FY2013 Annual Report

Information Processing Biology Unit

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

 

 
 

Abstract

Our unit is interested in understanding how cellular networks in the nervous system processes environmental information to regulate animal behaviors, including learning and memory. In order to survive, animals must closely monitor environmental and body fluid pH. However, little is known about how animals monitor alkaline pH in the environment at molecular and cellular levels. In this study, we have employed C. elegans as a model system. While C. elegans is attracted to mildly alkaline pH, it is repelled by strongly alkaline pH. In previous years, we have demonstrated that multimodal sensory neurons (ASH) sense strong environmental alkalinity through activation of transient receptor potential vanilloid type (TRPV) ion channels encoded by the osm-9 and ocr-2 genes. Furthermore, mildly alkaline pH is perceived by a gustatory sensory neuron, ASEL, in which GCY-14, a receptor-type guanylyl cyclase, serves as a cell-surface sensor molecule. This fiscal year, we have studied how C. elegas is attracted by mildly alkaline pH, and avoids strongly alkaline pH, and have found that the behavioral decision making is the result of competition between the two sensory neurons, ASEL and ASH.

In recent years, we also tried to develop protocols to study learning and memory in C. elegans, and found that the animal can learn and form long-term associative memory. It can learn to associate an appetitive or aversive olfactory stimulus with an unconditioned stimulus, and can retain the long-term associative memory. This fiscal year, we are continuing to elucidate neural networks responsible for the long-term associative memory.

We are also interested in understanding how neurons/cells detect extracellular information and transmit it into the cell. For the last 26 years, ligand-induced dimerization has been widely thought to be a common property of transmembrane signaling mechanisms for all known growth factor and cytokine receptors, among others. In previous years, however, we have found that growth factor receptors such as epidermal growth factor (EGF/ErbB) receptors and nerve growth factor (NGF) receptor have preformed, inactive, dimeric structures prior to ligand binding. We have proposed an alternative ‘rotation/twist’ model in that ligand binding to the extracellular domains induces a rotation or twist of the transmembrane domains parallel to the plane of the membrane. This activates the intracellular domains, which often encode or physically interact with enzymes such as kinases, by rearranging their dimeric structures. This fiscal year, we have also found co-operative interaction between EGF and it's receptor EGFR.

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 that control animal behaviors in response to external stimuli. These findings may also be invaluable in developing pharmaceuticals for human diseases such as cancers and mental diseases.

1. Staff

  • Cahyo Budiman (joint appointment with Skoglund unit)
  • Bunsho Itoh
  • Hiraku Miyagi
  • Takeshi Mise
  • Takashi Murayama
  • Hideki Muto
  • Hitomi Ohtaki
  • Toshihiro Sassa

2. Graduate Students

  • Mohamed Abdelhack
  • Viktoras Lisicovas
  • Erin Boldt (Intern)
  • Kazuto Kawamura (Rotation)
  • Georgeou Leonidas (Rotation)

3. Collaborations

  • Theme: 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

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 membranes in order to transmit external information, such as environmental changes and cell-cell communications, into the cell. Such information flow is fundamental for all organisms, from bacteria to humans. Dysregulation of cell surface receptor molecules causes a variety of impairments, including mental and developmental diseases and cancers in humans. (1) We wish to understand information processing through cellular networks. Specifically, we want to know how external information is sensed and transmitted through sensory neurons, how it is processed by the nervous system, and how it controls animal behaviors, including learning and memory. (2) We wish also to understand at the molecular level how external information is sensed and transmitted into the inside of neurons/cells by cell-surface receptors. We also seek to know how the information is processed, how it is transferred to other parts of the cells, and how it regulates other cellular activities.

4.2. Progress Report

4.2.1. Information processing by the nervous system

4.2.1.1. Alkalinity sensation in C. elegans.

4.2.1.1.1. Background.

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 where they are directly exposed to the external medium. These amphid neurons have roles in chemotaxis, thermotaxis, mechanosensation, osmotaxis, and dauer pheromone sensation.

Animals can survive only 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. Mildly alkaline pH in the environment is one of attractive cues for C. elegans. From pH 2.8-10.4, wild-type animals prefer higher alkaline pH ranges, but they avoid pH ≥11.0 and ≤4.0. However, neurons and cell-surface molecules that sense mildly and strongly alkaline pH have not been elucidated in C. elegans. Neural networks responsible for this worm’s attraction to mildly alkaline pH and aversion from strongly alkaline pH can also be efficiently analyzed, since wiring diagrams of all neurons have been reconstructed from electron micrographs of serial thin sections of the whole C. elegans body.

4.2.1.1.2. Sensation of mildly alkaline pH.

Previously, we devised an agar-plate assay with a linear pH gradient in order to investigate cellular and molecular bases for C. elegans’ chemotaxis toward mildly alkaline pH. Along the pH gradient from pH 6.8-8.5, 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 mildly alkaline pH, we performed a series of RNAi knock down experiments of genes encoding various channels, cell-surface receptors, and guanylyl cyclases, in conjunction with analysis of chemotaxis mutants. Among the genes analyzed, tax-2, tax-4 and gcy-14 were found to be involved in C. elegans’ chemoattraction to mildly alkaline pH. By imaging Ca2+ concentration changes using a Ca2+-sensing fluorescent protein, we also found that ASE-left (ASEL) is activated by a pH up-shift. The transmembrane receptor-type guanylyl cyclase (RGC) GCY-14, which is tagged with green fluorescent protein (GFP), was localized to ASEL sensory cilia, and in the gcy-14 mutant, ASEL did not respond to a pH up-shift. While wild-type ASI sensory neurons do not respond to pH up-shift, ASI neurons ectopically expressing GCY-14 were activated by a pH up-shift from pH 7.0-10.0. This suggests that GCY-14 is sufficient for alkaline pH sensation. These results indicate that GCY-14 acts as a mildly alkaline pH sensor, and an increased concentration of cGMP opens the cGMP-gated cation channel TAX-2/TAX-4 for activation of ASEL. We have further dissected the GCY-14 molecule in order to determine which part of it recognizes 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 mildly alkaline pH. Results of this experiment demonstrate 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 cilia. we have also tried to identify a histidine residue(s) in the extracellular domain that may play an essential role in alkaline-pH sensation. A mutated GCY-14 protein that substitutes Gln for His-174, was defective in rescue experiments of gcy-14 mutants, indicating that His-174 plays a critical role in alkaline pH sensation. Furthermore, when GCY-14 was ectopically expressed in alkaline pH-insensitive ASG chemosensory neurons, ASG was activated by pH up-shift from 6.8-10.0. This indicates that GCY-14 is a sensor molecule for mildly alkaline pH. These results together with other data collected in the past years have been published (Murayama et al., 2013).

4.2.1.1.3. Sensation of strongly alkaline pH.

During the work on mild alkalinity sensation described above, we found that C. elegans avoids higher pH than ~10.5 as a noxious stimulus in chemotaxis assays using agar plates divided into four quadrants. Using the plate assay, in previous years, we identified several genes that play pivotal roles in the avoidance behavior: che-2, dyf-3, osm-1, and osm-5, which are required for chemosensation, and osm-9 and ocr-2, which encode subunits of type-V transient-receptor potential (TRPV) channels. Rescue experiments that employed dyf-3 and osm-9 mutants by injecting the respective cDNA into ASH chemosensory neurons indicate that ASH is a major neuron responsible for strongly alkaline-pH sensation in C. elegans. This was confirmed by Ca2+ imaging in which a Ca2+ concentration increase was observed in ASH following stimulation with strongly alkaline pH. We further confirmed that ASH sensory neurons are responsible for nociception of strongly alkaline pH using genetic rescue experiments and laser microsurgery of the neurons. Furthermore, genetic rescue of osm-9 mutants by specifically expressing OSM-9 in ASH showed that TRPV channels play an essential role in sensing of high pH. Ca2+ imaging in vivo also revealed that ASH neurons were activated by high pH stimulation, but ASH of osm-9 or ocr-2 mutants were not. These results demonstrate that in C. elegans, high pH is sensed by ASH nociceptors through opening of OSM-9/OCR-2 TRPV channels. These results have been published (Sassa et al, 2013). In this fiscal year, furthermore, we have found that C. elegans mutants deficient in a G-protein a subunit, GOA-1, failed to avoid strongly alkaline pH with normal Ca2+ influx into ASH. These results suggest that GOA-1 regulates signal transmission downstream of Ca2+ influx through OSM-9/OCR-2 TRPV channels in ASH (Fig. 1; Sassa & Maruyama, 2013).

                        

Figure 1. Behavior and imaging analyses of mutants deficient in a G protein a subunit. (A) Avoidance indices of wild-type and mutant animals. Assays were carried out using Petri dishes with four quadrants as described previously.13 Error bars indicate the SEM of five independent assays. **p < 0.01. (B) Ca2+ imaging of ASH in wild-type and goa-1 animals upon stimulation with pH 11.2. The red line represents the period of time during which animals were stimulated with pH 11.2 buffer. Numbers of recordings are shown in parentheses, and light color shading denotes the SEM.

4.2.1.1.3. Decision-making in C. elegans chemotaxis.

As described above, monitoring of environmental and tissue pH is critical for animal survival, and C. elegans is attracted to mildly alkaline pH, but avoids strongly alkaline pH. However, little is known about how the behavioral switching or decision-making occurs. Genetic dissection and Ca2+ imaging have previously demonstrated that ASEL and ASH are the major sensory neurons responsible for attraction and repulsion, respectively. In this fiscal year, we have found that unlike C. elegans wild type, mutants deficient in ASEL or ASH were repelled by mildly alkaline pH, and were strongly attracted to alkaline pH, respectively. These results suggest that signals through ASEL and ASH compete to determine the animal’s alkaline-pH chemotaxis. Furthermore, mutants with two ASEL neurons were more efficiently attracted to mildly alkaline pH than the wild type with a single ASEL neuron, indicating that higher activity of ASEL induces stronger attraction to mildly alkaline pH. This stronger attraction was overridden by normal activity of ASH, suggesting that ASH-mediated avoidance dominates ASEL-mediated attraction. Thus, C. elegans chemotactic behaviors to alkaline pH seems to be determined by signal strengths from the sensory neurons ASEL and ASH, and the behavior decision-making seems to be the result of competition between the two sensory neurons (Figs. 2 & 3; Murayama & Maruyama, 2013).

                                                            

Figure 2. Chemotaxis assay of C. elegans to alkaline pH. Chemotaxis assays were carried out using agar plates in Petri dishes, 10 cm in diameter, divided into four quadrants containing either neutral or alkaline pH, as described previously.10 After being washed twice with deionized H2O, animals were placed at the center of assay plates, and allowed to move freely for eight minutes. A chemotaxis index (CI) was calculated using the equation CI = (Nalkaline – Nneutral) / (Nalkaline + Nneutral), in which Nalkaline and Nneutral are the numbers of animals in alkaline and neutral pH areas, respectively. The following mutant stains were used: OF226 che-1(p679) I, CX10 osm-9(ky10) IV, SP1603 dyf-3(m185) IV, OH7805 otIs204[ceh-36::lsy-6;elt-2::gfp] as ASELx2, OH2535 lsy-6(ot71) V as ASERx2. Error bars indicate the SEM (n = 6-8 assays).

                                      

 

Figure 3. Model neural networks that may be involved in alkaline pH sensing. Interneurons that regulate motor neurons for head movement are indicated by a yellow background, and interneurons that are reportedly involved in forward and backward locomotion are shown in blue and red, respectively. Arrows and I-shaped bars represent chemical synapses and gap junctions, respectively. Thickness of arrows and I-shaped bars indicate relative strengths of the connections.

4.2.1.2. Associative learning and memory in C. elegans.

4.2.1.2.1. Background.

C. elegans is an excellent model organism for the study of learning and memory. 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 and/or voltage-sensitive fluorescent proteins in living animals.

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 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 to use two defined chemical cues for conditioning of animals in associative learning paradigms.

4.2.1.2.2. Appetitive olfactory learning and associative long-term memory.

In previous years, we developed an associative learning paradigm in C. elegans by classical (Pavlovian) conditioning of the animals with 1-nonanol, as a conditioned stimulus (CS), and potassium chloride (KCl) as an unconditioned stimulus (US). Before conditioning, animals avoided nonanol, an aversive olfactory stimulus, and were attracted by KCl, an appetitive gustatory stimulus, in chemotaxis assays. In contrast, after eight-cycle massed (without intertrial intervals, ITI) or spaced (with 10-min ITI) training, animals were attracted to nonanol. Memory induced by the massed training was extinguished within three hours, while the spaced training induced memory that was retained for more than 12 hours. Animals treated with cycloheximide or actinomycin D failed to form long-term memory by the spaced training, whereas 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 STM/MTM and LTM, while mutations in crh-1 encoding the CREB transcription factor affected only formation of LTM. Last fiscal year, we determined sensory neurons that detect nonanol in C. elegans as a step toward elucidating the neural networks responsible for associative learning and memory with 1-nonanol and KCl. Olfactory avoidance assays of animals whose amphid AWB or ASH neurons were separately ablated by laser microsurgery, demonstrate that AWB is responsible for the perception of a low concentration, 0.1%, of 1-nonanol. This fiscal year, we have been trying to identify neurons required for learning and memory using rescue experiments of the mutants.

4.2.1.2.3. Aversive olfactory learning and associative long-term memory.

In previous years, we also developed protocols for classical conditioning of C. elegans with 1-propanol, as a CS and hydrochloride (HCl), pH 4.0, as a US. Before conditioning, worms were 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. 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/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 LTM. This work has recently been published (Amano & Maruyama, 2011). In this fiscal year, we have been trying to identify neurons required for learning and memory using rescue experiments of the mutants.

4.2.2. Information processing by neurons/cells

4.2.2.1. Background.

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/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 is likely 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 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. 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 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. 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 (Fig. 4) 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 4. ‘Rotation/twist’ model for molecular mechanism underlying activation of EGFR by EGF binding. EGFR exists as a dimer on the surface of living cells, in which the intracellular domains have a symmetric back-to-back, stable dimeric structure. In contrast, the extracellular domains have a relatively flexible, dimeric structure, and take stable tethered or flexible untethered structure. The ligand EGF binds to the untethered extracellular domain of EGFR. Two, ligand-bound, extracellular domains of the EGFR dimer interact each other through the 'dimerization arm' of the second subdomain of the extracellular domain. This interaction induces rotation or twist of the transmembrane domains of the receptor, which in turn, dissociates the asymmetric dimeric, intracellular domains. This dissociation is followed by rearrangement of the intracellular domains, which take an asymmetric, head-to-tail, structure. In this asymmetric structure, the kinases can phosphorylate its substrate tyrosines to initiate intracellular signaling cascades (Maruyama, I. N. Cells 3, 304-330, 2014) . 

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 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/twist of the receptor’s transmembrane domain parallel to the plane of the plasma membrane. Therefore, we have been continuing to test the “rotation/twist” 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/twist of transmembrane domains of preformed receptor dimers for the rearrangement of intracellular domains necessary for activation.

4.2.2.2.  Crystal structure of Tar with repellent.

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, and others have reported that the transmembrane domain shifts vertically 1-2 Å when aspartate binds. This suggests that the receptor structures without and with bound aspartate are very similar, consistent with our ‘rotation/twist’ model for the mechanism of Tar activity regulation by aspartate (Maruyama et al., 1995). Furthermore, the ‘rotation/twist’ model predicts that another cofactor for Tar, nickel, a repellent in E. coli chemotaxis, stabilizes the transmembrane domains in a distinct rotational orientation. That is, nickel induced rotation/twist of the transmembrane domains parallel with the plane of the cytoplasmic membrane. To test this model, this fiscal year we are still trying to crystalize the extracellular domain of Tar in the presence of Ni2+ or Co2+ ions.

4.2.2.3. Kinetic analysis of EGF interaction with EGFR.

To analyze kinetics of EGF interaction with EGFR, this fiscal year we have employed total internal reflection fluorescence microscopy (TIRFM) to observe single molecules of fluorescetly labeled EGF bound to EGFR on the cell surface. Using the technique, we are currently analyzing association and dissociation rates between EGF and EGFR at the single molecule level on the surface of living cells.

 

5. Publications

5.1 Journals

  1. Murayama, T., Takayama, J., Fujiwara, M. & Maruyama, I. N. Environmental Alkalinity Sensing Mediated by the Transmembrane Guanylyl Cyclase GCY-14 in C. elegans. Current Biology (2013) 23(11) :1007-12. 
  2. Sassa, T., Murayama, T. & Maruyama, I. N. Strongly alkaline pH avoidance mediated by ASH sensory neurons in C. elegans. Neuroscience Letters (2013)555:248-52 [Plenary Article awarded].  
  3. Murayama, T., Maruyama, I. N. Decision making in C. elegans chemotaxis to alkaline pH: Competition between two sensory neurons, ASEL and ASH. Communicative & integrative biology (2013) 6: e26633. 
  4. Sassa, T. & Maruyama, I. N. A G-protein α subunit, GOA-1, plays a role in C. elegans avoidance behavior of strongly alkaline pH. Communicative & Integrative Biology (2013) 6:e26668. 

5.2 Presentations

  1. Murayama, T., Takayama, J., Fujiwara, M. & Maruyama, I. Alkaline pH sensation mediated by GCY-14, a transmembrane guanylyl cyclase, in The 19th International C. elegans Meeting, UCLA, CA, USA (June 26-30, 2013)
  2. Mise, T., Matsunami, H., Sanehisa, S., Samatey, F. A. & Maruyama, I. Presentation Transmembrane signaling mediated by the E. coli aspartate receptor Tar, in The 13th Annual meeting of the protein Science Society of Japan, Tottori, Japan (June 12-14, 2013).
  3. Miyagi, H., Sassa, T. & Maruyama, I. Presentation Spontanous activation of EGFR by mutations in its intracellular region in the absence of boud lingand, in The 51st annual meeting of the biophysical society of Japan, Kyoto international conference center, Kyoto, Japan (October 28-30, 2013)
  4. Shindou, T., Ochi-shindou, M., Murayama, I., Sassa, T., Wickens, J. R. & Maruyama, I. Electrophysiological properties of ASE neurons of C. elegans in Neuroscience 2013 (Society for Neuroscience), San Diego Convention Center, CA, USA (November 9-13, 2013).

6. Meetings and Events

6.1 Seminar

  • Title: Loss of neuronal 3D chromatin organization causes transcriptional and behavioral deficits related to serotonergic dysfunction
  • Speaker: Satomi Ito, Institute for Integrated Cell-material Sciences (iCeMS), Kyoto University
  • Date: February 12, 2014
  • Venue: C015, Level C, Lab1, OIST
  • Title: Structural-Functional Studies on Photosystem II Complex from Thermophilic Cyanobacteria
  • Speaker: Faisal Koua, The OCU Advanced Research Institute of Natural Science & Technology (OCARINA) Osaka-City University
  • Date: March 3, 2014
  • Venue: D015, Level D, Lab1, OIST
  • Title: Mechanism how F1-ATPase varies motor torque reflecting energy level of ATPDate: March 3, 2014
  • Speaker: Eiichiro Saita, Department of Molecular Bioscience Kyoto Sangyo University
  • Date: March 13, 2014
  • Venue: D014, Level D, Lab1, OIST
  • Title: Biochemical studies on the multiplicity of 5’caps in small RNAs
  • Speaker: Rehab F. Abdelhamid
  • Date: March 27, 2014
  • Venue: C015, Level C, Lab1, OIST

7. Others

The articles about our research picked up by outside websites.