17. Susaki and Ueda. Cell Chemical Biology, 23(1):137-57 (2016).
18. Tatsuki et al. Neuron, 90(1) : 70–85 (2016).
19. Tainaka et al. Annual Revieew of Cell and Developmental Biology, 32, 713-741 (2016).
20. Ode et al, Molecular Cell, 65, 176–190 (2017).

Minor Changes but Major Future Impacts, Can Be Revealed by Extensive and HighThrough-Put In Vivo Visualization Using 4K/8K CMOS Sensors
Satoshi NISHIMURA
Center for Molecular Medicine, Jichi Medical University, Tochigi, JAPANTo capture rare/minor changes but with biologically important importance for future, we developed
multiscale imaging system in space and time dimensions which was optimized for living animals. Especially,
recently advanced technologies including 1:CMOS sensors with large pixel numbers (4 to 8K), 2:
highthroughput-image handling methods, and 3: low-power high-NA lens were integrated into one opticalsystem. Animals are hold in customized chamber during observation, and controlled by robot-like mechanicaldevices which can adujust animal positions in synchronization with CMOS shutters and XYZT scanning.
Large pixel numbers enhanced imaging fields and evaluated information per time. Recording time was also
increased to cover from milli-second to hours to identify key points during long pathological changes.
“Singularity points” can be identified form long-time recordings by reverse engineering techniques.
We identified tiny scratch on endothelium can induce massive platelet aggregations. Thrombus formationand inflammatory changes followed in later phase, which is one of the main components of cardiovascular
events. Neutrophils were accumulated to repair leak of blood into lymphatic stromal space after damage, andmacrophage recruitment followed. Platelet activation in millisecond order, and chronic inflammatory
reactions in hours are simultaneously evaluated by single imaging modality. We covered micro to macroscale in space and time dimension, and many massive pathological changes can be attributed to small triggers.
Singularity in life can be visualized, and will be the therapeutic target with minimum invasive ness.

Raman Microscopy for Molecular Imaging of Living Cells
Katsumasa FUJITA
Department of Applied Physics, Osaka University
Raman spectroscopy has been used for material analysis in the various research field. The capability ofdetection molecular vibrations enables to detect molecular species, conditions, and their environment in asample to understand the function and characteristic of target materials. The recent development oftechniques for highly sensitive Raman detection has expanded the application of Raman spectroscopy to thebiomedical field. In particular, Raman microscopy that can obtain spatial distributions of biologicalmolecules base on their vibrations provides various methodologies to analyze complicated biologicalphenomena. In our research, we have developed a Raman microscope that can rapidly image molecular
distribution in a live cell [1,2]and studied the application to the analysis of molecular dynamics during cellular
events, such as apoptosis, cell division and differentiation [3-7]. We measured Raman spectra during thosecellular events and found that Raman spectra can represent the cell state during the activities, indicating that
Raman microscopy can be used to investigate cell status without labeling. Since Raman microscopy canprovide comprehensive information of intracellular molecules, it can be a tool to characterize cells and findfunctional singularities that may exhibit significant contribution to the cellular event.
References:[1] A. F. Palonpon et al., Nat. Protoc., 8, 677 (2013).
[2] K. Hamada et al., J. Biomed. Opt. 13, 044027 (2008).
[3] M. Okada et al., Proc. Nat. Acad. Sci. USA, 109, 28 (2012).
[4] A. Hashimoto, et al., Sci. Rep., 5, 12529 (2015).
[5] T. Ichimura et al., PLOS ONE, 9, e84478 (2014).
[6] T. Ichimura et al., Sci. Rep. 6: 37562 (2016).
[7] L.-d. Chiu et al., Sci. Rep. 7: 43569 (2017)

Development of Techniques For Imaging Physiological Functions Toward
Visualization of Minority Cells
Takeharu NAGAI,
The Institute of Scientific and Industrial Research, Osaka University
If we carefully observe the cell population that at first glance looks uniform and homogeneous, we may findsmall number of heterogeneous cells with a different nature. Moreover, this minority cells would sometimes
significantly alter the behavior of the whole cell population. In this symposium, I would like to discuss 1)
possible mechanism by which variety output could be produced even in the cells with identical biomolecular
reaction networks, and 2) development of techniques for imaging physiological function at wide range ofspatiotemporal scale, which is indispensable for identification of minority cells.

Integrin Clusters: A Robust Way to Organize Adhesions for Cell Mechanics
Rishita Changede, Michael SHEETZ

Mechanobiology Institute, National University of Singapore, Singapore
Recent studies at the nanometer scale have revealed that relatively uniform clusters of adhesion proteins (50100 nm) constitute the modular units of cell adhesion sites in both cell-matrix and cell-cell adhesions. Super
resolution microscopy and membrane protein diffusion studies both suggest that even large focal adhesions
are formed of 100nm clusters that are loosely aggregated. Clusters of 20-50 adhesion molecules (integrins or
cadherins) can support large forces through avidity binding interactions but can also be disassembled or
endocytosed rapidly. Assembly of the clusters of integrins is force-independent and involves gathering
integrins at ligand binding sites where they are stabilized by cytoplasmic adhesion proteins that crosslink the
integrin cytoplasmic tails plus connect the clusters to the cell cytoskeleton (see clusters of paxillin in Fig. 1).
Cooperative-signaling events can occur in a single cluster without cascading to other clusters. Thus, theclusters appear to be very important elements in many cellular processes and can be considered as a critical
functional module.

Figure 1 PALM super-resolution image of paxillin clusters (50-100 nm in diameter) in purple overlaid
on a standard epi-fluorescence image of the same paxillin adhesions (diffuse white) in a spread fibroblast.
Bar is 1000 nm.

Quantitative cell biology of plant stomata
Takumi HIGAKI
Graduate School of Frontier Sciences, The University of Tokyo2
Manual evaluation of cellular structures is still a popular approach in cell biological studies. However,such approaches are laborious and are prone to error, especially when large quantities of image data need to
be analyzed. We have introduced an image analysis method that overcomes these limitations by quantificationand clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density arequantified by measurement of newly-developed, robust metric parameters from images. Thereafter, themicroscopic images are classified without supervision by clustering based on the metric patterns. Clustering
allows us to collectively investigate the large number of cytoskeletal structure images without laborious
inspection. Application of this framework to images of GFP-labeled actin cytoskeletons in plant guard cells
determined that microfilaments are radially oriented and transiently bundled in the process of diurnal stomatal
opening. The measurements also revealed that the artificial bundling of actin cytoskeletons suppressed thediurnal patterns of stomatal opening, suggesting that changes in the level of actin bundling are crucial for
promoting stomatal opening. In addition, in order to comprehensively grasp cell biological events in plantstomatal movement, we have acquired microscopic images of guard cells with various organelles markers(Figure 1). We visualized the average organelle distributions in guard cells using probabilistic mapping(Figure 2). Subtractive images of open and closed stomata showed distribution changes in some intracellular
structures including endoplasmic reticulum (ER). Time-lapse imaging showed that similar ER distributionchanges occurred during stomatal opening induced by laser oblation of neighboring epidermal cells,
indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

Figure 1. Representative images of guard cells.

Figure 2. Probability map images of guard cells.

Development of Bioluminescent Low Affinity Ca2+ Indicators Applicable to Analysis
of Ca2+ Dynamics in Endoplasmic Reticulum.
Nadim Hossain1, Kazushi Suzuki1, Megumi Iwano2, Tomoki Matsuda2 and Takeharu Nagai2
1

Graduate School of Engineering, Osaka University, 2ISIR, Osaka University
E-mail: nd6@sanken.osaka-u.ac.jp

Calcium ion (Ca2+) is a crucial intracellular signaling molecule concerned with many biological processes. Ca2+
release from major intracellular Ca2+ store endoplasmic reticulum (ER) which is one of the key factors for thisregulation through the controlling of cytosolic Ca2+ level. To investigate ER Ca2+ dynamics (having sub
milimolar (mM) range of Ca2+), a low affinity Ca2+ indicator is able to facilitate the Ca2+ imaging. Over the last
couple of decade’s variety of the fluorescence protein (FP) based Genetically Encoded Ca2+ Indicators (GECIs)
have been developed for live cell imaging. These Ca2+ indicators are the most promising tools for investigating
subcellular Ca2+ dynamics in cellular organelles. To monitor the Ca2+ dynamics in ER, we have successfully
developed a bioluminescent low affinity intensiometric genetically encoded Ca2+ indicators named CeNL(Ca2+).
It does not require any excitation light; therefore, it can be overcome the potential disadvantages (phototoxicity,
photobleching, and autofluorescence from the specimen) in fluorescence imaging. By using the CeNL(Ca2+) andpreviously developed high affinity green color GECI GeNL(Ca2+), we have successfully achieved thesimultaneous dual color Ca2+ imaging in ER and nucleus in mammalian cells. We are expecting that our low
affinity indicator may investigate neuronal cells and skeletal muscle Ca2+ imaging.

Development of a novel long-circulating ultrasound contrast agent
Lisa MUNAKATA1), Ryo SUZUKI1), Mutsumi SUGII1), Johan UNGA1), Hitoshi URUGA1),
Tadamitsu SHIMA1), Daiki OMATA1), Kazuo MARUYAMA1)
1)

Teikyo University, Faculty of Pharma-Sciences, Laboratory of Drug Delivery System, Tokyo, JapanINTRODUCTION
Ultrasound (US) imaging is comparatively inexpensive, minimally invasive and provide real-timevisualization. It is important to understand the vascular structure of tumor tissue. However, it is difficult totake the US imaging of blood flow in small vessel of tumor tissue. To solve this, microbubbles as US
contrast agents have been developed. In Japan, the contrast agent Sonazoid is approved. It is applied for
diagnostics of liver cancer as negative staining by being uptaken by kupffer cells in the liver, but not intumor. However, because the blood clearance of Sonazoid is fast due to the liver accumulation, it would not
be suitable for other tumors and disorders. In this study, we tried to develop novel microbubbles for long
circulation to observe dynamics of blood flow.
METHODS
Preparation of Lipid Bubbles (LB)

Bubbleswere

preparedfromliposomesofdistearoyl

phosphocholine (DSPC), distearoyl phosphatidylglycerol (DSPG) and 1,2 – distearoyl – sn – glycerol – 3 –phosphoethanolamine – N - [methoxy (polyethylene glycol) - 2000] (DSPE-MPEG(2k)), which were
homogenized together with C3F8 gas. Then, the bubbles were freeze-dried and rehydrated before use.
Assessment of stability of LB in blood Mice (ddY, female, 6 weeks), anesthetized with isoflurane, wereintravenously administrated LB or Sonazoid (1 × 107 particles / 20 PL). Then the stability in blood wasassessed by comparing intensity of US imaging in kidney. The intensity of US imaging was analyzed by
ImageJ software. Then, the time until half of maximum intensity (T1/2) was calculated.
RESULTS AND DISCUSSION
T1/2 increased with increasing DSPG content. The longest T1/2 was achieved with LB containing
60 mol% DSPG, T1/2 of LB containing 90% DSPG was shorter compared to 60% DSPG containing LB. In
addition, we compared T1/2 on kidney between 60% DSPG containing LB and Sonazoid. T 1/2 of LB
containing 60% DSPG was longer than that of Sonazoid. From these results, it was thought that 60% DSPG
containing LB could escape from reticuloendothelial system such as liver.
CONCLUSION
We succeeded to develop the long circulating microbubble by adding DSPG to the shell of LB.
And there was optimal rate of DSPG content of 60 mol%. We expect that our long circulating LB could bea useful US diagnostic agent for various types of tumor where Sonazoid is not applicable.
ACKNOWLEDGMENTS
This research was partially supported by the MEXT-Supported Program for the Strategic
Research Foundation at Private Universities and the Strategic Research Program for Brain Sciences fromJapan Agency for Medical Research and Development.

Preparation of acoustic nanodroplets using a microfluidity device
and evaluation of their physical properties
Tadamitsu SHIMA 1), Tomoyuki NAOI 1), Ryo SUZUKI 1) , Yusuke ODA 1), Johan UNGA 1)
Lisa MUNAKATA 1), Hitoshi URUGA 1), Mutsumi SUGII 1), Daiki OMATA 1), Kazuo MARUYAMA 1)
1)

Teikyo University, Faculty of Pharma-Sciences, Laboratory of Drug Delivery System, Tokyo, JapanINTRODUCTION
Nanodroplets containing liquid perfluoropentane (PFP) that can be turned to gas by ultrasound irradiation
have been proposed as new technology for diagnosis and treatment of cancer. However, the optimum
production method has not been established, because of non-uniform size and difficulties to scale up.
Therefore, to make uniform nanodroplets with a method that allows large scale manufacturing is mostimportant for clinical application. In order to solve these problems, we focused on a mixing technology using
a microfluidics device that should give uniform formulations and is easy to scale up. In this study, we
compared the particle size of nanodroplets produced using a microfluidics device with nanodroplets producedby conventional ethanol injection method. We also evaluated the phase change of nanodroplets from liquid
to gas and in vivo kinetics.
METHODS
Lipid and PFP were dissolved in ethanol, and mixed with water with a microfluidics device or by injection
into water while stirring in a test tube. Ethanol was removed using ultrafiltration. The nanodroplet sizes were
measured with dynamic light scattering. The phase change from liquid droplets to gas bubbles was observedwith an ultrasound imaging device. Furthermore, the DiR as fluorescent probe was added to the suspensionof nanodroplets and it was intravenously injected in tumor-bearing mice. The in vivo kinetics was evaluatedby observation at 1, 3, 6, 12, 24 and 48 hours after administration of the nanodroplets by In Vivo Imaging
System (IVIS).
RESULTS AND DISCUSSION
Nanodroplets had an average size of about 200 nm and a uniform size distribution. The prepared nanodroplets
were reproducible with microfluidics device. Observation with ultrasound imaging showed echo signal
enhancement which occurred by ultrasound irradiation. This shows that the nanodroplets had become gas
bubbles. Enhancement of echo signal was not seen with the control formulation not containing PFP. Thissuggests that the microfluidics device is suitable tool for making the phase change nanodroplets. IVIS
imaging showed that the nanodroplets were accumulated in tumor tissue. Therefore, we expect that
nanodroplets could be a new ultrasound imaging agent.

Visualization of insulin secretion from rat pancreatic INS-1 cells
by using a fluorescent membrane probe.
Megumi Kawakubo 1), Miyuki Sibata 2), Eriko Nagayama 2), Reina Ikaga 3), Naoko Iida-Tanaka 2)
1)

Department of Health and Nutritional Science, Graduate School of Studies in Human Culture, Otsuma
Women’s University, 2) Department of Food Science, Otsuma Women’s University,
3)

Graduate School of Humanities and Sciences, Ochanomizu University

Conventional fluorescence observation of insulin secretion in living cells has been achieved by labelinginsulin itself or insulin granules with fluorescent proteins, in which genetic engineering was needed. In
this study, we proposed a simple method by using a fluorescent membrane probe, FM1-43, and aimed todevelop the method suitable for simultaneous observation with other parameters such as cellular,
endoplasmic reticulum and mitochondrial Ca2+ concentrations. FM1-43 is a fluorescent dye reversibly
binding to the cell membrane. The number of fluorescent vesicles in cytosol is expected to increase
gradually during insulin secretion, because a part of lipid bilayer in insulin granules constantly returns
back into the cytosol after transitionally fusing to plasma membrane and being stained by FM1-43.
After stimulation with glucose, the intracellular fluorescent intensity of FM1-43 in rat pancreatic INS-1
cells gradually increased as expected (Fig.1). The rate of intensity increments which is expected tocorrespond to the insulin secretion during the time, regularly oscillated and the oscillation frequency wasin good agreement with the one of real insulin concentration in the extracellular fluid (Fig.2). Looking at
the area under the curve of the increments rate, the number of fluorescent vesicles in the cells greatly
increased at around 5 and 25 minutes after glucose stimulation, also in good agreement with the patternof insulin concentration changes in the extracellular fluid. By using this method, it is expected that thesecretory rhythm of individual cells can be captured, and simultaneous observation of insulin secretionand other intracellular parameters will be possible.

(A)

(B)

(C)

Fig.1. Fluorescent images of INS-1 cells labeled by
FM1-43. The images were obtained before (A) andafter 19 (B) and 39 (C) minutes of stimulation withglucose.

Fig.2. Time course of the increments rateof the intracellular fluorescent intensity(black line) and the amount of insulin
excreted in the extracellular fluid (grayline).

Knockdown of perilipin 4 affects on lipid droplet formation andmitochondrial morphology in 3T3-L1 adipocytes.
1)

Chiori HASEGAWA , Miyu KOTANI1), Reina IKAGA2), Hikaru TANAKA3), Naoko Iida-TANAKA1)
1)
2)
3)

Department of Food Science, Otsuma Women’s University,

Graduate School of Humanities and Sciences, Ochanomizu University,

Department of Pharmacology, Toho University faculty of pharmaceutical Sciences

Adipocytes have endocrine functions and secrete hormones and cytokines to regulate metabolic states ofwhole body. Adipocyte enlargement resulted from lipid droplet accumulation could be a switch to signal
pathway for secretion of cytokines, increasing inflammation around adipocytes and then causing

lifestyle-related diseases. The switching mechanism, however, is still unclear. Perilipins are a family ofproteins which localizes on the lipid droplet surface and play an important role in lipid droplet formationand maturation. Perilipins may also be involved in communication with surrounding organella. We
observed that mitochondria formed a network around lipid droplets during differentiation and maturationof 3T3-L1 to adipocytes. In this study, we focused on perilipin 4, as a clue to clarify the relationshipbetween lipid droplets, and created the cell lines (shPLIN4) from 3T3-L1 cells, in which the expressionof perilipin 4 mRNA was knocked down to ca. 40%.
In shPLIN4, the lipid droplets were smaller but much more than those in the control cells (shCont) andmitochondria were shorter and thinner (Fig.1A and C), indicating that the knockdown of perilipin4
affected both of lipid droplets formation and mitochondrial energy states. The mRNA expression of
enzymes in glycolysis and electron transfer system of shPLIN4 was lower than those of shCont, so the
ATP synthesis could be decreased. However, the expression level of AMPK in shPLIN4 was also
decreased. An possible explanation of these results isthat knockdown of perilipin 4 reduced the expression

(A)

(B)

(C)

(D)

level of AMPK, possibly lowering the whole energy
production. Treatment with AICAR, an AMPK activator,
recovered the mRNA levels of the enzymes and themitochondrial morphology in shPLIN4 (Fig 1D).
On the other hand, the expression of MCP-1, an
inflammatory cytokine, in shPLIN4 was much moreincreased compared to shCont, and AICAR treatment did
not recover it. It is most likely that the perilipin 4
knockdown affects the inflammatory states in 3T3-L1
adipocytes through a different pathway from the AMPK
signaling.

Fig.1. Fluorescent images of mitochondrialabeled with MitoTracker Deep Red in
shPLIN4 (C and D) and shCont (A and B).
The cells in panel B and D were treatedwith 1mM AICAR over night.

Structure of Protein A and its derivative in solution studied by small-angle x-ray
scattering
T Masaji Shinjo1), Kaoru Ichimura2), Masaki Kojima7) and Hiroshi Kihara3-6)
1)
3)

Kansai Medical Univ., 2) Nagahama Inst. of Bio-Sci. & Technol,

Himeji-Hinomoto College, 4) Hinomoto Gakuen Educational Foundation, 5) SR Center,

Ritsumeikan Univ. 6) SR Research Center, Nagoya Univ., 7) Tokyo Univ. of Pharmacetutial Sci.
Protein A (PA) is a 56 kDa surface protein with five homologous Ig-binding domains found in the cellwall of S. aureus. Each domain folds into a three-helix bundle, and is able to bind to IgGs. PA releases Igs
in acidic pH, suggesting its pH-dep. conformational change. We, then, start investigating the structure of PA
and its derivative, C5, in both of neutral and acidic pHs by SAXS. Results show: PA and C5 form globule
conformations, and their Rg values are 3.73 ± 0.02 nm within experimental errors both at neutral and acidic
pHs. However, Kratky plots and P(r) suggest the conformation of PA at acidic pH is different from the othercases. Experiments were done at PF BL 6A with the approval No. of 2013G101. Authors are grateful to thesample supply from KANEKA CORP.
At the symposium, we will show results of PA and C5 with Fc fragment Ig.

Analysis for antigen phagocytotic reaction regulated by cellular signal transduction

Shintaro YOSHIMURA1, Kouta FUJIKURA2, Shunya OKANO1, Toshinori NAKAYAMA4 and Kahoko
HASHIMOTO1, 2
1) Chiba Institute of Technology, Department of Life and Environmental Sciences, Faculty of Engineering,2) Graduate School of Engineering, 3) Department of Life Science, Faculty of Advanced Engineering, 4)
Chiba University, Graduate School of Medicine
In immune system, antigen presenting cells act as an important role both for the host defense and for
immunological tolerance. Macrophage performs transrational effect on innate immunity and adaptive
immunity. Like dendritic cell, macrophage is also called the professional antigen presenting cells, however,
it is indicated that antigen processing potency of macrophage is predominant. In this study, we analyzed theantigen phagocytotic reaction, which activates cellular signal transduction. By using fluorescent dye labeled
ovalbumin, macrophage cell line: RAW264.7 was analyzed for antigen phagocytotic manner with confocal
imaging study and western blotting. When macrophage showed endocytosis of antigen proteins, such as
ovalbumin, the signal of NF-κB was activated to induce cellular transcription. NF-κB activation was also
observed when antigen signal had been disappeared, recognized by the fluorescent intensity of ovalbumin.
We propose that antigen presenting cells, especially macrophage, may be activated when endocytosis isinitiated and also when antigen degradation is initiated. To understand whether different types of antigen, ordifferent size of antigen may occur the different patterns of cellular signal activation, we have compared theovalbumin phagocytosis with BODIPY conjugated E.coli phagocytosis. Understanding of the mechanism ofphagocytotic time dependent pattern would contribute the therapeutic timing of allergy and inflammation.
Fig.1
0

15

60

150 (min)Fig.2

Macrophage cell line: RAW264.7 was treated with fluorescent dye labeled ovalbumin; Alexa 594 ovalbumin,
the fluorescent intensity of antigen was recognized from 15 min to 120 min. The signal was disappeared at
150 min (Fig.1). Phospho-NF-κB band was also recognized at the early phase of phagocytosis (Fig.2).

Single-molecule observation of actin polymerization
using linear zero-mode waveguides
Soichiro Fujii1), Ryo Iizuka1), Masamichi Yamamoto1), Makoto Tsunoda1),
Takashi Tanii2), & Takashi Funatsu1)
1)Grad. Sch. Pharm. Sci., Univ. Tokyo, 2)Fac. Sci. Eng., Waseda Univ.

Actin is a ubiquitous cytoskeletal protein, which is essential for the structure and function of eukaryotic cells.
Actin polymerization occurs through three phases called nucleation, elongation, and steady-state phases. A
previous study showed that the ends of actin filaments grow and shorten more rapidly than expected frommeasured rate constants of monomer association and dissociation [1]. There are several possible
polymerization mechanisms to explain this interesting result, but detailed polymerization mechanism is notknown. In this study, we aimed to investigate the oligomeric state of actin incorporated into the filament ends
using single-molecule fluorescence imaging in the actin polymerization process. Actin polymerization occurs
when the concentration of G-actin monomer in solution exceeds 100 nM (critical concentration). Therefore,
single-molecule fluorescence imaging at such a high concentration is required. However, single-molecule
fluorescence imaging of actin polymerization is impossible under total internal reflection fluorescencemicroscopy (TIRFM). It is because the signal-to-noise ratio (SNR) cannot exceed 3 at concentrations above
100 nM due to the size of the excitation region. In contrast, SNR can exceed 3 by using linear zero-mode
waveguides (ZMWs) that suppress background light by narrowing the excitation region. Hence, we observed
actin polymerization using single-molecule fluorescence imaging with linear ZMWs (Fig. 1). As a result, wefound that the monomer associated with filament ends in the elongation phase, but small oligomers (dimer
to trimer) were also added to the filaments in the steady-state phase. Furthermore, no cooperative binding ofactin at the ends of the filament was observed.

Fig. 1

Experimental setting with linear zero-mode waveguides

Reference
1.

Fujiwara I., Takahashi S., Tadakuma H., Funatsu T., & Ishiwata S. Microscopic analysis ofpolymerization dynamics with individual actin filaments. Nat. Cell Biol. 4, 666–673 (2002).

Development of a method of heating a single cell using gold nanoparticles
Takaaki Honda1), Kohki Okabe1, 2), Takashi Funatsu1)
1)Grad. Sch. Pharma., Univ. Tokyo, 2) JST, PRESTO

Temperature is a fundamental physical quantity, which has significant effects on cellular functions. Recent
study has shown that the intracellular temperature distribution is heterogeneous. For example, mitochondrial
chemical stimulation brought local heat generation; and there was an intracellular temperature gradient near
this organelle. These results suggested that local temperature changes might regulate various biochemical
reactions in a cell. Thus, understanding the relationship between intracellular temperature and cell functions
may contribute to obtaining better insights into cell functions. However, the mechanism and the significanceof intracellular heat generation are still unknown. Therefore, it is desired to examine cell response to
intracellular local heating. In this study, we have developed a method to control the intracellular local
temperature for investigation of temperature-dependent cell functions.
Here, we report a simple technique to heat a single cell using gold nanoparticles (GNPs), which efficiently
convert light to heat. While heating the cell using GNPs, we measured temperature in a cell by using amolecular thermometer, fluorescent nanogel thermometer (FNT). At low temperature, the fluorescenceintensity of FNT is weak while at high temperature, the intensity is strong. We irradiated GNPs incorporated
into COS7 cells with a visible laser and observed temperature rise in cells by FNT. By adjusting the amountof GNPs and the laser power, we could quantitatively control the intracellular temperature rise. Furthermore,
we succeeded in inducing a cell response by this heating method; we observed stress granules (SG) formation
upon heating, which is responsible for the suppression of translation during stress. Through the heatgeneration by GNPs, SG formation was induced. These results show that GNPs have a potential for the means
to heat the intracellular local region and this method is useful for the comprehension of cell responses to local
temperature change in the cells. Our method will contribute greatly to investigate intracellular thermogenesisbased thermal biology.

Microfabrication techniques for observing neuronal transmission pathway
Hideyuki TERAZONO1), Akihiro HATTORI1), Masao ODATA1), Kenji MATSUURA1), Kenji YASUDA 1,2)
1)

WASEDA Biosci. Res. Inst. Singapore (WABIOS), Waseda Univ. JAPAN, 2) Dept. Physics, Waseda Univ.
JAPAN

Neuronal circuits in vivo and in vitro consist of huge number of cells and form complexed neurotransmission
pathways. Therefore, it is difficult to understand how neuronal communication works at network level. If weare able to reconstruct simple neuronal circuits and regulate the transmission pathway, the mechanism of how
the neuronal circuit stores memories and processes information at the neuronal circuit level will probably
become clear. To this end, we developed a microfabrication technique to create artificial neuronal circuits
composed of individual neurons using agarose and an infrared laser (Agarose-microfabrication technique).
Using this technique, we successfully created simple neuronal circuits with controlled synaptogenesis. By
combining electrophysiological technique, we further demonstrated that the artificial neuronal circuit showedoriginal firing pattern depending on the circuit structure. The electrophysiological technique allows us todetect the frequency of neuronal activity in detail. On the other hand, bioimaging technique enables us toobserve the transmission pathway of the neuronal circuit. We developed another microfabrication technique
to observe the neuronal transmission pathway using bioimaging technique. To study the neuronaltransmission pathway, a 2.5-dimensional (2.5D) microfabrication technique using an ultraviolet curing resin
was developed. The structure formed by 2.5D microfabrication technique is steric, which allows us tococultivate neurons and glias that are required to keep the structure of neuronal circuit for a long time. As aresult, the 2.5D technique allowed us to co-cultivate neurons and glias for a long time while the designed
structure is maintained. Furthermore, a combination of the 2.5D technique and the agarose microfabrication
technique allowed us to guide the synaptogenesis. In summary, the new microfabrication technique withbioimaging shows possibilities for observing principles of neuronal networks such as neuronal plasticity at
network level.

Circulating model

Figure 1.

Interaction model

Agarose microfabrication techniqueFigure 2. Combination technique of 2.5D
and agarose microfabrication technique

RGB-color fluorescent genetically-encoded ATP indicators forenergy metabolism research
Satoshi ARAI1,2), Takashi TSUBOI3), Tetsuya KITAGUCHI1,2)
1) WASEDA Bioscience Research Institute in Singapore (WABIOS), Singapore, 2) Comprehensive

Research Organization, Waseda University, Japan, 3) Department of Life Sciences, Graduate Schoolof Arts and Sciences, The University of Tokyo, Japan.
Adenosine triphosphate (ATP) is used for various cellular activities (for example, cell motility,
exocytosis and development). For understanding of the dynamics of ATP at cellular level, fluorescentgenetically-encoded indicators would be a powerful tool for optically detecting intracellular ATP. Recent
years, we engineered red, green, and blue (RGB-color) intensiometric indicators using single fluorescentproteins where the fluorescence intensity at a single wavelength is enhanced upon sensing ATP. The
expanded color usability of these indicators enabled monitoring the ATP dynamics in different organelles
at same cell at the same time. Here, we imaged the dynamics of cytoplasmic and mitochondrial ATP ofcells derived from a wide variety of species, including mammalian cells, plants, and worms. Furthermore,
coupling with additional fluorescent indicators, we visualized the dynamic interplay of ATP, cAMP, andCa2+ in the brown adipocyte during an uncoupling event. We do believe that the toolset of indicators will
facilitate future research into energy metabolism.

Concomitant monitoring of local heating and heat-triggered cargo release fromthermosensitive liposomes by fluorescence imaging
Keitaro Sou1,2), Satoshi Arai1), Li Yan Chan3), Chi-Lik Ken Lee 3)
1)

Waseda Bioscience Research Institute in Singapore (WABIOS), Singapore, 2) Organization for University
Research Initiatives, Waseda University, Tokyo Japan, 3) Department for Technology, Innovation &Enterprise, Singapore Polytechnic, Singapore
Control of cargo release from nanoparticles is an important technology for maximizing the benefits of

the nanoparticles for biological and biomedical applications such as bioimaging and drug delivery systems.
Heat stimulus is a potent tool to trigger the release of cargo from the nanoparticles, for which a light energy
is a practical source to generate local heating at a target region. Herein, we attempt to apply thefluorescence imaging techniques for concomitant monitoring of the heat-triggered cargo release fromlipid-based nanoparticles (liposomes) and local heating by photothermal conversion of water with a 980 nm
near-infrared (NIR) laser irradiation. Liposome formulation consisting of 1,2-dipalmitory-sn-glycero-3phosphocholine (DPPC) with an anionic lipid and PEG-lipid as stabilizers encapsulating hypertonic
solution with lipid membrane shows that a gel to liquid-crystalline phase transition at around 40 oC
effectively triggers the release of cargo from liposomes at temperature above 40 oC with NIR irradiation.
Our proof of concept has been demonstrated in a cancer cell with microscopic monitoring the actual“intracellular temperature” using a fluorescent thermosensor. An intracellular thermometry revealed that itwas not until the intracellular temperature reached around 40 oC by NIR irradiation that the release of the
cargo started gradually, showing the good agreement with the result from the extracellular in vitro study.
This targeted release of cargo from thermosensitive liposomes based on a photothermal effect using NIR
laser offers a potent nanoscale platform for the on-demand release of drugs in intracellular space with local
heating. The intracellular thermometry facilitates the quantitative monitoring and control of the heating at
the cellular level. Furthermore an extended concept for macroscopic temperature monitoring using thethermosensitive liposomes was examined using NIR laser-induced heating of water in a hydrogel matrix.
The temperature distribution in hydrogel by photothermal conversion can now be traced by fluorescence inreal time, with the use of thermosensitive liposomes that release the fluorescence cargo at different
threshold temperatures. These liposome platforms, equipped with temperature-sensing capability, extend
the concept of temperature monitoring for heat-triggered drug release, as well as thermotherapy. Thus
fluorescence imaging techniques are powerful tool for concomitant real-time monitoring for precise control
of local temperature and drug release triggered by heat stimulus at target region.

Evaluation of cell-to-cell conduction on one-dimensional line of cardiac muscle cells
network for in vitro predictive cardiotoxicity measurement beyond cell-based drug
discovery electrophysiology
Kenji Matsuura1), 2), Moe Iwamura3), Naoki Takahashi3), Yuki Yamanaka3), Natuki Seki4),
Masao Odaka1), 2), Akihiro Hattori1), 2), Hideyuki Terazono1), 2) and Kenji Yasuda1-4)
1)

WASEDA Biosci. Res. Ins. in Singapore (WABIOS), 2) Org. Univ. Res. Initiative, Waseda Univ., Japan
3)

Dept. Phys. & Appl. Phys. Graduate School of Advanced Sci. & Eng., Waseda Univ., Japan4)

Dep. of Phys. & Appl. Phys. School of Advanced Sci. & Eng., Waseda Univ., Japan

In recent years, technologies to evaluate the effect of drugs to human cardiac muscle cells have beenpromoted vigorously. As the one of interested questions in this field is whether the in vitro assay can
predict the clinical issues such as the “Torsade de pointes (TdP)”, which is the potentially deadly cardiac
arrhythmia cause by the asynchronous beating behavior of neighboring cells. Hence, we have proposed an
on-chip cell-to-cell conduction screening assay of cardiomyocytes to overcome the limitations of theconventional cell-based in vitro assays. In the assay, we set a thousand of cardiomyocytes
one-dimensionally on the rectangular shaped agarose microchambers on the multi electrode array chip, andmeasured the propagation of signal in cardiac muscle cell network and measured the increase andfluctuation of their propagations and durations field potentials (FPD)(Fig. 1). The result showed that theevaluation of delay and fluctuation cell-to-cell conduction have a potential to predict risks of arrhythmia
more precisely beyond the conventional in vitro predictive cardiotoxicity assays.




(b)

(a)
Figure 1.

(c)

(a) Schematic of an on-chip multi electrode array system. (b) Schematic of a MEA chip.

(c) Clusters of cardiac muscle cells which were lined-up on a surface of MEA chip.

Real time on-chip imaging cell sorter for shape recognition of target cells in droplets
Mathias Girault1), Hyonchol Kim2), Kenji Matsuura3,4), Masao Odaka3,4), Akihiro Hattori3,4), Hideyuki
Terazono3,4) and Kenji Yasuda3-5)
1)
2)

French National Center for Scientific Research, Bordeaux, CRPP, Pessac, France

Biomedical Research Institute, National Institute of Advanced Industrial Sci. and Tech., Ibaraki, Japan
3)
4)

Org. Univ. Res. Initiatives, Waseda Univ., Japan,

WASEDA Bioscience Research Institute in Singapore (WABIOS)

5)

Dept. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan

Automatic recognition of cells is an important challenge in numerous biological applications such as cluster
cell analysis. Although fluorescence based methods can detect cells with accuracy, microfluidic on-chip
imaging cell sorters offer several advantages in terms of recognition especially with regards to cells withcomplex morphologies. In this context we developed a label-free microfluidic droplet-sorting system basedon image recognition of cells in droplets (Fig. 1). To test the applicability of this method, a mixture of two
species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) weresuccessfully identified and discriminated at a rate of 10 Hz (91 ± 4.5% and 90 ± 3.8%, respectively) (Fig.
2). The results indicate that imaging cell sorter can isolate single target cells from a mixture of cells withhigh accuracy without any staining.

Fig. 1: Example of cell detection (red square) in theFig.2: The panels a and b show parts of theregion of interest (green rectangle) before a droplet

collection channels after droplet screening

sorting process. Diameter of droplet is 80µm.

process. White and black arrows show thedroplets

containing

tricornutum

andrespectively.

Phaeodactylum

Dunaliellatertiolecta,

On-chip multi-imaging flowcytometry
for non-labeled detection of cell clusters in blood with imaging biomarkers
Moe Iwamura1), Yuki Yamanaka1), Kento Iida2), Masao Odaka3.4), Akihiro Hattori3.4),
Kenji Matsuura3.4),Hideyuki Terazono3.4) and Kenji Yasuda1-4)
1) Dept. Phys. & Appl. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan,
2) Dept. Phys. & Appl. Phys., Schl. Adv. Sci. & Eng., Waseda Univ., Japan,
3) Org. Univ. Res. Init., Waseda Univ., Japan, 4) WASEDA Bioscience Res. Ins. in Singapore (WABIOS)

Detecting the circulating tumor cells (CTCs) in blood is important issue for understanding the metastasis as
non-invasive liquid biopsy. However, conventional molecular biomarkers were not effective enough to
identify the CTCs. Here, we have developed an on-chip multi-imaging cell analysis flowcytometry, in whichtarget cells are identified by their morphological characteristics without any label of molecular biomarkers
such as EpCAM antibody (Fig.1). Firstly, we fabricated poly-dimethylpolysiloxane (PDMS) microstructuresin the cell sorting chip to evaluate the reproducibility, and found that the microstructures shrank at a constantrate of 80% at the first exposure step of SU-8 from photo-masks in the microfabrication process. Hence, we
improved the procedure and succeeded in stably producing precise designed microchannel chips by designing
the channel with compensations of the shrinkage. Secondly, we examined the efficacy of the system using
the obtained blood samples of rats, in which prostate cancer cell line was implanted, and we detected that thefrequency of CTC clusters increased exponentially over time after implantation (Fig.2). These results suggestthat the function of the system is suitable for imaging biomarker identification with up to 10,000 samples/s
resolution and was enough to measure the time course change of CTC clusters.

Figure 1. Overview of the on-chip multi-imaging cell analysis flowcytometry. (a) Schematic image.
(b) Design of the microchip. (c) Image of the microchip.

Figure 2. Frequency of cellular area. (a) Histograms of total cell area, S, of cancer cell-implanted.
(b) The time course frequency of cells which have over 200 μm2 areas.

Time course change of circulating tumor cell clusters in blood
evaluated by imaging biomarkers of on-chip multi-imaging cell sorter
Masao Odaka1,2), Moe Iwamura3), Akihiro Hattori1,2), Kenji Matsuura1,2), Hideyuki Terazono1,2),
Yuki Yamanaka3), Kento Iida4), and Kenji Yasuda1-4)
1)

WASEDA Biosci. Res. Ins. in Singapore (WABIOS), 2) Org. Univ. Res. Initiatives, Waseda Univ., Japan,
3)

Dept. Pure & Appl. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan,
4)

Dept. Pure & Appl. Phys., Schl. Adv. Sci. & Eng., Waseda Univ., Japan,

Technology for analyzing diagnostic conditions and symptom of patients has rapidly advanced by liquidbiopsy of blood, in which molecular and physical biomarkers are applied. Especially, it is expected toestablish a diagnostic method accurately confirming cancer metastasis using a small volume of patient
blood to evaluate the effectiveness of chemotherapy and post-surgery checking. Here, we have developed
an on-chip imaging cell sorter to identify and evaluate their time course change of circulating tumor cells(CTCs) based on their morphological characteristics of clusters, which is the third and more precise index
of identification of CTCs against conventional molecular or physical biomarkers (Fig. 1). As a practicalapplication, we applied our imaging cell sorter for recognition of CTCs of human patient blood. We
succeeded in identifying CTCs specifically as the clustered cells and found the correlation between thenumber of CTC clusters and chemotherapy effectiveness, which was not detectable for Cell Search® assay.
Moreover, we confirmed the number of cell clusters in healthy blood and those of infection disease patientswere negligible small. The results indicate the effectiveness and advantages of this non-labeled imaging
biomarkers for practical CTC detections in chemotherapy and post-surgery checking.

Figure 1. Typical time course change of CTC clusters in blood after implanted into rat. Size (SC), cellularperimeter (PC), and the number of cells in the cluster are indicated.

Origin of cardiomyocyte cluster beating rythms:
Elucidation of selection rule of interbeat intervals of cardiomyocyte network   from single cells
Naoki Takahashi1), Natuki Seki2), Hideyuki Terazono3.4) , Masao Odaka3.4),
Akihiro Hattori3.4), Kenji Matsuura3.4) and Kenji Yasuda1-4)
1)Dept. Phys. & Appl. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan, 2) Dept. Phys., Schl. Adv.
Sci. & Eng., Waseda Univ., Japan, 3) Org. Univ. Res. Init.., Waseda Univ., Japan, 4) WASEDA Bioscience
Research Institute in Singapore (WABIOS)

The synchronization behavior of cardiomyocytes is one of the most characteristic dynamics of thecommunity effect of cell networks. However, the selection rule of interbeat interval (IBI) in thesynchronization of cardiomyocytes has not been clarified. Here, we examined the hidden mechanism of IBI
selection of the cell cluster from variety of different IBI of component cells during their clustering. First,
we compared the IBIs and its stabilities (coefficient of variation; CV) of cardiomyocyte clusters of mouseembryo and those of isolated single cells, which were acquired from the cluster after its IBI measurementwith trypsination. Some isolated cardiomyocytes showed faster IBI than the clusters. In addition, the IBI of
the clusters has stability equivalent to the most stable isolated cells. These results showed experimentallythat only the concept of conduction in cardiomyocyte network is not enough to explain IBI selection rule ina spontaneous beating cell networks.Furthermore, we suggest that the IBI of cardiomyocyte network is
determined depending on the stability of the IBI of the component cells before synchronization.

Figure 1.Dish holder for cell sedimentation.

.
Figure 2. An example of beating properties of isolated single cardiomyocytes obtained from thebeating cluster.

Minimum width of artificial neuronal circuit patterns
for stable elongation of neurites
1)

Takahito Kikuchi , Shota Aoki2), Masao Odaka3,4), Akihiro Hattori3,4), Kenji Matsuura3,4),
Hideyuki Terazono3,4), and Kenji Yasuda1-4)
1)Dept. Pure & Appl. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan,
2)

Dept. Pure & Appl. Phys., Schl. Adv. Sci. & Eng., Waseda Univ., Japan,

3) Org. Univ. Res. Init., Waseda Univ., Japan, 4) WASEDA Bioscience Res. Ins. in Singapore (WABIOS)

Development of microfabrication technology has enabled us to manipulate neurons and construct a living
neuronal network in micropatterned structures as desired. Moreover, establishments of stepwise
microfabrication technique have allowed us to control axon-dendrite polarity in cultured neurons andconstruct a living neuronal circuit. However, we still need to understand the characteristics of neurite
elongation and differentiation for more precise and reliable control of polarity of neurites. Here, we havedeveloped an in situ photothermal microfabrication system for stepwise and flexible micropatterning during
cell cultivation, which consists of four major parts; a cultivation dish coated with an agarose thin layer, a1064/1480-nm dual infrared laser, a phase-contrast microscope with a charge-coupled device (CCD) camera
with image analyzer, and a semi-automated micropatterning computer (Fig. 1). We found the minimum
requirement of micropattern width for neurite elongation: In 3 μm in width, 19% of neurites were elongatedlonger than 100 μm in the channel, whereas in 12 μm in width, 71% were elongated (Table1 and Fig. 2). The
results indicate the existence of minimum microchannel width for steady neurite extension.
Table 1. Relation between width of channel and ratio of elongation.

Fig. 1. Design (a) and procedures (b, c) of in situ photothermal

Fig. 2. Neurite elongation in each width of

microfabrication method. Microstructures of agarose aremicrochannel. Arrowheads, edges of elongatedfabricated by spot heating of IR laser during cultivation.

neurites.

Studies on spatiotemporal adaptive regulation mechanism ofmacrophage phagocytosis: response of multiple stimulations due tophysical contacts with multiway optical tweezers
Yoshiki Nakata1), Yuya Furumoto2), Masao Odaka3,4), Akihiro Hattori3,4), Kenji Matsuura3,4),
Hideyuki Terazono3,4), and Kenji Yasuda1-4)
1)Dept. Pure & Appl. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan,
2)
Dept. Pure & Appl. Phys., Schl. Adv. Sci. & Eng., Waseda Univ., Japan,
3)
Org. Univ. Res. Init., Waseda Univ., Japan, 4) WASEDA Bioscience Res. Ins. in Singapore (WABIOS)
Macrophages have an intelligent function of recognition and response to antigens on their surface.
However, their adaptive regulation mechanism in spatiotemporal viewpoint especially under competing
condition due to multiple stimulations has not been investigated. Here, we examined a series of various
time courses (temporal aspect) and contact positions (spatial aspect) of multiple stimulations forphagocytosis (Fig.1). We observed alveolar macrophages by on-chip single cell measurement assay, inwhich single cells are isolated within each microchamber in the cultivation microchip, and plurality ofantigens can be handled simultaneously to contact to membrane surface of macrophages with two-way
optical tweezers (Fig. 2). Consequently, a series of multiple phagocytosis by four antigens occurred
according to antigen (zymosan) contacted order regardless of their positions (Fig. 3). Therefore,
macrophages memorized the order of contacted zymosans at least 4 points. The results indicate that
phagocytosis is not independent local phenomenon on cell membrane and macrophage memorizes the timecourse order of contacted antibody (at least 4 points).
Tc: contacting time

1
2nd

Fig.1. Spatiotemporal guiding of antigens. Order andcontact positions of multiple stimulations.Strictly control
the position and the timing of zymosan stimulation.
MacrophageTf    

Ts

d

Tc

2

3rd

4th

PC CCD CameraTcg

Tf: finished time 

e

f

Ts

Tf    

h

Tc

3

Cell

i

Tf    

Ts



k



Zymosan
4

Dish

c

b

Ts: starting time

Order of phagocytosis occurred1a

ZymosanstTc

Obj.100
0 

5

Laser Transmitter (1064nm)

10

15



Ts

 Tf     

20

25

Relative times

Fig.2. Two-way optical tweezers and observation assay.

Fig.3. Order of phagocytosis images. The moment of

We caught a zymosan by optical tweezers, and carried on

antigens contact (a,d,g,j), starting phagocytizing (b,e,h,k),a macrophage.

and finished (c,f,i,l).

Development of on-chip dual measurement system for quasi-in vivo screening ofcardiotoxicity using extracellular field potential recording and optical displacement
analysis of single cells
Akihiro Hattori1, 2), Naoki Takahashi3), Masao Odaka1, 2), Kenji Matsuura1, 2),
Hideyuki Terazono1, 2) Natsuki Seki4) and Kenji Yasuda1-4)
1)

WASEDA Biosci. Res. Ins. in Singapore (WABIOS), 2) Org. Univ. Res. Initiative, Waseda Univ., Japan,

3)

Dept. Phys. & Appl. Phys., Grad. Schl. Adv. Sci. & Eng., Waseda Univ., Japan, 4)Dept. Phys., Schl. Adv.
Sci. & Eng., Waseda Univ., Japan

In cardiac toxic assessment in drug development, adding to the electrophysiological measurement isimportant methodology, quantitative evaluation of mechanophysiological responses in cardiomyocytes
has becoming more important for precise prediction of cardiotoxicity especially in anticancer drug
development. For the evaluation of correlation between electrophysiological effects andmechanophysiological effects of drugs simultaneously, we have developed the on-chip dual
measurement

assay

that

records

extracellularfield

potentials

andbeatingmotionsof

one-dimensionally lined-up cardiomyocyte networks simultaneously. Human cardiomyocytes wereapplied into rectangular microchambers fabricated on the multi electrode array (MEA) chip, andlabeled with fluorescent polystyrene beads to detect beating motion of cardiomyocytes network by
1/100 s high-speed camera. We succeeded in simultaneous recording of extracellular field potentials
and motion of cardiomyocytes, and found that beating rate of cardiomyocytes and force generated bycontraction of cardiomyocytes were changed significantly in the presence of drug. The results indicatethat the compositive analysis of electrophysiological properties and mechanophysiological properties
should become an effective biomarker of cardiotoxicity estimation.

Fig. 1 Representative measurement parameters of the dual measurement system. (a) Analysis
parameter of field potential (FP) recording. (b) Analysis parameter of optical imaging.

Molecular structure-based analysis of drug resistance of influenza virus B
Yusuke Kato1,2), Haruka Hishiki3), Yosuke Kameoka4), Reiko Itoh5), Tomohiro Someya6), Nobue Inoue7),
Mana Haraki8), Tomomichi Kurosaki9), Shochi Suzuki5), Tomoko Ogawa10),
Naruhiko Ishiwada11), and Kazuo Suzuki5, 12)*1)

Institute for Enzyme Research, Tokuhshima University, 2) Institute for Health Sciences, Tokushima Bunri

University, 3) Chiba University Graduate School of Medicine, 4) A-CLIP Institute, 5) Asia International
Institute of Infectious Diseases Control, Teikyo University, 6) Department of Pediatrics, Goi Hospital, 7)
Department of Pediatrics, Yamanouchi Hospital, 8) Mana Children’s Clinic, 9) Kurosaki Children’s Clinic, 10)
Chiba Prefectural Institute of Public Health, 11) Department of Infectious Diseases, Chiba Medical
Mycology Research Center, 12) Department of Immunology, National Institute of Infectious Diseases*Presentation
Neuraminidase inhibitors have been prescribed for the treatment of both influenza A and B viral
infections in children. Infection with influenza virus occasionally displays prolonged period of the highfever despite the treatment with the neuraminidase inhibitors. Such prolonged fever suggests resistance ofthe viruses to the neuraminidase inhibitors. It has been reported that mutations in the genomes of influenza
viruses are related with the drug resistance. Here, we studied association between prolonged fever andmutations in influenza B from child patients.
RNA was isolated from fixed nasopharyngeal swab of 206 patients with influenza in 2013/2014 season.
The samples from ten patients having fever over 38oC for more than 48hr were analyzed to determine
neuraminidase sequences. Structural models reflecting the gene mutation of neuraminidase of influenza B
virus (BNA) were built and analyzed.
Patients infected with influenza B exhibited longer period of fever on average than those with influenza
A. Sequences of BNAs from the patients with prolonged fever period differed from that of the vaccine
strain (B/Massachusetts/02/2012). Two of the BNA sequences contained mutations for potential drug
resistance: S99N, T106I, K125T and S295R in the sample M2-1 and I262M, V271T, K/E272Q, E320K,
D342G and M375K in the K41-1. Structural models of BNA with the mutations suggested that R295 of theM2-1 and Q272 and K375 of the K41-1 may be responsible for the drug resistance.
Drug-resistant mechanism of BNA was clarified by using structural model analysis. Proper use ofneuraminidase inhibitors against influenza virus infection is necessary in clinical setting.

A microbead supported membrane-based fluorescence imaging assay reveals
intermembrane receptor-ligand complex dimension with nanometer precision
Kabir H Biswas1), Jay T Groves 1,2)
1)

Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore,2)

Department of Chemistry, University of California, Berkeley, California 94720, USA

Abstract Body Receptor-ligand complexes spanning a cell-cell interface inevitably establish a preferredintermembrane spacing based on the molecular dimensions and orientation of the complexes. This
couples molecular binding events to membrane mechanics and large-scale spatial organization ofreceptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method toprecisely

determineintermembrane

receptor-ligand

dimensionatadhesionsestablishedbyreceptor-ligand binding between apposed membranes in vitro. Adhesions were reconstituted between
planar and silica microbead supported membranes via specific interaction between cognate
receptor:ligand pairs (EphA2:EphrinA1 and E-cadherin:anti-E-cadherin antibody). Epi-fluorescenceimaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead,combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane
receptor-ligand dimension with better than 1 nm precision. An advantage of this assay is that no
specialized equipment or imaging methods are required.

Investigation of membrane interaction mechanism of Lti30 using Imaging Fluorescence
Correlation Spectroscopy
Anjali Gupta1, Pia Harryson2, Thorsten Wohland1
1

Department of Biological Sciences and Centre for Bioimaging Sciences, National University ofSingapore, Singapore
2

Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, 7
Stockholm University, 106 91 Stockholm, Sweden
Email:a0123870@u.nus.edu
Dehydrins are intrinsically disordered plant proteins whose expression is upregulated underdesiccation and cold stress conditions. Lti30 is a cold-induced dehydrin composed of six archetypicalK-segments each with two adjacent histidines. This protein requires protonation of histidine residues
to interact with the membrane so it is proposed that these histidine residues act as pH switch andinfluence Lti30’s membrane binding abilities. Literature suggests that Lti30 becomes structured oninteracting with membrane and it interacts actively with anionic membrane lipids via electrostatics
and weakly interacts with zwitterionic lipids. In this work, the effect of Lti30, K-segment, and Ksegment with two histidines on membrane diffusion has been studied with the help of Imaging
Fluorescence correlation spectroscopy. Imaging FCS method allows measuring the effect of protein
on hundreds of contiguous pixels and hence large areas on the membrane. Each measurement
provides a diffusion and concentration map over the whole region of interest which can then be
developed into a time-lapse movie. Effect of these protein/peptides have been studied on three
supported lipid bilayer systems namely DOPC, DOPC: DOPS (4:1) and DOPC: DPPC (1:1) at fourpH conditions of 5.8, 6.3, 7.4 and 9.0 as they are mechanistically important pH conditions for Lti30.
With these experiments, for the first time, we show that Lti30 in addition to anionic lipids can interact
actively with specific types of zwitterionic lipids as well. It is also found that on protonation Lti30 has
a tendency to aggregate over the membrane which can have important implications for membranestability.

Measuring Oxygen Concentrations in Bacterial Biofilms Using Transient State
Monitoring by Single Plane Illumination Microscopy
Andreas Karampatzakis1, Scott A. Rice2, Yehuda Cohen2, Thorsten Wohland1
1
2

NUS Centre for Bio-Imaging Sciences, National University of Singapore.

Singapore Centre on Environmental Life Sciences Engineering (SCELSE), N.T.U. Singapore.

Biofilms are structured bacterial communities in which cells are held together within a matrix
formed by self–secreted polymeric compounds. They are notoriously hard to remove once formed, and are
responsible for up to 80% of all bacterial infections. Oxygen is required for respiration in aerobic bacteria
and hence plays an essential role in the generation of energy, necessary for cell maintenance and growth.
The distribution of oxygen within a biofilm can be heterogeneous and is expected to change during theirlife cycle. Most commonly, measurements of oxygen are carried out using electrodes which are invasive innature and may alter the micromechanical properties of the sample, giving rise to artefacts.
Here, we apply Transient State (TRAST) monitoring, a non-invasive method that can measure therelative populations of molecules in the triplet states by averaging the fluorescence intensity undertime-modulated illumination. We use a Single Plane Illumination Microscope (SPIM), which allows opticalslicing, suitable for imaging thick samples such as biofilms (Figure 1a). We present, for the first time, amap of triplet relaxation times measured inside a Pseudomonas aeruginosa biofilm, at the μm range (Figure1b). We further investigate the effect of microenvironmental factors (viscosity, oxygen uptake) on the
behavior of fluorescence, to establish a protocol for quantitative oxygen concentration measurements. Our
results indicate that completely anoxic zones lie within the colonies, and oxygen concentration gradientsare formed near the boundaries of the biofilm that extend outside the areas of high cell density (Figure 1c).

Fig 1. Imaging a P. aeruginosa biofilm microcolony (~80 h post–inoculation). (a) SPIM image. (b) Triplet
relaxation time map. (c) Oxygen concentration map.

Plasma membrane domain confinement of secreted signaling protein Wnt3 revealed
by SPIM-FCS in live zebrafish
Xue Wen NG1), Cathleen TEH 2), Vladimir KORZH 2), Thorsten WOHLAND 1,3)
1)Department of Chemistry and NUS Centre for BioImaging Sciences, National University of Singapore,
Singapore, 2)Institute of Molecular and Cell Biology, Agency for Science, Technology and Research,
Singapore, 3)Department of Biological Sciences, National University of Singapore, Singapore

The embryonic development of a zebrafish is tightly regulated by multiple genes in a concerted manner.
The WNT3 gene is one such gene that encodes the secreted signaling protein Wnt3 which activates theWnt signaling pathway that is responsible for modulating vital developmental processes such asanterior-posterior axis specification and neurogenesis. Wnt3 is transported intracellularly to the cell
membranes of Wnt-producing cells upon undergoing post-translational palmitoylation at the endoplasmic
reticulum by membrane-bound O-acyltransferase, Porcupine. By comparing the amino acid sequence ofWnt3 with identified highly-conserved palmitoylation sites of other Wnt proteins, the lipid modificationsof Wnt3 most likely occur at sites C80 and S212. Previously, our group demonstrated the importance ofpalmitoylation of Wnt3 by Porcupine for the membrane localization and secretion of Wnt3 as well as
proper brain development in live Wnt3-EGFP transgenic zebrafish embryos through Porcupine inhibition
experiments. In this study, we applied single plane illumination microscopy-fluorescence correlation
spectroscopy (SPIM-FCS) on the same Wnt-EGFP transgenic line to determine the membrane
distribution of Wnt3. SPIM-FCS conducts FCS measurements in an imaging approach that offers hugestatistics of data due to its multiplexing capability which generates spatial maps of diffusivity andconcentration. In addition, sub-resolution membrane organization of fluorescent probes was also deducedby performing the FCS diffusion law analysis on SPIM-FCS measurements which studies the spatial
dependence of diffusion of the probes. Overall, our results illustrate the association of Wnt3 withcholesterol-dependent plasma membrane domains that is influenced by the inhibition of palmitoylationand the reduction of several lipid components via drug treatments in the cerebellum of live zebrafish
embryos.

Theoretical and Simulation Studies for Radiation Damage from Free Radicals
and Hydrogen Bubbles in Cryo-Electron Microscopy
1)Ting-Hang Pei and 1,2)N. Duane Loh

1. Centre for BioImaging Sciences, Department of Biological Sciences, National University ofSingapore, Singapore 117543, Singapore.
2. Department of Physics, National University of Singapore, Singapore, Singapore 117543, Singapore.
Email: DBSPEIT@nus.edu.sg, duaneloh@nus.edu.sg

Abstract: Radiation damage is an important consideration for improving sample contrast and resolution in
cryo-electron microscopy (cryoEM). It was proposed [1] that hydrogen bubbles are an important sign ofsample damage in cryo electron microscopy: incident electrons dissociate H2O molecules which produceshydrogen and hydroxyl radicals. The latter is expected to modify the hydrogen bonds in biomolecules, which
produce hydrogen gas that nucleate into high-pressure bubbles. Knowing the pressure of such hydrogen
bubbles, and how to reduce them are hence important for detecting and mitigating sample damage.
Here we investigate the claim that such hydrogen bubbles could have internal pressures of 1000 atm [2].
Furthermore, we explore the prospect of interpreting electron-induced damage of two-dimensional protein
crystals from changes in their weak continuous diffraction intensities (i.e. inter-Bragg diffraction) [3].

Reference:
1. Glaeser, R. M., Chapter Two – Specimen Behavior in the Electron Beam; Methods in Enzymology(eds.) (Elsevier Inc., 2016).
2. R. Leapman and S. Sun, “Cryo-electron energy loss spectroscopy: observations on vitrified hydrated
specimens and radiation Damage,” Ultramicroscopy 59, 71-79 (1995)
3. Kartik Ayyer et al., “Macromolecular diffractive imaging using imperfect crystals,” Nature 530, 202206 (2016)

Deconstructing the factors involved in the modulation of mobility of molecules in microcolonies
of Pseudomonas aeruginosa biofilms
Jagadish Sankaran1,2), Nicholas John Tan Jie Hao4), Kumaravel Kandaswamy5,6), Yehuda Cohen4,5), Scott A
Rice4,5) and Thorsten Wohland1,2,3)
1)

Centre for BioImaging Sciences, National University of Singapore, Singapore 117557
Departments of Biological Sciences and 3)Chemistry, National University of Singapore, Singapore 117346
4)
School of Biological Sciences and 5)Singapore Centre for Environmental Life Sciences Engineering, Nanyang
Technological University, Singapore 637551
6)Interdisciplinary Graduate School, Nanyang Technological University, Singapore 639798
2)

Pseudomonas aeruginosa (PA) exists in two different life styles, which are the freely swimming unicellular
planktonic form, and the surface attached multicellular form called biofilm. The bacterial cells in a PA biofilm
are embedded in a self-produced matrix composed of different polysaccharides, DNA, proteins and phages. PA
biofilm development has been categorized into three different stages. During the first stage, the bacteria attach
itself to the substrate. During the second stage, the biofilms undergo maturation and form mushroom shaped
microcolonies. During the last stage, the microcolonies disperse and release freely swimming planktonic
bacteria. Cyclic diGMP has been identified as a soluble second messenger involved in bacterial communication
during the dispersal stage. Single Plane Illumination Microscopy-Fluorescence Correlation Spectroscopy(SPIM-FCS) enables one to quantitate the diffusion coefficient at contiguous locations of a biofilm. Our results
suggest that the diffusion coefficient of fluorescent probes decrease with increasing size of the microcolony.
Only positively charged molecules localize into the biofilm and exhibit different mobilities at different
locations of the biofilm. Phosphodiesterases that cleave cyclic diGMP have been shown to promote dispersal of
biofilms. Hence using a bacterial strain with a phosphodiesterase gene under the control of arabinose induction,
we have monitored the real-time changes in diffusion properties at contiguous locations of a microcolony
before and after dispersal. Our results aid in deciphering the individual role of size, charge and bacterial cell
physiology, in contributing to the diffusion limitation in PA biofilms.

Strain Maps Reveal Mechanical Properties of Embryos during Morphogenetic
Movements
D. Bhattacharya1,2, J. Zhong2,3, A. Kabla4, P. Matsudaira1,2,3
1Singapore-MIT Alliance for Research and Technology, National University of Singapore
2MechanoBiology Institute, Singapore, National University of Singapore
3Center for BioImaging Sciences, Dept. of Biological Sciences, National University of Singapore
4Department of Engineering, Cambridge University
The body plan of multicellular organisms is shaped through collective cell behaviors, and is known asmorphogenesis, or morphogenetic movements. An important morphogenetic event during early embryo
development is the convergence and extension at mid-gastrulation stage, where cells collectively movefrom the ventral side of the embryo towards the dorsal axis, underlying the neural tube formation and thesomatogenesis. The biophysical and biochemical mechanisms of early development have been
intensively studied at the cellular level, but due to the technical challenge for quantifying the force anddeformation the global mechanics at the tissue level remains unknown. Here we combine in-toto imaging
by light-sheet microscopy and computational methods to calculate the strain map of zebrafish
development from 7.5-12 hpf, representing the tissue deformation, during mid-gastrulation. Keyparameters for the dynamics include: 1. vector field of collective cell migrations, 2. mechanical curl ofsheets of cells, which describes the domain rotation, and 3. strain, which measures the tissue deformation,
have been calculated over the entire embryo. Furthermore, based on these results we have identified
strain associated with convergent extension and pre-somatogenesis events. The strain maps are a firststep in the quantitation of the mechanical forces that power morphogenesis.

A Method to Quantify Co-localization in Biological Images
Shiwen ZHU1, Roy E. WELSCH2, and Paul T. MATSUDAIRA1,3
1 Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #10-01 CREATE TowerSingapore 138602
2 Sloan School of Management, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
3 Department of Biology Science, National University of Singapore, Singapore 117543

Quantitative co-localization analysis with fluorescent microscopy is a common approach to assess thespatial co-ordination of molecules and thus to understand their functions in biological processes. However,
the results of co-localization analysis might not be consistent due to various imaging conditions and it
raises the problems in comparison and interpretation of the results from different methods We propose anovel method to separate a co-localization event into two aspects: co-occurrence and intensity correlation,
which are usually combined as one parameter in other quantitative co-localization analyses. By examiningco-localization through both co-occurrence and intensity correlation, the co-localization analysis provides
accurate and interpretable results. Furthermore, the co-occurrence pixels can be visualized in an additional
image channel to provide an intuitive impression of the quantity and locations of the co-localization eventsoccurring.

Tension-dependent dynamics of adherens junction components
during cell junction deformations
Yusuke HARA1,2), Murat SHAGIROV1), Kok Hwee LIM1,2), Yusuke TOYAMA1,2,3)
1)

Mechanobiology Institute, Singapore 2)Temasek Life sciences Laboratory 3)Department of Biological
Science, National University of Singapore

At the apical side of epithelial tissues, the cell-cell boundaries form framework of the tissue, anddeformations in these boundaries, e.g. boundary contraction and elongation, and associated forces form themechanical basis of tissue morphogenesis. In such highly mechanical process, the adherens junction (AJ)
plays important roles for maintenance of tissue integrity, force transmission to neighbors, and regulation ofcell shape changes or rearrangements. Although it has been studied that the AJ has ability to react against
applied forces, we less know that how the AJ structures behave in boundary deformations that dynamically
evolve in time and in space. To understand the AJ's mechanotransduction mechanism and answer how the AJ
dynamics is involved in tissue morphogenesis, elucidations of mechanics underlying different boundary
dynamics, tracking their transitions, and observations of responces of the AJ molecules are essential.
Using amnioserosa cells, which exhibit rapid cell boundary oscillations (contraction and elongation; 1
period = 2~5 min) during Drosophila dorsal closure, we demonstrated that cell junctional tensions closely
correlated with boundary dynamics and shapes. Based on the correlations, we developed a method to noninvasively estimate the junctional tension over time, and found that the tension dynamically fluctuates during
boundary oscillations. Furthermore, we showed that an adherens junction (AJ) molecule, vinculin,
dynamically accumulates to or dissociates from oscillating boundary in a junctional-tension-dependent
manner (Hara et al., Curr. Biol., 2016).
Interestingly, our recent observations indicate that the other AJ components (E-cadherin, α-catenin, etc.)
also dynamically fluctuate its density and stability in response to the junctional tension changes. It suggeststhat the AJ molecules have abilities to respond rapidly against tension changes in order to modify the modeof cell boundary deformation or reorganize cell-cell junctions during tissue morphogenesis.
In this presentation, I will provide an overview of mechanics underlying cell boundary deformations in
Drosophila epithelium, and highlight the short-term dynamics of AJ components responding to rapid tension
changes.

E-cad

E-cad, Actin
HIGHER MOBILITY

p120

β-cat

?

Rapid
tensionchange(~5 min)

Vinc
α-cat

E-cad

Vinc
α-cat

p120

β-cat

?

Higher junctional tension

Lower junctional tension(e.g. contraction)(e.g. elongation)

Basolateral protrusion and apical contraction cooperatively drive Drosophila germband extension
Zijun Sun1, Christopher Amourda1, Murat Shagirov1, Yusuke Hara1, Timothy E. Saunders1,2,3, and Yusuke
Toyama1,2,4,*
1 Mechanobiology Institute, National University of Singapore, T-Lab, 5A Engineering Drive 1, Singapore
117411
2 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore
117543
3 Institute for Molecular and Cell Biology, Agency for Science Technology and Research, 61 Biopolis Dr,
Singapore 138673
4 Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore
117604Throughout development, tissues undergo complex morphological changes, resulting from cellularmechanics that evolve over time and in three-dimensional space. During Drosophila germ-band extension(GBE), cell intercalation is the key mechanism for tissue extension, and the associated apical junction
remodelling is driven by polarized myosin II-dependent contraction. However, the contribution of thebasolateral cellular mechanics to GBE remains poorly understood. Here, we characterize how cells
coordinate their shape from apical to basal side during rosette formation, a hallmark of cell intercalation.
Basolateral rosette formation is driven by cells mostly located at the dorsal/ventral part of the rosette. Thesecells exhibit actin-rich wedge-shaped basolateral protrusions and migrate towards each other. Surprisingly,
the formation of basolateral rosettes precedes that of the apical rosettes. We further show that basolateralrosette formation is independent of apical contractility, but requires Rac1-dependent protrusive motility.
Furthermore, we identified Src42A as a regulator of basolateral rosette formation. Our data show that inaddition to apical contraction, active cell migration driven by basolateral protrusions plays a pivotal role inrosette formation and contributes to GBE.
(Nature Cell Biology 19(4), 375-383, 2017)

Geometric constraints induce cellular skew and apical-to-basal
neighbor exchange in curved epithelial tissues
Anqi Huang1,*, Kok Haur Ong2,*, Jean-Francois Rupprecht1,*, Jianmin Yin1,*, Christopher P. Amourda1,
Matteo Rauzi4,¡, Lars Hufnagel4, Maria Leptin4, Weimiao Yu2, Timothy E. Saunders1,2,3
1 Mechanobiology Institute, National University of Singapore, Singapore
2 Institute of Molecular and Cell Biology, A*Star, Biopolis, Singapore
3 Department of Biological Sciences, National University of Singapore, Singapore
4 European Molecular Biology Laboratory Heidelberg, Heidelberg, Germany* These authors contributed equally (presented alphabetically)
Organ and tissue formation are complex three-dimensional processes involving cell division, growth,migration, and rearrangement, which all occur within physically constrained regions. However, analyzing
such processes in three dimensions in vivo is challenging. Here, we focus on the process of cellularizationin the anterior pole of the early Drosophila embryo to explore how cells compete for space under geometricconstraints. Using microfluidics combined with fluorescent microscopy, we extract quantitative informationon the three dimensional epithelial cell morphology. We observe a cellular membrane rearrangement inwhich cells exchange neighbors along the apical-basal axis. Such apical-to-basal neighbor exchanges areobserved more frequently in the anterior pole than in the embryo trunk. Further, cells within the anterior poleskew toward the trunk along their long axis relative to the embryo surface, with maximum skew on theventral side. We construct a vertex model for cells in a curved environment that reproduces the observed
cellular skew and demonstrates that geometric constraints can induce an asymmetric force on cells in theanterior. Overall, cell deformation and rearrangements appear to play important roles in cell packing in thehighly curved three-dimensional environment of the Drosophila embryo

in toto Quantitative Imaging Tools to study Drosophila Organogenesis
A. Singh, S. Tlili, K. Karkali, J. Munoz, E. Martin-Blanco and T. E. Saunders
How complex 3D organs are formed in a live organism is poorly understood- particularly in terms of its
control on size and function. Understanding its dynamic behavior, its shape and complex functions in livingorganism brings many challenges. We use Drosophila embryos as a model organism and in toto quantitative
imagining tools to study organogenesis - in particular, we focus on the ventral nerve cord formation during
embryogenesis. The ventral nerve cord is made from two types of cells, neurons and glial cells. The glial
cells provide mechanical support to neurons and they maintain ion-homeostasis. The ventral nerve cordspans nearly the entire embryo length and to access information about its three-dimensional topology
requires in toto imaging tools, such as light-sheet microscopy. We use quantitative image analysis –specifically three-dimensional PIV - to extract the dynamic changes in the ventral nerve cord shape along its
entire length in both wildtype and mutant backgrounds. Such an approach is allowing us to build up one of
the first complete atlases of internal, three-dimensional organ formation.

Influence of the biomechanical signalling on apico-basal polarisation
De Mets Richard1, Weng Zhe1, Yue Zhang1, Viasnoff Virgile 1,2
1 MechanoBiology Institute, National University of Singapore, Singapore
2 ESPCI ParisTech, Paris, France
Aims and/or Background: Cell capacity to answer to biochemical, topographical and rheologicalparameter of the environment has been extensively studied in the past years. One of its observablebehaviour is the cell polarity. Depending on the cell type, the cell is able to change its cell shape inorder to create actin structure allowing the cell, for example, to migrate in a preferential direction(mesenchymal cell line) or to extend its membrane to increase surface contact with the external media(epithelial cell line). Polarised epithelial cells form three different types of contact with theirsurrounding environment. At the basal side, facing the tissue, transmembrane protein called integrins
create link between the extracellular matrix (ECM) and the actin cytoskeleton (focal adhesion). On theside, transmembrane cadherins family maintain the integrity of the tissue by connecting the cell with
their neighbours (cell-cell junction). At the very tip of the cell-cell junction near the lumen, a special
structure called tight junction made by the interaction of transmembrane claudins is in charge of thecontrol of transport of ion within the intercellular space. The tight junction is connected to an actin
reinforced structure called actin belt via the ZOs proteins. A bad location of one of the protein in thetight junction can lead to different diseases going from bacterial or virus infection to cancer (1).
The goal of this study is to understand better the tight junction establishment and thebiochemical and mechanical cues influencing its formation via a bottom-up approach using
biocompatible
tune-able
microniches,andlaser-based
micropatterningtechnics.
Methods: Using laminated microniches, we were able to study the effect of biochemical andmechanical cues on apico-basal polarization. We manage, by coating with laser double patterningtechnics in 2D or by incubating separately the protein on the bottom and the side of the microwell, todistinguish the effect of ligand (Laminin, E-cadherin) signalling, combined with dimensionality, on
the tight junction formation. The read-out that we choose to observe is the localisation of different
apical markers (Podocalyxin, Moesin), tight junction markers (Claudin-3, Par-3, ZO-1, N-WASP),
and some structural markers (F-Actin, E-cadherin) on all those different configurations.
Results: We observed, by comparing apico-basal polarity markers and tight junction markers
localisation in 2D and 3D with the same kind of ligand signalling, that the dimensionality is importantto recruit Claudin-3, Par-3 and ZO-1 on the side of the microwell (Figure). We hypothesized that thisresult may be due to a difference of tension on the cell edge between 2D and 3D configuration.
Experiments with magnetic micro-beads has been planned in order to pull on the apical pole of thecell and see the effect on protein localisation.
We also observed than the double signalling (ECM, E-cadherin) in 3D microniches isessential to obtain a recruitment of Par3 on the extremity of the lateral side of the cell. However, wefailedtoobserveany
goodrecruitment
of
N-WASPonthosesystems.
Conclusion: With this study, we can conclude than the double signaling in a 3D environment isessential to obtain a good localization of Par-3 on the edge of the cell. We also observe the importanceof three-dimensionality to control good tight junction markers localization.
(1) Forster & al, HistoChem Cell Biol (2008)

A

B

C

D

E

F

Figure 1: Protein localization depeding on the types of cues that the cell is experiencing. A 3D
microwell with Ecadherin on the side, Laminin at the bottom. B 3D microwell with Ecadherin
everywhere. C 3D microwell with Laminin everywhere. D Epithelial cell in a monolayer. E 2D
micropattern of Ecadherin. F 2D micropattern of Laminin.

A novel DNA binding mode of H-NS drives chromosome
compaction and gene silencing in single cells
Yunfeng Gao1, Yong Hwee Foo1, Ricksen S. Winardhi1, Qingnan Tang1,
Jie Yan1,2 and Linda J. Kenney1,3,4*
1

Mechanobiology Institute, National University of Singapore; 2Department of Physics, National Universityof Singapore; 3Jesse Brown Veterans Administration Medical Center, Chicago, IL; 4Department ofMicrobiology, University of Illinois-Chicago, Chicago, IL
Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise
mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. HNS possesses two functional domains, an N-terminal oligomerization domain and a C-terminal DNA
binding domain separated by a flexible linker. The linker is suspected to play an important role inpromoting H-NS and DNA interaction. We compared the localization of wt H-NS and a linker deletionmutant ΔL with the nucleoid in bacteria cells using super-resolution microscopy (single-molecule
localization microscopy; SMLM). It was observed that in the ΔL linker mutant, the H-NS protein does
not localize with the nucleoid, while the wt H-NS form foci that overlap with the nucleoid. Using
DBSCAN, we show that the sizes of the clusters are 273 ± 108 nm, with 26.4% of the H-NS inside theseclusters. The cell lengths of the ΔL linker mutant are longer, with larger nucleoids, suggesting that
nucleoid compaction is affected. Next, we applied single-particle tracking-photoactivatable localization
microscopy (sptPALM) in live cells to quantitate the amount of binding between wt H-NS / ΔL with thenucleoid. We showed that 95% of the wt H-NS are bound to the nucleoid, while in the ΔL linker mutant,
only 21% are bound, 55% having weak interaction and 24% not bound. These observations identify thelinker as being essential for the binding of H-NS to DNA and for the appearance of foci in superresolution images. Supported by the RCE in Mechanobiology from the Ministry of Education, Singapore

Remodeling of adhesion and modulation of mechanical tensile
forces during apoptosis in Drosophila epithelium
Xiang Teng1,2), Lei Qin2, Roland Le Borgne3,4) and Yusuke Toyama1,2,5,*)
1)

Department of Biological Sciences, National University of Singapore, Singapore.,2)

3)

Mechanobiology Institute, National University of Singapore, Singapore.

CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Rennes, France.
4)

Universite Rennes 1, Faculte de Medecine, Rennes, France.
5)

Temasek Life Sciences Laboratory, Singapore.

Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue
homeostasis (a). During apoptosis within a tissue, the adhesions between dying and neighboring non-dying
cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosincables formed in apoptotic and neighboring cells drives cell extrusion (b). To date, the coordination
between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic
cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue
replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs)
between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including Ecadherin. Concurrently, surrounding tissue tension is transiently released (c). Contraction of a supracellular actomyosin cable, which forms in neighboring cells (d), brings neighboring cells together andfurther reshapes tissue tension toward the completion of extrusion (e). We propose a model in which
modulation of tissue tension represents a mechanism of apoptotic cell extrusion.