FY2017 Annual Report

Abstract

Our team is dedicated to
(1) methodology development for single-molecule imaging and manipulation at nanometer precisions in living cells, with a special attention paid to high time resolutions (ultrafast single fluorescent-molecule tracking), as well as
(2) revealing meso~nano-scale processes in signal transduction in/on the cell membrane and the formation and remodeling of the neuronal network, by using the developed single-molecule techniques.
The smooth liaison between physics/engineering and biomedicine is a key for our research. Based on unique insights we develop by applying single-molecule tracking methods to nano~meso-scale processes occurring in signal transduction and neuronal network formation/modulation, we intend to develop new types of systems molecular biology.
In the same context, 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 various plasma membrane functions.

1. Staff

Dr. Amine Betul Nuriseria Aladag, Post Doctoral Scholar
Dr. An-An Liu, JSPS Research Fellow
Dr. Peng Zhou, Post Doctoral Scholar
Dr. Irina Meshcheryakova, Technician
Ms. Limin Chen, Technician
Ms. Aya Nakamura, Technician
Ms. Yuri Nemoto, Technician
Mr. Taka-aki Tsunoyama, Technician
Mr. Alexey Yudin, Technician
Mr. Shogo Miyagi, Technician (Part-time)
Mr. Masaya Negawa, Technicien (Part-time)
Ms. Miwako Tokuda, Research Unit Administrator
Dr. Akihiro Kusumi, Professor

2. Collaborations

2.1 Revealing metastable signaling molecular complexes by single-molecule imaging

- Description: Developing ultrafast 3D single-molecule imaging, and applying it to 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 of Advanced Studies, Kyoto University
      Dr. Nao Hiramoto-Yamaki, Postdoctoral Research Associate, Institute of Advanced Studies, Kyoto University
      Dr. Akio Tsuboi, Professor, Nara Medical University
      Dr. Kenichi Suzuki, Professor, G-CHAIN, Gifu University

2.2 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
      Mr. Bo Tang, PhD candidate
      Ms. Meng-ni Bao, PhD candidate
      Ms. Dan-dan Fu, PhD candidate
      Ms. Jing Li, PhD candidate
College of Chemistry and Molecular Sciences, Wuhan University, P. R. China

2.3 Unraveling single-molecule dynamics of neurotransmitter receptors in neurons

- Description: Examining dynamic monomer-oligomer equilibrium of neurotransmitter receptors and their entrance-exiting dynamics into and out of the synapses

- Type of collaboration: Joint research

- Researchers:
      Dr. Jyoji Morise, Assistant Professor, Graduate School of Medicine, Kyoto University
      Dr. Shogo Oka, Professor, Graduate School of Medicine, Kyoto University
      Dr. Kenichi Suzuki, Professor, G-CHAIN, Gifu University
      Dr. Takahiro Fujiwara, Associate Professor, Institute of Advanced Studies, Kyoto University

2.4 Revealing regulation systems of synapse dynamics based on nanoscale morphological analysis

- Description: Revealing dynamic regulation systems for molecular compositions in the synapse using 3D reconstruction electron microscopy and super-resolution fluorescence microscopy

- Type of collaboration: Joint research

- Researchers:
Dr. Shigeo Okabe, Professor, Graduate School of Medicine, The University of Tokyo
Dr. Yasunori Inoue, Professor, Graduate School of Engineering, Kyoto University

3. Activities and Findings

3.1 Revealing metastable signaling molecular complexes by developing new single-molecule imaging methods

In this project, we develop new single fluorescent-molecule imaging-tracking methods and new fluorescent probes, including ultrafast (world’s fastest) single-molecule imaging, methods for suppressing photobleaching and photoblinking for very long single molecule tracking (several minutes) in living cells, and new fluorescent lipid probes that behave very much like their parent endogenous lipid molecules. By applying the developed methods and probes, we try to reveal the dynamics and formation mechanism of metastable signaling molecular complexes in the context of synaptic signaling, Fcepsilon signaling, focal adhesion architecture and signaling, and GPI-anchored proteins’ raft-based signaling. One of the major developments on this front in the FY2017 is described below.

Revealing raft-based sphingomyelin interactions by developing new fluorescent sphingomyelin analogs

Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs.

Here, we developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethyleneglycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations.

Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.

3.2 Unraveling the regulation mechanisms for the synaptic structural plasticity by observing dynamics, assembly, and function of neuronal receptors using super-resolution single-molecule imaging and tracking

In this project, we try to understand the mechanisms by which structural synaptic plasticity is induced. To accomplish this goal, we examine, at the level of single molecules, molecular interactions and dynamics in hippocampal neurons in culture. In particular, we examine the dynamic equilibrium of monomers, dimers, oligomers, and clusters of neurotramsmitter receptors and other neuronal molecules, the molecules’ cooperative interactions, including the possibility of phase separation, as well as their entrance-exiting dynamics into and out of the synapses. One of the major advancements on this front in the FY2017 is described below.

Unraveling dynamic meso-scale anchorage of GPI-anchored receptors, Prion Protein and Thy1, in the neuronal plasma membrane using super-resolution single-molecule tracking

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homodimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown.

Here, using single fluorescent-molecule imaging, we found that both prion protein and Thy1, a control glycosylphosphatidylinositol (GPI)-anchored protein, exhibited very similar intermittent transient immobilizations lasting for a few seconds within an area of 24.2 and 3.5 nm in diameter in CHO-K1 and hippocampal neurons cultured for 1- and 2-weeks, respectively. Prion protein molecules were immobile during 72% of the time, approximately 1.4× more than Thy1, due to prion protein’s higher immobilization frequency. When mobile, prion protein diffused 1.7× slower than Thy1. Prion protein’s slower diffusion might be caused by its transient interaction with other prion protein molecules, whereas its brief immobilization might be due to temporary association with prion protein clusters.

Prion protein molecules might be newly recruited to prion protein clusters all the time, and simultaneously, prion protein molecules in the cluster might be departing continuously. Such dynamic interactions of normal prion protein moleculeswould strongly enhance the spreading of misfolded prion protein.

3.3 Examining and refining our working hypothesis, in which the bulk plasma membrane could be largely considered to be hierarchically organized in three-tiered meso-scale domains, particularly in the context of signal transduction

As described in the home page of our web site, we think the concept of three-tiered meso-scale domain architecture of the plasma membrane provides an excellent perspective on the mechanisms for various functions of the plasma membrane. “Meso” means “between” and the meso-scale generally speaks to the scale between nanometer and micrometer. Often, the actual scale of meso is between 3 and 300 nm. It is an interesting scale where non-living molecules turn into living cells.

The first and most basic tier in this hierarchical architecture is the membrane compartments, formed due to the partitioning of the entire plasma membrane by the actin-based membrane skeleton. We think it is the most basic tier because it is everywhere throughout the plasma membrane and it dominates the movements of all the molecules associated with the plasma membrane.

The second tier is the raft domains, which are localized within the membrane compartments.

The third tier is dynamic protein complexes, with lifetimes often of the order of 0.1 seconds. And so, these are metastable or transient molecular complexes.

Of course, in the real plasma membrane, these three domains coexist in a single membrane and work in concert to enable various plasma membrane functions.

We at the Kusumi lab are examining and refining this working hypothesis. By performing such research, we hope to obtain better perspectives on how the plasma membrane is organized or poised to perform various plasma membrane functions, particularly the signal transduction. One of the major advancements on this front in the FY2017 is described below.

Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. We examined cortical actin structures and dynamics in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging.

The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner.

Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters.

The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface.

Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”.

4. Publications

ORIGINAL ARTICLES

A. Makino, M. Abe, R. Ishitsuka, M. Murate, T. Kishimoto, S. Sakai, F. Hulin-Matsuda, Y. Shimada, T. Inaba, H. Miyatake, H. Tanaka, A. Kurahashi, C.-G. Pack, R. Kasai, S. Kubo, N. L. Schieber, N. Dohmae, N. Tochio, K. Hagiwara, Y. Sasaki, Y. Aida, F. Fujimori, T. Kigawa, K. Nishibori, R. G. Parton, A. Kusumi, Y. Sako, G. Anderluh, M. Yamashita, T. Kobayashi, P. Greimel, and T. Kobayashi. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Nieman-Pick type C. FASEB J. 31, 1301-1322 (2017). doi: 10.1096/fj.201500075R

M. Kinoshita, K. G. N. Suzuki, N. Matsumori, M. Takada, H. Ano, K. Morigaki, M. Abe, A. Makino, T. Kobayashi, K. M. Hirosawa, T. K. Fujiwara, A. Kusumi (Co-Corresponding author), and M. Murata. Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs. J. Cell Biol. 216, 1183-1204 (2017). doi: 10.1083/jcb.201607086

S. Wakayama, S. Kiyonaka, I. Arai, W. Kakegawa, S. Matsuda, K. Ibata, Y. L. Nemoto, A. Kusumi, M. Yuzaki, and I. Hamachi. Chemical labeling for visualizing native AMPA 1 receptors in live neurons. Nat. Commun. 8, 14850 (2017). doi:10.1038/ncomms14850

Y. L. Nemoto, R. J. Morris, H. Hijikata, T. A. Tsunoyama, A. C. E. Shibata, R. S. Kasai, A. Kusumi (Co-Corresponding author), and T. K. Fujiwara. Dynamic meso-scale anchorage of GPI-anchored receptors in the plasma membrane: prion protein vs. Thy1. Cell Biochem. Biophys. 75, 399-412 (2017). doi:10.1007/s12013-017-0808-3

Y. M. Shirai, T. A. Tsunoyama, N. Hiramoto-Yamaki, K. M. Hirosawa, A. C. E. Shibata, K. Kondo, A. Tsurumune, F. Ishidate, A. Kusumi (Co-Corresponding author), and T. K. Fujiwara. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy. PLoS ONE 12, e0188778 (2018). doi: 0.1083/jcb.201607086

INVITED REVIEW ARTICLES

K. G. N. Suzuki, H. Ando, N. Komura, T. Fujiwara, M. Kiso, and A. Kusumi. Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging. Biochim. Biophys. Acta - General Subjects (Review in a special issue on Neuro-glycoscience) 1861, 2494-2506 (2017). doi: 10.1016/j.bbagen.2017.07.012

N. Komura, K. G. N. Suzuki, H. Ando, M. Konishi, A. Imamura, H. Ishida, A. Kusumi, and M. Kiso. Synthesis of fluorescent gangliosides for the studies of raft domains. Methods Enzymol. 597, 239-263 (2017). doi: 10.1016/bs.mie.2017.06.004.

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

INVITED PRESENTATIONS

A. Kusumi. Very transient molecular complexes enable signal transduction: findings by single-molecule tracking. IUPAC International Congress on Analytical Sciences 2017. Haikou, Hainan Province, China. May 7, 2017.

A. Kusumi. Single-molecule view of the plasma membrane organization for signal transduction. Closing Plenary Lecture. Federation of European Neuroscience (FENS) Regional Meeting 2017. Pécs, Hungary. September 2017.

A. Kusumi. Single-molecule tracking detection of very transient signaling molecular complexes. The Second Adriatic Symposium on Biophysical Approaches in Biomedical Studies. The Mediterranean Institute for Life Sciences, Split, Croatia. September 2017.

A. Kusumi. Signal transduction by transient molecular complexes: findings by single-molecule tracking. 5th European Joint Theoretical/Experimental Meeting on Membranes (EJTEMM2017). Jagiellonian University, Krakow, Poland. 6-8 December 2017.

A. Kusumi. Signal transduction by transient molecular complexes: findings by single-molecule tracking. Biophysics and Systems Biology Seminar Series. University of California, Irvine. February 22, 2018.

ORAL PRESENTATIONS (Selected for Oral Presentation)

T. A. Tsunoyama, K. G. N. Kenichi, T. K. Fujiwara, and A. Kusumi. Super-long single fluorescent-molecule tracking revealed tension-dependent dynamic anchorage of integrin for cell adhesion. The 62nd Annual Meeting of Biophysical Society of U.S.A. 2017, San Francisco, U.S.A. February 18, 2018.

P. Zhou, R. S. Kasai, K. M. Hirosawa, A. Yudin, Y. M. Shirai, T. K. Fujiwara, and A. Kusumi. Transient hetero-dimerization of opioid receptors (GPCRs) and their formation mechanisms revealed by single-molecule tracking. The 62nd Annual Meeting of Biophysical Society of U.S.A. 2017, San Francisco, U.S.A. February 19, 2018.

ORAL PRESENTATIONS (General)

A. Yudin, T. K. Fujiwara, T. A. Tsunoyama, and A. Kusumi. Dynamic mesoscale anchorage of GPI-anchored receptors prion protein and Thy1 in the cell membrane as revealed by single molecule tracking. The 55th Annual Meeting of Biophysical Society of Japan 2017, Kumamoto, Japan. September 19, 2017.

A. Liu, Y. Kudo, S. Liu, K. Suzuki, T. Fujiwara, D. Pang, S. Leppla, and A. Kusumi. Actin polymerization signal emitted at the raft nanodomains of the clusters of the anthrax-toxin-receptor complex: a single-molecule study. The 55th Annual Meeting of the Biophysical Society of Japan. Kumamoto, Japan. September 19, 2017.

POSTER PRESENTATIONS

Y. L. Nemoto, R. J. Morris, H. Hijikata, T. A. Tsunoyama, A. C. E. Shibata, R. S. Kasai, A. Kusumi, and T. K. Fujiwara. Dynamic mesoscale anchorage of GPI-anchored receptors prion protein and Thy1 in the cell membrane as revealed by single molecule tracking. The 55th Annual Meeting of Biophysical Society of Japan 2017, Kumamoto, Japan. September 21, 2017.

Y. L. Nemoto, R. J. Morris, H. Hijikata, T. A. Tsunoyama, A, C. E. Shibata, R. S. Kasai, A. Kusumi, and T. K. Fujiwara. Dynamic meso-scale anchorage of GPI-anchored receptors, prion protein and Thy1, in the plasma membrane; detection by single-molecule imaging. Consortium of Biological Sciences 2017, Kobe, Japan. December 8, 2017.

A. Yudin, T. K. Fujiwara, T. A. Tsunoyama, and A. Kusumi. Evaluating the permeability across the actin-based compartment barrier in the plasma membrane from single-molecule trajectories. The 62nd Annual Meeting of Biophysical Society of U.S.A., San Francisco, U.S.A. February 18, 2018.

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

The 85th Membrane Seminar

     Speaker: Prof. Thomas Blanpied, Department of Physiology, University of Maryland School of Medicine
     Title: Nanostructure and alignment control function of single synapses
     Date: 9 March 2018
     Venue: Central Building C209

7. Other

Nothing to report.