FY2021 Annual Report

Membrane Cooperativity Unit
Professor Akihiro Kusumi


We at the Membrane Cooperativity Unit are working hard to reveal how the dynamic platforms for signal transduction and the synapses for the neuronal transmission form and function in the plasma membrane. For this purpose, we take a unique approach (in addition to other more conventional approaches). Namely, we develop new and unique methods for single-molecule imaging and manipulation at nanometer precisions in living cells, with a special attention paid to high time resolutions (world’s fastest single fluorescent-molecule imaging). The smooth liaison between physics/engineering and biomedicine is a key for our research. 

The plasma membrane is the outermost membrane of the cell, and thus it encloses the entire cell. It is critically important for the cell - the fundamental unit of life - because it defines the space for it. The plasma membrane exchanges information, energy, and substances with the outside world, and we pay special attention to the mechanism for signal transfer from outside to inside the cell, a function generally called “signal transduction”. In the signal transduction process, the plasma membrane works like a sensor + computer + effector.

The Membrane Cooperativity Unit strives to understand how the plasma membrane works at very fundamental levels, based on unique insights we obtain by applying single-molecule imaging-tracking methods. More specifically, we are now revealing the mechanisms by which the metastable molecular complexes and meso-scale membrane domains, including membrane compartments, raft domains, and protein oligomers, form and work in concert to enable signal transduction and synapse formation/modulation in/on the plasma membrane. 

1. Staff

  • Dr. Amine Betul Nuriseria Aladag, Post Doctoral Scholar
  • Dr. HooiCheng Lim, Post Doctoral Scholar
  • Dr. Taka-Aki Tsunoyama, Post Doctoral Scholar
  • Dr. Peng Zhou, Post Doctoral Scholar 
  • Dr. Tim Yeh, Visiting Professor (on sabbatical leave from the University of Texas at Austin)
  • Dr. Irina Meshcheryakova, Technician
  • Mr. Jun-Seok Lee, Technician
  • Ms. Limin Chen, Technician 
  • Ms. Hiroko Hijikata, Technician
  • Ms. Aya Nakamura, Technician
  • Ms. Yuri Nemoto, Technician
  • Mr. Hugo Musset, Rotation Student
  • Mr. Esteban Gabriel Fregoso Fernandez, Rotation Student
  • Mr. Jerome Theodore Tinker, Rotation Student
  • Mr. Takatoshi Kinjo, Research Intern
  • Mr. Tatsuhiro Nishi, Research Assistant (Part-time)
  • Ms. Yuka Nakadomari, Research Assistant (Part-time)
  • Mr. Ryoga Maeda, Research Assistant (Part-time)
  • Mr. Yuta Kogi, Research Assistant (Part-time)
  • Mr. Yusuke Higa, Research Assistant (Part-time)
  • Mr. Rin Nakama, Research Assistant (Part-time)
  • Ms. Sachie Matsuoka, Research Unit Administrator
  • Dr. Akihiro Kusumi, Professor

2. Collaborations

2.1 Revealing the dynamics, structure, and function of metastable signaling molecular complexes by single-molecule imaging

  • Description: Developing ultrafast 3D single-molecule imaging, and applying it to revealing the dynamics and formation mechanism of the signaling complex in synaptic signaling, Fcepsilon signaling, focal adhesion architecture and signaling, and GPI-anchored proteins’ raft-based signaling
  • Type of collaboration: Joint research
  • Researchers: 
    • Dr. Takahiro Fujiwara, Associate Professor, Institute for Integrated Cell-Material Sciences (iCeMS), Institute of Advanced Studies, Kyoto University
    • Dr. Kenichi Suzuki, Professor, G-CHAIN, Gifu University

2.2 Unraveling the large-scale molecular-species selective diffusion barriers in the axonal initial segment in the neuron using ultrafast single-molecule imaging

  • Description: By applying ultrafast single-molecule imaging and ultrafast single-molecule localization microscopy developed by us, we try to unravel the large-scale molecular-species selective diffusion barriers in the axonal initial segment in the neuron
  • Type of collaboration: Joint research
  • Researchers: 
    • Dr. Takahiro Fujiwara, Associate Professor, Institute for Integrated Cell-Material Sciences (iCeMS), Institute of Advanced Studies, Kyoto University

2.3 Elucidation of dynamics and formation mechanisms of cellular signaling complexes by developing new single particle tracking methods

  • Description: Developing fluorescent probes for their applications to single-molecule imaging in living cells, and by using the developed probes, elucidating dynamics and formation mechanisms of cellular signaling complexes induced by various intercellular signaling molecules and alien antigens, including (non-pathogenic) viruses 
  •  Type of collaboration: Joint research
  •  Researchers:
    • Dr. Dai-Wen Pang, Professor
    • Dr. An-An Liu, lecturer
    • Ms. Dan-dan Fu, PhD candidate

College of Chemistry and Molecular Sciences, Wuhan University, P. R. China

2.4 Elucidating the functions of plasma membrane compartmentalization

  • Description: Elucidating how the signal transduction functions of the plasma membrane is regulated using the actin-based compartmentalization of the plasma membrane, using ultrafast single-molecule imaging-tracking and super-resolution microscopy 
  • Type of collaboration: Joint research
  • Researchers: 
    • Dr. Pakorn Tony Kanchanawong, Professor, Mechanobiology Institute, The National University of Singapore

2.5 Development of deep-learning methods for single-molecule imaging experiments and analysis 

  • Description: Developing AI-based methods for performing single-molecule imaging and for analyzing single-molecule imaging data
  • Type of collaboration: Joint research
  • Researchers:
    • Dr. Kazuhiro Hotta, Professor, Mechanobiology Institute, Department of Electrical and Electronic Engineering, Faculty of Engineering, Meijo University

2.6 Revealing the mechanisms for the synapse formation and long-term potentiation by combining super-resolution microscopy and single-molecule imaging 

  • Description: To discover the mechanisms for functional and structural synaptic plasticity underlying learning and memory, by the combined use of super-resolution microscopy and single-molecule imaging
  • Type of collaboration: Joint research
  • Researchers:
    • Dr. Michisuke Yuzaki, Professor, Graduate School of Medicine, Keio University 


3. Activities and Findings

3.1 Development of ultrafast single-molecule imaging and ultrafast PALM/STORM (Fujiwara et al. 2021a, b; bioRxiv).

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the time resolutions of single-molecule imaging-tracking of ≤0.033 (0.1) ms, with a localization precision of 30 (20) nm, fastest achieved thus far, which is now limited by photophysics of the fluorophores (thus the ultimate rate achievable with available fluorescent dye molecules). Ultrafast PALM and STORM with a data collection rate of 1 kHz for a large 640x640-pixel view-field, again limited by the fluorophore photophysics were also developed. Using the developed ultrafast camera system, we unequivocally revealed that molecules in the plasma membrane (PM) undergo hop diffusion in both the apical and basal PMs, due to the actin-induced membrane skeleton meshwork and that the focal adhesion (FA) is a fluid partitioned into ~74-nm compartments, supporting the model of the actin-induced compartmentalized archipelago of FA-protein islands. By simultaneously performing ultrafast PALM and single-molecule imaging in living cells, we revealed that integrins undergoing hop diffusion in the FA zone intermittently associate/dissociate from the FA-protein islands, dynamically linking the FA-protein islands to the extracellular matrix.

3.2 Discovery of the signal integration platform, iTRVZ (Tsunoyama et al. 2022 bioRxiv)

We found a nanometer-scale liquid-like protein platform for integrating the signals downstream from GPI-anchored receptors and receptor-type tyrosine kinases, inducing the non-linearly amplified PLCg-IP3-Ca2+ signal outputs via the mutual activations of focal adhesion kinase and Src-family kinases. The platform employs some of the focal adhesion proteins, including integrin, talin, RIAM, VASP, and zyxin, but is distinct from focal adhesions, and is thus termed iTRVZ. The iTRVZ formation is driven by the protein liquid-liquid phase separation and the interactions with the raft domains in the plasma membrane and cortical actin. iTRVZ non-linearly integrates the two distinctly different receptor signals, and thus works as an AND logic gate and noise filter. Dwell lieftimes of the iTRVZ constituent molecules and signaling molecules recruited to iTRVZ are in the range of 0.1 ~ 2 s, whereas the iTRVZ lifetime is ~10 s, allowing dynamic signal regulation and secure signal switching off.


4. Publications

4.1 Journals

Original Articles

  1. B. Tang, E.-Z. Sun, Z.-L. Zhang, S.-L. Liu, J. Liu, A. Kusumi, Z. Hu, T. Zeng, Y.-F. Kang, H.-W. Tang, and D.-W. Pang. Sphingomyelin-sequestered cholesterol domain recruits formin-binding protein 17 for constricting clathrin-coated pits in influenza virus entry. J. Virol. In press (2022). doi: 10.1128/JVI.01813-21 
  2. S. Godó, K. Barabás, F. Lengyel, D. Ernszt, T. Kovács, M. Kecskés, C. Varga, T. Z. Jánosi, G. Makkai, G. Kovács, B. Orsolits, T. K. Fujiwara, A. Kusumi, and I. M. Abraham. Single-molecule imaging reveals rapid estradiol action on the surface movement of AMPA receptors in live neurons. Front. Cell Dev. Biol. 9: 708715 (2021). doi: 10.3389/fcell.2021.708715
  3. K. Barabás, J. Kobolák, S. Godó, T. Kovács, D. Ernszt, M. Kecskés, C. Varga, T. Z. Jánosi, T. Fujiwara, A. Kusumi, A. Téglási, A. Dinnyés, and I. M. Abraham. Live-cell imaging of single neurotrophin receptor molecules on human neurons in Alzheimer’s disease. Int. J. Mol. Sci. 22: 13260 (2022). doi: 10.3390/ijms.222413260

Manuscripts in the preprint server bioRxiv

  1. T. K. Fujiwara, S. Takeuchi, Z. Kalay, Y. Nagai, T. A. Tsunoyama, T. Kalkbrenner, K. Iwasawa, K. P. Ritchie, K. G. N. Suzuki, and A. Kusumi. Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM. bioRxiv 10.26.465864 (2021); https://doi.org/10.1101/2021.10.26.465864
  2. T. K. Fujiwara, S. Takeuchi, Z. Kalay, Y. Nagai, T. A. Tsunoyama, T. Kalkbrenner, K. Iwasawa, K. P. Ritchie, K. G. N. Suzuki, and A. Kusumi. Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion. bioRxiv 10.26.465868 (2021); https://doi.org/10.1101/2021.10.26.465868.
  3. T. A. Tsunoyama, C. Hoffmann, B. Tang, K. M. Hirosawa, Y. L. Nemoto, R. S. Kasai, T. K. Fujiwara, K. G. N. Suzuki, D. Milovanovic, and A. Kusumi. iTRVZ: Liquid nano-platform for signal integration on the plasma membrane.  bioRxiv 10.1101/2021.12.30.474523; https://biorxiv.org/cgi/content/short/2021.12.30.474523v1 (2021).


4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

Oral Presentations

  1. Y.L. Nemoto, R.S. Kasai, H. Hijikata, T.A. Tsunoyama, K. Naito, N. Hiramoto-Yamaki, T.K. Fujiwara, A. Kusumi. Synaptic anchorage of AMPA receptors mediated by their direct binding to PDZ proteins and dynamic interactions with TARPγ-2, The Annual Meeting of the Biophysical Society of Japan, Online, from Nov.25-27, 2021

  2. Saahil Acharya, Taka A. Tsunoyama, Irina Meshcheryakova, Aya Nakamura, Hiroko Hijikata, Yuri Nemoto, Takahiro K. Fujiwara, Akihiro Kusumi. Oligomerization of neuronal receptors is essential for assembly and function of the synapse, The Annual Meeting of the Biophysical Society of Japan, Online, from Nov.25-27, 2021


5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

6.1 Seminar Series by Prof. Tim Yeh

  • Date: Apr. 26, May 10, May 17 and May 24
  • Venue: OIST Campus Center Building C209 
  • Speaker: Dr. Tim Yeh (University of Texas at Austin) 
  • Themes
    • 4pi microscopy including iPALM, 4pi-SMS, multifocus microscopy, light-field microscopy
    • Hyperspectral imaging and FRAP
    • SIM, STED, SMLM (sptPALM)
    • Rapid 3D volumetric imaging in tissues using multiphoton microscopy
    • Optical tweezers
    • Living-cell imaging techniques


7. Other

Nothing to report.