FY2021 Annual Report

Principal Investigator: Ichiro Maruyama
Research Theme: Information Processing by Life



In order to survive, animals must closely monitor environmental changes, and must keep the memory as experiences to adjust their behavior to the environment. We are interested in understanding how neuronal networks process environmental information to regulate animal behaviors, including decision-making, learning and memory. We are also interested in the effect of aging on the cognitive and mechanical behaviors. In previous years, we developed protocols to study associative learning and memory in the nematode Caenorhabditis elegans as a model organism. C. elegans is innately attracted to propanol, a short-chain alcohol, and avoids acid such as pH 4.0. After repeated conditioning C. elegans with propanol and acid, it associates the two stimuli and avoid propanol. C. elegans retains the memory up to 24 hours as long-term associative memory. We have been trying to identify neuronal circuits responsible for the memory trace in the C. elegans nervous system by using multiple techniques including genetic rescue experiments, Ca2+ imaging and electrophysiological analyses of neuronal activity. Through forward genetic screens, we isolated C. elegans mutants that progressively lose locomotor ability in early adulthood.

We are also interested in understanding how neurons/cells detect extracellular information and transmit it inside the cell. For the last three decades, ligand-induced dimerization has been widely assumed to be a common property of transmembrane signaling by receptors for all known growth factors and cytokines among others. In previous years, however, we found that receptors for epidermal growth factor (EGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) adopt a preformed, yet inactive, dimeric structure prior to ligand binding. We have also found that a receptor-type guanylyl cyclase, GCY-14 in C. elegans, adopts an obligatory homodimeric form on the cell surface of a sensory neuron. Based on the results, we proposed an alternative allosteric model, the ‘rotation model’, for the activation of preformed cell surface receptor dimers, where ligand binding to the extracellular domains induces rotation of the transmembrane domains parallel to the plane of the membrane. This transmembrane rotation rearranges the intracellular domain dimers for activation. Optical single molecule observation showed that preformed EGF receptor dimer is activated by a single ligand binding event, consistently with negative cooperativity of ligand binding to the EGF receptor.

These results provide insights into the molecular mechanism underlying information transfer from the outside of neurons/cells to the inside, as well as an understanding of neuronal networks responsible for cognitive and mechanical behaviors. The findings may also be invaluable in developing pharmaceuticals for human diseases such as cancers and mental diseases.

1. Staff

  • Higor Alves Iha (until November 30, 2021)
  • Takashi Murayama
  • Hitomi Ohtaki
  • Eiichiro Saita
  • Mihiro Shibutani (from April 12, 2021)
  • Tsukasa Shimojima
  • Rajiv Singh 
  • Suteera Viboonyasek

2. Graduate and other students

  • Dong Cao (until December 17, 2021)
  • Hinako Namihira (December 13, 2021 - March 31, 2022)

3. Collaborations

3.1 Functional analyses of small G protein and their downstream proteins

  • Type of collaboration: Joint research
  • Researchers:
    • Ken-ichi Kariya, University of the Ryukyus
    • Tsuyoshi Asato, University of the Ryukyus
    • Kimiko Nonaka, University of the Ryukyus

3.2 Study of signal transduction pathways that regulate cellular functions

  • Type of collaboration: Joint research
  • Researchers:
    • Ken-ichi Kariya, University of the Ryukyus
    • Tsuyoshi Asato, University of the Ryukyus
    • Kimiko Nonaka, University of the Ryukyus

3.3 Search of the new cancer stem cell marker of spinocellular carcinoma

  • Type of collaboration: Joint research
  • Researchers:
    • Ken-ichi Kariya, University of the Ryukyus
    • Tsuyoshi Asato, University of the Ryukyus
    • Kimiko Nonaka, University of the Ryukyus

4. Activities and Findings

4.1 Project Aims

All forms of life are separated from their environments by cell membranes, and all neurons/cells have cell-surface receptor proteins that span the membrane in order to transmit external information, such as environmental changes and cell-cell communications, inside the cell. Such information flow is fundamental for all forms of life, from bacteria to humans. Dysregulation of cell surface receptor molecules causes a variety of impairments, including mental and developmental diseases and cancer in humans. (1) We wish to understand the information processing through cellular networks. Specifically, we want to know how the external information is sensed and transmitted through sensory neurons, how it is processed by the nervous system, how it controls animal behaviors, including decision-making, learning and memory, and how aging affects the cognitive and behavioral capabilities. (2) We wish also to understand at the molecular level how the external information is sensed and transmitted into the inside of neurons/cells by cell-surface receptors.

4.2. Information processing by the nervous system


C. elegans is a model organism suited for the study of complex behaviors and cognition. The hermaphrodite nervous system consists of only 302 neurons, and neural circuits, chemical synapses, and electrical gap junctions of these invariant neurons have completely been reconstructed from serial thin sections of electron micrographs. The worm body is transparent throughout its life so that neural activities can directly be observed using Ca2+-sensitive or voltage-sensitive fluorescent proteins in living animals. C. elegans provides a unique opportunity to study lifespan and healthspan due to the availability of genetic techniques and its relatively short lifespan.

In 1975, associative learning in C. elegans was first suggested from the finding that worms return to their temperature of cultivation if they had food at that temperature. Most of learning paradigms in C. elegans are based on pairing chemical cues with food or starvation. Rather than pairing chemical cues with food or starvation, however, it would be preferable for subsequent analysis of neural circuits responsible for associative learning and memory to use two defined chemical cues for conditioning of animals in associative learning paradigms.

Previously, we found that C. elegans is attracted to mildly alkaline pH, but is repelled by strongly alkaline pH. This is reasonable as mildly alkaline pH is a signal for the presence of food, while strongly alkaline pH is harmful for the animal. We are interested in how the animal's behavioral switch (decision-making) is regulated by the nervous system and how aging. 

In previous years, we have developed protocols for classical conditioning of C. elegans with 1-propanol, as a conditioned stimulus (CS) and hydrochloride (HCl), pH 4.0, as an unconditioned stimulus (US). Before conditioning, animals were innately attracted to 1-propanol, and avoided HCl in chemotaxis assays. However, after spaced or massed training, animals were either not attracted at all or were repelled by propanol on the assay plate (aversive associative learning). The memory after the spaced training was retained for 24 hours, while the memory after the massed training did not even persist for 3 hours. Animals pretreated with transcription and translation inhibitors failed to form the memory by spaced training, whereas the memory after massed training was not significantly affected by inhibitors and was sensitive to cold-shock anesthesia. Furthermore, memory after spaced training was reasonably disrupted by extinction learning, in which the worm is repeatedly exposed to the CS in the absence of US. Therefore, memories after spaced and massed training can be classified as LTM and STM, respectively. Consistently, like other organisms including AplysiaDrosophila, and mice, C. elegans mutants defective in nmr-1, encoding an NMDA receptor subunit, failed to form both long-term associative memory (LTM) and short-term associative memory (STM), while mutations in crh-1 encoding the CREB transcription factor affected only LTM (Amano & Maruyama, 2011). We have also developed protocols for appetitive associative learning in C. elegans using 1-nonanol as a CS and KCl as a US. The animal avoids 1-nonanol before conditioning, and is attracted to 1-nonanol after conditioning with both 1-nonanol and KCl.  Again, C. elegans mutants defective in nmr-1, encoding an NMDA receptor subunit, failed to form both LTM and STM, while mutations in crh-1 encoding the CREB transcription factor affected only LTM (Nishijima & Maruyama, 2017).

As described above, we have previously found that C. elegans is attracted to mildly alkaline pH, but is repelled by strongly alkaline pH (Murayama et al., 2013; Sassa et al., 2013). Mildly alkaline pH is mainly detected by  the receptor guanylyl cyclase GCY-14 expressed on the cilial surface of the gustatory sensory neuron ASEL, while strongly alkaline pH is sensed by polymodal ASH sensory neurons.

Toward the eluciation of the neuronal networks that are involved in the associative learning and memory as well as in the decision-making, we have established an electrophysiological method for the measurement of neuronal activity responsible for the cognition. When measured electrophysiological activity of a major gustatory sensory neuron, ASEL, upon stimulation with NaCl, we unexpectedly discovered that ASEL actively sends electrial signals  from its cilium to cell body through dendrite (Shindou et al., 2019). This does make sense since by setting the threshold that evokes regenerative depolarization in dendrites of sensory neurons, the animal can respond to only significant environmental changes. This result is surprizing because high-impedance neurons with short processes such as C. elegans neurons are thought to transmit electrical signals by passive propagation.

The causes of adult-onset neuromuscular disorders such as amyotrophic lateral sclerosis (ALS) and myopathies remain largely unknown. C. elegans is a model organism that has been used to identify key genes involved in complex biological processes such as aging. For example, a C. elegans forward genetic screen led to the identification of a mutation in the age-1 gene that causes a 40% extension of mean lifespan. Approximately 65% of human disease genes have a counterpart in C. elegans, making it a promising animal model to find new disease-relevant genes. Taking a forward genetic approach can lead to findings that contribute to basic knowledge as well as clinical applications. Toward the goal, we developed the “Edge Assay” for the isolation of C. elegans mutants that progressively lose the abilities. Amongst mutants isolated by using the “Edge Assay”, those defective in the elpc-2 gene, which encodes Elongator complex protein component-2, and the hda-3 gene, which encodes a class I histone deacetylase, showed shortened locomotor healthspan (Kawamura and Maruyama, 2019; Kawamura and Maruyama, 2020). One of the ELPC-2 functions is the modification of tRNA that regulates translation efficiency and protein folding. C. elegans loses production of molecular chaperones at an early stage of adulthood. Therefore, misfolded proteins that are produced by inefficient tRNA modifications in elpc-2 mutants may begin to accumulate during early adulthood and cause a progressive decline in locomotor ability. Regulation of proper tRNA modifications during aging may represent a new avenue to promote proteostasis and locomotor healthspan. The histone deacetylase HDA-3 can affect the transcriptional expression of many downstream genes, which include genes involved in maintaining locomotor healthspan during later stages of reproductive adulthood.

Progress Reports

As described above, we accidentally found that one of major gustatory sensory neurons sends degenerative all-or-none signals upon stimulation with an up-step of environmental salt concentrations. We have extended this discovery to most, if not all, of olfactory and gustatory sensory neurons (Figure 1). These results support our hypothesis that the C. elegans sensory neurons actively send all-or-none signals by setting thresholds to detect significant environmental changes.

Figure 1. Electrophysiological analysis of the AWC olfactory sensory neuron.

4.3 Information Processing by Neurons/Cells


The bacterial cell-surface receptor, Tar, recognizes aspartate molecules in the environment, and directs bacterial cells toward higher concentrations of the attractant as a nutrient, or toward lower concentration of repellents, such as nickel and cobalt ions. This transmembrane signaling is mediated by Tar in its homodimeric form on the cell surface. We have previously shown that Tar activity is regulated by its ligands, which bind to the extracellular domain of the receptor and lock/freeze the rotational movement of the receptor’s transmembrane domains parallel to the plane of the membrane (Maruyama et al., 1995). This locking/freezing of the rotation at one position by the attractant is likely to inhibit the associated histidine kinase CheA, while the locking/freezing at another position by the repellent seems to activate the kinase activity (the rotation model).

We also analyzed the molecular mechanism underlying activation of the human epidermal growth factor receptor (EGFR) family of cell-surface receptor tyrosine kinases, also known as ErbB or HER (Moriki et al., 2001). EGF/ErbB receptors play a pivotal role in the development of organisms, and are frequently implicated in human cancers. Furthermore, these receptors also regulate neural activities, and mutations of these receptor genes are frequently associated with mental diseases. The receptor family consists of four members, EGFR/ErbB1, ErbB2/Neu/HER2, ErbB3/HER3 and ErbB4/HER4, and has a large (~620 amino acid) extracellular ligand-binding region, a single, transmembrane alpha-helix, and an intracellular region containing the tyrosine kinase and its regulatory domain. They form a network of homo- and heterodimers. ErbB2 can only be regulated indirectly, and is thought to be the preferred heterodimerization partner for other ErbB receptors. ErbB3, on the other hand, must associate with an ErbB family member that has an active tyrosine kinase in order to respond to its own ligand, neuregulin (NRG).

Ligand-induced dimerization has widely been thought to be a property common to the transmembrane signaling by all known growth factor receptors including the EGF/ErbB receptors (Ligand-induced Dimerization Model). According to the model, receptor dimerization is responsible for autophosphorylation of the intrinsic kinase activity, which is mediated by an intermolecular process. The model holds that ligand binds to the monomeric form of the receptor, inducing its dimerization and resulting activation. However, it remains controversial whether the receptor actually has a monomeric or dimeric structure prior to ligand binding.

Using chemical cross-linking and sucrose density-gradient centrifugation, we recently discovered that in the absence of bound ligand, EGFR has the ability to form a dimer and that the majority (>80%) of receptors exist as preformed dimers on the cell surface. We also analyzed receptor dimerization by inserting cysteine residues at strategic positions along the longitudinal axis of the alpha-helical extracellular juxtamembrane region. Mutant receptors spontaneously formed disulfide bridges and transformed NIH3T3 cells in the absence of ligand, depending upon the positions of the cysteine residues inserted. Kinetic analysis of disulfide bonding indicates that ligand binding induces flexible rotation or twist of the juxtamembrane region of the receptor in a plane parallel with the lipid bilayer. The binding of an ATP competitor to the intracellular kinase domain also induced similar flexible rotation/twist of the juxtamembrane region. All disulfide-bonded dimers had flexible ligand-binding domains with the same biphasic affinities for ligand as the wild type. Based on these results, we have proposed an alternative ‘rotation/twist’ model (Figure 2) for the molecular mechanism of EGF receptor activation, in which ligand binding to the flexible extracellular domains of the receptor dimer induces rotation/twist of the juxtamembrane regions, hence the transmembrane domains, rearrange the kinase domains for the receptor activation. Indeed, this rotation/twist model (Moriki et al., 2001; Tao and Maruyama, 2008) is consistent with recent results by others in which the receptor kinase, transmembrane and unactivated extracellular domains are shown to have homodimeric structures.

Figure 2. “Rotation model” for molecular mechanism underlying activation of type-1 transmembrane cell-surface receptors including EGFR (Maruyama, 2015).

To support the ‘rotation’ model, we have recently determined preformed, homo- and heterodimeric structures of EGFR and ErbB2 at physiological expression levels (~104 molecules per cell), using fluorescence microscopy, fluorescence resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy (FCCS) (Liu et al., 2007). When fluorescent protein (FP)-fused EGFR and ErbB2 were expressed on the cell surface of Chinese hamster ovary cells at physiological expression levels, FRET was detected between the donor and acceptor FPs in the absence of ligand. Furthermore, cross-correlation between FPs separately fused to EGFR or ErbB2 was also observed by FCCS, indicating that EGFR and ErbB2 molecules diffuse together as homo- or heterodimers in the cell membrane. These results demonstrate that prior to ligand binding, the cell-surface receptors can spontaneously form homo- and heterodimers, irrespective of their expression levels ranging from ~2 x 104 to ~5 x 106 molecules per cell.

Furthermore, we have been analyzing preformed homo- and heterodimeric structures between all the members, EGFR, ErbB2, ErbB3, and ErbB4, of the receptor family by employing bimolecular fluorescence complementation (BiFC) assay. We have found that all members display preformed, yet inactive, homo- and heterodimeric structures in the absence of bound ligand (Tao & Maruyama, 2008). Ligand-independent dimerization of EGF/ErbB receptors occurs in the endoplasmic reticulum (ER) before newly synthesized receptor molecules reach the cell surface. Furthermore, we have also found that ErbB3 was localized in the nucleus when expressed alone or together with ErbB4. When coexpressed with EGFR or ErbB2, however, ErbB3 was located in the plasma membrane. These results indicate that all the EGF/ErbB receptors exist as homo- and heterodimers before ligand binding, consistent with the ‘rotation/twist’ model. ErbB receptors exist as dimers on the cell surface, mainly through interaction between their transmembrane domains, intracellular kinase domains and C-terminal tails. Receptor dimers have flexible extracellular domains, and perhaps can take two major conformations, closed (tethered) and open (untethered) states. Ligand binding to the open form of the receptor dimer stabilizes the extracellular domains, resulting in approximately 140° rotation or twist of the transmembrane domains about its helix axis parallel to the plane of the cell membrane, dissociate the symmetric back-to-back kinase domains, and then rearrange the kinase domains to take head-to-tail asymmetric configuration for the receptor activation.

As described above, different cell-surface receptors, bacterial Tar and human EGF/ErbB receptors, seem to be similarly regulated by their ligands in order to transmit the extracellular information to the inside of the cell. Ligand binding regulates the rotation of the receptor’s transmembrane domain parallel to the plane of the plasma membrane. Therefore, we have been continuing to test “the rotation model” for the molecular activation mechanism of other cell-surface receptors, including Tar, EGF/ErbB receptors and neurotrophin receptors. Indeed, we have recently elucidated that the neurotrophin receptor TrkA is present as a preformed, yet inactive, dimer in living cells (Shen & Maruyama, 2011). We have also previously found that the intracellular domain of the EGF/ErbB receptors plays a crucial role in the spontaneous formation of receptor dimers (Tao & Maruyama, 2008). Thus, an increasing number of studies demonstrate that many transmembrane receptors, which include receptors previously thought to be activated by ligand-induced receptor dimerization, exist as preformed, yet inactive, homo- and heterodimers in living cells. These receptors include the aspartate receptor Tar, EGF/ErbB receptor family members, erythropoietin receptor (EpoR), growth hormone receptor (GHR), Toll-like receptor-9 (TLR9), natriuretic peptide receptor A (NPRA), nerve growth factor (NGF) receptor TrkA, and brain-derived neurotrophic factor (BDNF) receptor TrkB. In the cases of Tar, EGFR, GHR, and NPRA, it has been proposed that ligand binding induces or stabilizes the rotation of transmembrane domains of preformed receptor dimers for the rearrangement of intracellular domains necessary for activation (Figure 2; Maruyama, 2015).

Progress report

Structural analysis of EGFR.

We have successfully purified full-length EGFR to homogeniety by affinity chromatography. Gel filtration analysis of the purified receptor demonstrates that the receptor has a homodimeric structure before and after ligand bidning. Cryo-electron tomography analysis of the purified receptor also showed homodimeric structures, where after ligand binding, the extracellular domains of the receptor become significantly more compact than those of unliganded receptors. Consistent with the homodimeric structure, optical single molecule observation revealed that the preformed EGF receptor dimer is activated by a single ligand binding event. Furthermore, receptor tetramerization, dimerization of the dimers, requires the receptor phosphorylation.

Structures of the bacterial aspartate receptor.

Three-dimensional (3D) structures of the extracellular ligand-binding domain of the bacterial aspartate receptor Tar, with and without bound aspartate, were determined 20 years ago. It has been reported that the transmembrane domain of Tar shifts vertically 1-2 Å upon binding of aspartate, an attractant for the bacteria. Based on this result, “The piston model” has been proposed as a molecular mechanism underlying activation of Tar by aspartate binding. An alternative “rotation model” has been proposed, in which repellent binding induces the rotation of the transmembrane parallel to the plane of the membrane (Maruyama et al., 1995). According to “the rotation model,” Tar with and without bound aspartate take the similar structure to each other, the most stable structure among rotationally flexible structures. As descried above, this is consistent with the 3D structures of the extracellular domain of Tar with and without bound aspartate. To test “the rotation model”, we are currently trying to determine 3D structures of the full-length Tar reconstituted in nanodics.


5. Publications

5.1 Journals

  1. Shibutani, M, Vibulyaseck, S, and Maruyama, I.N. Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis Elegans. JoVE 

5.2 Oral and Poster Presentations

  1. Murayama, T. and Maruyama, I.N. (2021). Electrophysiological properties of amphid sensory neurons in C. elegans. 23rd International C. elegans Conference Online (June 21-25)

6. Meetings and Events

6.1 Seminar

Title: Phage Display Libraries of Monosaccharide-Modified α-Helix Peptides for Carbohydrate-Binding Proteins

  • Speaker: Ichiro Maruyama
  • Date: May 27, 2021
  • Venue: University of the Ryukyus School of Medicine