FY2011 Annual Report
Information Processing Biology Unit
Professor Ichi Maruyama
We are interested in an understanding of how cells/neurons detect extracellular information and transmit it to the inside of the cell. For the last 25 years, ligand-induced dimerization has widely been thought to be a property common to the transmembrane signaling mechanism of all known growth factor and cytokine receptors among others (dimerization model). In previous years, however, we have found growth factor receptors such as epidermal growth factor (EGF/ErbB) receptors and nerve growth factor (NGF) receptor have preformed, yet inactive, dimeric structures prior to ligand binding, and have proposed an alternative ‘rotation/twist’ model in that ligand binding to the extracellular domains induces the rotation or twist of the transmembrane domains in order to activate the intracellular domains, which often encode or physically interact with enzymes such as a kinase or guanylyl cyclase, by rearranging its dimeric structure. This fiscal year, we have also shown that in living cells, the TrkB receptor for brain-derived neurotrophic factor (BDNF) also exists as a homodimer prior to ligand binding.
Our unit is also interested in an understanding of how the nervous system processes extracellular information as cellular networks to regulate animal behaviors including learning and memory. Animals can survive in a narrow pH range by monitoring the pH in the environment and their body fluids. However, little is known about how animals monitor alkaline pH in the environment at molecular and cellular levels. In this study, we have employed the nematode Caenorhabditis elegans (C. elegans) as a model system. While C. elegans is attracted to low-alkaline pH, C. elegans is repelled from high-alkaline pH. In previous years, we have found that GCY-14, a membrane receptor-type guanylyl cyclase, plays as a sensor molecule for the environmental mild alkalinity. In this fiscal year, we have further demonstrated that GCY-14 acts in the form of preformed dimers as a sensory molecule expressed on the surface of a gustatory sensory neuron.
In the past years, we have also tried to develop protocols for the study of learning and memory in C. elegans, and found that the animal can learn and form associative long-term memory. The animal can learn to associate an appetitive or aversive olfactory stimulus with an unconditioned stimulus, and can retain the associative long-term memory. This fiscal year, we are continuing to elucidate neural networks responsible for the associative long-term memory.
These results provide us insights into an understanding of a molecular mechanism underlying information transfer from the outside of cells/neurons to the inside, as well as an understanding of neuronal networks that control animal behaviors in response to external stimuli. These findings may also be invaluable for the development of pharmaceuticals for human diseases such as cancers and mental diseases.
- Bunsho Ito, Ph.D. (since January 10th, 2012)
- Yoko Kudeken, B.A.
- Takeshi Mise, Ph.D. (since August 1st, 2011)
- Hiraku Miyagi, M.S.
- Takashi Murayama, Ph.D.
- Saori Nishijima, Ph.D.
- Toshihiro Sassa, Ph.D.
- Regulation of Cellular Functions by Signaling Molecules
- Type of collaboration: Joint research
- Prof. Ken-ichi Kariya, University of the Ryukyus School of Medicine, Okinawa, Japan
3. Activities and Findings
3.1. Project Aims
All forms of life are separated from non-life by cell membranes, and all cells/neurons have cell-surface receptor proteins that span the membranes in order to transmit external information, such as environmental changes and cell-cell communications, to the inside of the cell. Such information flow is fundamental for all organisms from bacteria to humans. Dysregulation of the cell surface receptor molecules often causes a variety of impairments including mental and developmental diseases and cancers in humans. (1) We wish to understand at the molecular level how the external information is sensed and transmitted into the inside of cells/neurons by cell-surface receptors, and how the information is processed, transferred to other parts of the cells and regulates other cellular activities. (2) We wish also to understand information processing at higher levels through cellular networks; namely, how the external information is sensed and transmitted through sensory neurons, processed by the nervous system, and how it controls animal behaviors including learning and memory.
3.2. Progress report
3.2.1. Information processing by cells/neurons
The bacterial cell-surface receptor Tar recognizes aspartate molecules in the environment, and brings bacterial cells toward the higher concentration of the attractant as a nutrient, or the lower concentration of repellents such as nickel and cobalt ions. This transmembrane signaling by Tar occurs within homodimeric receptor molecules on the cell surface. We have previously shown that the Tar activity is regulated by its ligands, which bind to the extracellular domain of the receptor and lock/freeze the rotational/twist movement of the receptor’s transmembrane domains (Maruyama et al., 1995). This locking/freezing of the rotation/twist at one position by the attractant seems to inhibit the associated histidine kinase Che A, while the locking/freezing at another position by the repellent seems to activate the kinase activity (the rotation/twist model).
We also analyzed a molecular mechanism underlying the activation of the human epidermal growth factor receptor (EGFR) family of cell-surface receptor tyrosine kinases, also known as ErbB or HER. These 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 residues long) extracellular ligand-binding region, a single transmembrane -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 mechanism of all known growth factor receptors including the EGF/ErbB receptors (Dimerization Model). According to the model, receptor dimerization is responsible for autophosphorylation of the intrinsic kinase activity, which is mediated by an intermolecular process. Namely, ligand binds to the monomeric form of the receptor, and induces its dimeric form for the activation. Prior to ligand binding, however, it still remains controversial whether the receptor has a monomeric or dimeric structure.
We have recently found by chemical cross-linking and sucrose density-gradient centrifugation that in the absence of bound ligand EGFR has an ability to form a dimer and the majority (>80%) of the receptor exists as a preformed dimer on the cell surface. We also analyzed the receptor dimerization by inserting cysteine residues at strategic positions about the -helix axis of the extracellular juxtamembrane region. The 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 the disulfide bonding indicates that ligand binding induces flexible rotation or twist of the juxtamembrane region of the receptor in the 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 the disulfide-bonded dimers had flexible ligand-binding domains with the same biphasic affinities for the ligand as the wild type. Based on these results, we have proposed an alternative ‘rotation/twist’ model for the molecular mechanism of the 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, and rearrange the kinase domains for the receptor activation. Indeed, this rotation/twist model (Moriki et al., 2001; Tao and Maruyama, 2008) is consistent with the homodimeric structure of the receptor kinase, transmembrane and unactivated extracellular domains that have recently been determined by others.
To support the ‘rotation/twist’ 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 EGFR and ErbB2 fused with a fluorescent protein (FP) were expressed on the cell surface of Chinese hamster ovary cells at physiological expression levels, FRET was detected between the donor and acceptor FPs. 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 the unactivated 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, and have found that all the members display preformed, yet inactive, homo- and heterodimeric structures in the absence of bound ligand (Tao & Maruyama, 2008). The ligand-independent dimerization of the 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 to the plasma membrane. These results indicate that all the EGF/ErbB receptors have homo- and heterodimeric structures before ligand binding, and are consistent with the ‘rotation/twist’ model. The ErbB receptors exist as a dimer on the cell surface, mainly through interaction between the intracellular kinase domains and C-terminal tails. The receptor dimers have flexible extracellular domains, and presumably can take two major conformations with low and high affinities for ligand. Ligand binding to the high affinity receptor stabilizes the extracellular domains, induces approximately 140-degree rotation or twist of the transmembrane domains about its helix axis in parallel to the cell membrane, dissociate the symmetric back-to-back kinase domains, and then rearrange the kinase domains to take head-to-tail asymmetric conformation 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. Namely, their ligand binding regulates the rotation/twist of the receptor’s transmembrane domain in parallel to the plane of the plasma membrane. Therefore, we have been continuing to test the “rotation/twist” model for the molecular mechanism of the activation of other cell-surface receptors including Tar, EGF/ErbB receptors and neurotrophin receptors as described below. Indeed, 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 for the spontaneous formation of the 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 are 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. In the cases of Tar, EGFR, GHR and NPRA, it has been proposed that ligand binding induces or stabilizes the rotation/twist of the transmembrane domains of the preformed receptor dimers for the rearrangement of the intracellular domains for activation.
126.96.36.199. Crystal structure of Tar with a repellent.
The three-dimensional structures of the extracellular ligand-binding domain of the bacterial aspartate receptor Tar with or without bound aspartate were determined 20 years ago, and it was found by others that the transmembrane domain vertically shifted 1-2 Å in distance when compared the two structures with and without bound aspartate. This might suggest that the receptor structures with and without bound aspartate are very similar to each other, consistent with our ‘rotation/twist’ model for the mechanism of the Tar activity regulation by aspartate (Maruyama et al., 1995). Furthermore, the ‘rotation/twist’ model predicts that another cofactor for Tar, nickel as a repellent in E. coli chemotaxis, stabilizes the transmembrane domains in a distinct rotational orientation. Namely, the repellent nickel could induce the rotation/twist of the transmembrane domains in parallel with the plane of the cytoplasmic membrane. To test this model, we are currently trying to determine the three dimensional structure of the extracellular domain of Tar in the presence of bound nickel ions.
Figure 1. Crystals of the periplasmic domain of the E. coli aspartate receptor Tar. Bars, 100 micrometer.
188.8.131.52. EGFR domains required for the spontaneous dimerization in the absence of bound ligand.
Three-dimensional structures of the intracellular domain of EGFR recently determined by others suggest that the intracellular domain may play roles in the spontaneous dimer formation. Through analyses of deletion mutants, indeed, we have found that the intracellular domain of EGFR plays a crucial role for the dimerization in the absence of ligand. We are currently trying to identify domains and amino-acid residues required for the dimerization by constructing point and deletion mutant receptors.
184.108.40.206. Preformed, yet inactive, dimeric structures of receptors for neurotrophic factors.
The nerve growth factor (NGF) family of neurotrophins that includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/5 play a central role in the development, growth, and maintenance of the nervous system. These neurotrophins interact with two types of cell surface receptors, the tropomyosin-related kinase (Trk) family of tyrosine kinase receptors, TrkA, TrkB and TrkC, and the p75 neurotrophin receptor (p75NTR). Trk receptor subtypes bind mature neurotrophins with different specificities: TrkA and TrkC preferentially bind NGF and NT-3, respectively, while TrkB mainly binds BDNF and NT-4. The p75NTR binds all mature neurotrophins with approximately equal low affinity and has been demonstrated to bind the proneurotrophins with high affinity. These receptors are present on the same cell, and modulate the responses of neurons to neurotrophins.
NGF-induced activation of TrkA is thought to be mediated by receptor dimerization. Cross-linking studies revealed the formation of TrkA homodimers upon NGF binding to PC12 cells and fibroblasts ectopically expressing TrkA receptors. Because NGF, as well as other neurotrophins, exist in solution as stable homodimers, it is thought that a single NGF dimer bridges two TrkA monomers. This model was supported by the symmetric crystal structure of the NGF complex with the extracellular IgG domain of TrkA. However, NGF mimetics and Fab fragments of antibody against TrkA, which are structurally incapable of dimerizing the receptor, could activate TrkA. Furthermore, an asymmetric chimeric NGF/Neurotrophin-4 heterodimer also was found to activate TrkA receptor. TrkA receptor molecules expressed in Xenopus Laevis oocytes were found to be present as oligomers in the plasma membrane in the absence of NGF. These previous results led us to examine whether neurotrophin receptors exist as monomers or preformed dimers on the cell surface prior to ligand binding. In the past years, we have analyzed structures of the TrkA receptor for NGF by chemical crosslinking, bimolecular fluorescence complementation (BiFC) and luciferase fragment complementation assays. These analyses demonstrated that before ligand binding, TrkA exists as homodimers in living cells (Shen & Maruyama, 2011). Using Brefeldin A, which disassembles the Golgi apparatus and blocks anterograde transport of the receptors from endoplasmic reticulum (ER) to Golgi, furthermore, it was found the preformed dimers were formed in ER before reaching Golgi. This work provides new insights into understanding of transmembrane signaling by receptors for neurotrophic factors.
This fiscal year, we have also analyzed the structure of brain-derived neurotrophic factor (BDNF) receptor TrkB in living cells, and found that the receptor also exists as a preformed, yet inactive, homodimeric structure prior to BDNF binding (Shen & Maruyama, 2012).
Figure 2. A confocal microscopy image of Chinese hamster ovary (CHO) cells expressing TrkB fused with green fluorescent protein (GFP). Bar, 10 micrometer.
3.2.2. Information processing by the nervous system
220.127.116.11. Alkalinity sensation in C. elegans.
Like other animals, C. elegans detects various environmental cues such as tastes and odors mainly through its amphid sensilla. The amphids are the largest chemosensory organs, and each amphid includes 12 sensory neurons (ADF, ADL, AFD, ASE, ASG, ASH, ASI, ASJ, ASK, AWA, AWB, and AWC) with ciliated dendrites as well as one sheath and one socket cell. Apart from AFD, the sensory dendrites of 11 neurons penetrate the sheath-cell ending, and the cilia of eight of these neurons, except those of AWA, AWB, and AWC, extend into the doughnut-like pore created by the socket cell and are directly exposed to the external medium. These amphid neurons have roles in chemotaxis, thermotaxis, mechanosensation, osmotaxis, and dauer pheromone sensation.
Animals can survive in a narrow pH range, and continuously monitor the pH of environment and body fluids. However, little is known about how animals monitor alkaline pH in the environment. Environmental mild alkalinity is one of attractive cues for C. elegans. Between pH 2.8 to pH 10.4, wild-type animal prefers higher alkaline pH ranges, while the animal avoids higher alkalinity than pH ~11.0 and acidic conditions lower than pH ~4.0. However, neurons and cell-surface molecules that sense low and high alkaline pH ranges have not been elucidated in C. elegans. Neural networks responsible for worm’s attraction to mild alkalinity and aversion from strong alkalinity can also be efficiently analyzed since the wiring diagrams of all neurons have been reconstructed from electron micrographs of serial thin sections of the C. elegans whole body.
18.104.22.168.2. Mild-alkalinity sensation.
In previous years, we have devised an agar plate assay with a linear pH gradient in order to investigate cellular and molecular bases for C. elegans chemotaxis toward low-alkaline pH. Along the pH gradient from pH 6.8 to pH 8.5, wild-type animals were attracted to higher pH regions, whereas che-1 mutants defective in chemosensory ASE neurons were not. To search for a molecular sensor for the low-alkaline pH, we have performed a series of RNAi knock down of genes encoding various channels, cell-surface receptors and guanylyl cyclases in conjunction with analysis of chemotaxis mutants previously known. Among the genes analyzed, tax-2, tax-4 and gcy-14 have been found to be involved in the C. elegans chemoattraction to the low-alkaline pH. By imaging of Ca2+ concentration changes using a Ca2+-sensing fluorescent protein, furthermore, we found that ASE-left (ASEL) is activated by pH up-shift. GFP-tagged GCY-14, a transmembrane receptor-type guanylyl cyclase (RGC), was localized to ASEL sensory cilia, and in the gcy-14 mutant, ASEL did not respond to pH up-shift. While ASI and ASG sensory neurons did not respond to pH up-shift, furthermore, ASI and ASG neurons ectopically expressing GCY-14 were activated by pH up-shift from pH 7.0 to pH10.0. This demonstrates that GCY-14 is sufficient for the alkaline pH sensation. These results indicate that GCY-14 acts as an alkaline pH sensor, and an increased concentration of cGMP opens the cGMP-gated cation channel TAX-2/TAX-4 for the activation of ASEL.
This fiscal year, we have further dissected the GCY-14 molecule in order to determine which part of the protein recognizes the environmental alkaline pH. GCY-14 consists of an extracellular domain, a membrane-spanning alpha-helix, and intracellular kinase-homology and guanylyl cyclase domains. We swapped these domains one by one with those of other alkaline-pH insensitive RGCs, DAF-11 and ODR-1, to identify a domain that detects the low-alkaline pH. The results demonstate that the extracellular domain is responsible for the sensation. Furthermore, the results also indicate that GCY-14 acts as a homodimer on the cell surface of ASEL sensilla. We have also identified a histidine residue in the extracellular domain that plays an essential role in the alkaline-pH sensation.
Figure 3. C. elegans immobilized by microfluidic devices by which the animals can be externally stimulated by various solutions, such as different pH and salts, shown by red, green and yellow.
22.214.171.124.3. Strong-alkalinity sensation.
During the work on mild alkalinity sensation described above, we have found that C. elegans avoids higher pH than ~11.0 using quadrant-plate assay (Fig. MA). Using the plate assay, in previous years, we have identified several genes that play pivotal roles in the avoidance behaviour: che-2, dyf-3, osm-1 and osm-5 required for chemosensation; and mutants, osm-9 and ocr-2 encoding subunits of the type-V transient-receptor potential (TRPV) channel. Rescue experiments dyf-3 and osm-9 mutants by injecting the respective cDNA into the chemosensory neuron ASH indicate that ASH is a major neuron responsible for the high alkaline pH sensation in C. elegans. This was confirmed by Ca2+ imaging in which upon stimulation with high alkaline pH, a Ca2+ concentration increase was observed in ASH.
126.96.36.199. Associative learning and memory in C. elegans
C. elegans is an excellent model organisms for the study of learning and memory. The hermaphrodite nervous system consists of only 302 neurons, and the neural circuits, through 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 fluorescent proteins in living animals.
Associative learning in C. elegans has first been 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 or cultivation temperature 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 that two defined chemical cues are used for conditioning of animals in associative learning paradigms.
188.8.131.52.2. Appetitive olfactory learning and associative long-term memory
In previous years, we have developed a learning paradigm in C. elegans by classical (Pavlovian) conditioning of the animals with nonanol, as a conditioned stimulus (CS), and potassium chloride (KCl) as an unconditioned stimulus (US). Before the conditioning, animals avoided nonanol, an aversive olfactory stimulus, and were attracted by KCl, an appetitive gustatory stimulus, in chemotaxis assay. After eight-cycle massed (without intertrial intervals, ITI) or spaced (with 10-min ITI) training, in contrast, animals were attracted to nonanol. Memory induced by the massed training was extinguished within three hours, while the spaced training induced the memory that was retained for more than 12 hours. Animals treated with cycloheximide or actinomycin D failed to form the long-lasting memory by the spaced training, whereas the memory induced by the massed training was not significantly affected. These results indicated that the memory formation by the spaced training, but not by the massed training, required protein synthesis and mRNA transcription. Therefore, the memories induced by the massed and spaced training are classified as short-term/middle-term (STM/MTM) and long-term (LTM) memories, respectively. In support of this, C. elegans mutants defective in nmr-1 encoding an NMDA receptor subunit failed to form both of the STM/MTM and LTM, while mutations in crh-1 encoding the CREB transcription factor affected only on the formation of the LTM. The last fiscal year, we determined sensory neurons that detect nonanol in C. elegans towards the elucidation of neural networks responsible for the associative learning and memory between nonanol and KCl. Olfactory avoidance assays of animals whose amphid AWB or ASH neurons were separately killed by laser microsurgery demonstrate that AWB is responsible for the perception of a low concentration, 0.1%, of nonanol.
Figure 4. Diagrams showing steps for associative conditioning of C. elegans by using nonanol as a CS and KCl as a US. These steps are repeated several times with or without the rest interval for training.
This fiscal year, we have also tried to develop an alternative learning paradigm by classical conditioning of the animals with nonanol, as a CS, and mild-alkaline pH as a US. As described above in the Mild-alkalinity sensation section (184.108.40.206.2.), mild-alkaline pH is an attractive cue for the animal, and could therefore serve as a US for the conditioning. Indeed, we have successfully conditioned the animals by spaced training 10 times with 10-min ITI. This paradigm using the mild-alkaline pH as a US would be better than the paradigm using KCl as a US since the animal has associative memory of the KCl concentration with food during the cultivation.
220.127.116.11.3. Aversive olfactory learning and associative long-term memory
We have also continuously been developing protocols for classical conditioning of animals with propanol, as a CS and hydrochloride (HCl), pH 4.0, as a US. Before the conditioning, worms were attracted to propanol, and avoided HCl in chemotaxis assay. After spaced or massed training, by contrast, animals were either not attracted at all or were repelled from propanol on the assay plate. The memory after the spaced training was retained for 24 h, while the memory after the massed training was no longer observable within 3 h. Animals pretreated with transcription and translation inhibitors failed to form the memory by the spaced training, whereas the memory after the massed training was not significantly affected by the inhibitors and was sensitive to cold-shock anesthesia. Furthermore, the memory after spaced training was reasonably disrupted by extinction learning, which is repeated exposed to the CS in the absence of US. Therefore, the memories after the spaced and massed trainings can be classified as LTM and STM/MTM, respectively. Consistently, like other organisms including Aplysia, Drosophila, and mice, C. elegans mutants defective in nmr-1 encoding an NMDA receptor subunit failed to form both LTM and STM/MTM, while mutations in crh-1 encoding the CREB transcription factor affected only the LTM. This work has recently been published (Amano & Maruyama, 2011).
4.2 Oral and Poster Presentations
Hiraku Miyagi, Aini S. Adenan, Zacharie Taoufiq, and Ichiro Maruyama: Spontaneous autoactivation of the EGF receptor induced by mutations in its C-terminal negatively charged residues. Experimental Biology 2011, Water E. Washington Convention Center, Washington, DC, USA (April 9-13, 2011).
Saori Nishijima, and Ichiro Maruyama: Appetitive olfactory learning and associative long-term memory in C. elegans. 18th International C. elegans Meeting, University of California, Los Angeles, California, USA (June 22-26, 2011).
Toshihiro Sassa, Takashi Murayama, and Ichiro Maruyama: High alkaline pH sensation in C. elegans. 18th International C. elegans Meeting, University of California, Los Angeles, California, USA (June 22-26, 2011).
Toshihiro Sassa, Takashi Murayama, and Ichiro Maruyama: High alkaline pH sensation in the nematode Caenorhabditis elegans. 34th Annual Meeting of the Japan Neuroscience Society, Pacifico Yokohama, Kanagawa, Japan (September 14-17, 2011).
Saori Nishijima, and Ichiro Maruyama: Appetitive olfactory learning and associative long-term memory in the nematode Caenorhabditis elegans.34th Annual Meeting of the Japan Neuroscience Society, Pacifico Yokohama, Kanagawa, Japan (September 14-17, 2011).
Jianying Shen and Ichiro Maruyama: Nerve growth factor receptor TrkA exists as a preformed, yet inactive, dimer in living cells. 56th Annual Meeting of Biophysical Society, San Diego Convention Center, San Diego, California, USA (February 25-29, 2012).
Ichiro Maruyama: Calcium imaging of single neurons of C. elegans in a microfluidic device. Lab-on-a-chip European congress, Edinburgh Conference Centre, Heriot-Whatt University, Edinburgh, Scotland (28-29 March, 2012).