Symposium

Symposium 22 The neuronal signaling in the lipid-rafts: focusing on the protein palmitoylation シンポジウム22 パルミトイル化に基づく脂質ラフトのシグナル伝達機構

SY22-1 Unraveling of raft organization and signal transduction mechanisms by high-resolution single-molecule imaging 高精度1分子観察によるラフト組織化とシグナル制御機構の解明 Suzuki Kenichi（鈴木健一） G-CHAIN, Gifu Univ. Raft domains have been drawing extensive attention as a signaling platform. However, raft structure and function are still very controversial. This is partially due to lack of raft lipid probes which behave like the parental molecules in cell membranes. Here, we developed probes of raft lipids such as gangliosides and sphingomyelin, which behave like the parental molecules for the first time ever in collaboration with Kiso/Ando lab. at Gifu Univ. and Murata lab. at Osaka Univ., respectively. Surprisingly, single-molecule observations (colocalization and FRET) revealed that all the fluorescent ganglioside probes (GM1, GM2, GM3, GalNacGD1a, GD1b, GD3, GT1b, GQ1b) transiently formed homodimers. However, gangliosides did not form heterodimers in general. The ganglioside homodimers recruited other raft associated molecules such as sphingomyelin. We found that the homodimers were induced by specific glyco-chain interactions and stabilized by cooperative raft-lipid interactions. Gangliosides are known to regulate activity of EGF receptors (EGFRs), but the mechanisms have been unknown. By observing single molecules of gangliosides and EGFRs, we investigated the mechanisms. GM3 interacted with EGFRs much more extensively than other gangliosides. Surprisingly, we found that GM3 homodimers associated with N-linked glycan of EGFR monomers, which suppressed EGFR dimerization. We also found that after stimulation, enhanced affinity of liganded EGFRs overcame the GM3-dimer affinity to EGFRs, and thus, stable EGFR dimers were formed and activated, triggering Grb2 recruitment and the intracellular downstream signaling.		SY22-2 Palmitoylation-dependent regulation of neurotransmitter receptors in excitatory synapses 興奮性シナプスにおける神経伝達物質受容体のパルミトイル化による膜局在制御 Hayashi Takashi（林崇） Dept. Biochem. Cell. Biol., Natl. Inst. Neurosci., NCNP S-palmitoylation is the post-translational covalent attachment of lipid to proteins via thioester bonds that can direct proteins, including many neuronal receptors and ion channels, to specific regions of the membrane, such as membrane lipid rafts. Like phosphorylation, this process is labile and reversible, which ensures dynamic regulation of palmitoylated proteins in synapses.Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic AMPA receptors expression controls strength of excitatory synaptic transmission and plasticity. Excess synaptic AMPA receptors leads to epilepsy in response to seizure-inducible stimulation. Our previous studies revealed that a key modification of AMPA receptors trafficking is the reversible post-translational palmitoylation at their C-termini. To clarify the meaning of palmitoylation-dependent regulation of AMPA receptors in vivo, we generated GluA1 palmitoylation-deficient knock-in mice. These mutant mice showed elevated seizure susceptibility and seizure-induced neuronal activity, without affecting normal synaptic transmission, gross brain structure and behavioral appearance at basal level. Disruption of the palmitoylation site was accompanied with up-regulation of GluA1 phosphorylation and protein expression in the cerebrum. Furthermore, GluA1 palmitoylation functioned to suppress excessive spine enlargement above a certain size in LTP. Our findings presented here indicate that abnormality in GluA1 palmitoylation is liable to lead hyperexcitability, resulting in epileptic seizure. Thus, proper palmitoylation-dependent regulation of AMPA receptors trafficking is important for the maintaining E/I balance in the normal brain.

SY22-3 A membrane protein enriched in lipid rafts via palmitoylation, acts as a signaling molecular in the developing neuron 神経細胞の形態形成シグナリングにおけるGPM6aのパルミトイル化と脂質ラフト集積の意義 Igarashi Michihiro（五十嵐道弘） Dept Neurochem & Melec Cell Biol, Niigata Univ Grad Sch Med Dent Sci Lipid rafts are known to be the specialized membrane domains with low fluidity, due to its enrichment of sphingolipids and cholesterol. In the hematic or the immune cells, these domains are recognized as the hotspots of the signaling. Even in the neuron which are involved in more complex signaling pathways are probably not exceptional. However, much higher concentrations of sphingolipids and cholesterol than in the other cells, the views and the functions of neuronal lipid rafts are considerably confused for the present. Here, we focused on a glycoprotein, GPM6a, which is the most abundant membrane protein in the growth cone membrane. With no definite physiological roles. In the adult brain, GPM6a is known to be one of the major palmitoylated proteins. We identified its palmitoylated sites. Using RNAi and KO of GPM6a, we found that this protein is involved in the neuronal polarity determination. Surprisingly, GPM6a without palmitoylation did not have any roles for polarity determination, although this protein was expressed still in the membrane. The downstream molecules of GPM6a were also enriched in the lipid rafts at the time of polarity determination. Using the superresolution microscopy, cholesterol and GPM6a underwent the specialized clathrin-independent endocytosis for signaling. Taken together, GPM6a is concentrated in the lipid rafts via palmitoylation and accumulates its downstream molecules at the step of the neuronal differentiation. (Ref) Honda et al.: J Neurosci 37:4406 [‘17]; ibid. J Biol Chem [’17]; Nozumi et al.: Cell Rep 18: 2003 (’17); Ito et al.: Neurosci Res (’18)		SY22-4 EMARS method for lipid raft analysis: its significance and perspectives EMARS法による脂質ラフト分子解析とその意義 Kotani Norihiro（小谷典弘）¹，中野貴成¹，井田唯¹，伊藤吏那¹，橋爪幹¹，山口亜利沙²，瀬尾誠³，荒木智之¹，北條泰嗣¹，本家孝一²，村越隆之¹ ¹The Department of Biochemistry, Saitama Medical University ²Department of Biochemistry, Kochi University Medical School ³Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University Recent progress of morphological observation techniques such as fluorescence microscopy has made it possible to physiologically observe a lipid raft structure. In contrast, the Detergent Insoluble Membrane (DIM) method, which has mainly used for simple biochemical analysis of lipid rafts, requires the destruction of sample cells or tissues. Also, there is a problem to collect and analyze the lipid raft structure together in spite of having structural and functional diversities of each lipid raft domain. Since the diversity of lipid rafts is presumed to be important for cell function such as signal transduction platforms, it is essentially necessary to individually analyze each lipid raft domain. Based on these points, it has been considered that a novel biochemical method complementing the DIM method is necessary to elucidate the physiological functions of lipid raft structure.We previously developed a method, called “Enzyme-mediated activation of radical sources (EMARS)” method, to specifically label molecular complexes on the cell membrane under physiological conditions (Kotani N. et al. Proc. Natl. Acad. Sci. USA (2008)). This is the technique of specifically labeling a group of molecules proximity to a selectable given molecule without destroying cells or tissues, therefore, variety of dynamic lipid raft molecules can be individually analyzed under physiological condition. In this session, we introduce the outline of the EMARS method, the experimental inside of lipid raft analysis using the EMARS method, and the applications and perspectives of lipid raft analysis for neural cells and tissues (e.g. acute brain slices).