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セマフォリンによるCaenorhabditis elegans表皮細胞での小胞輸送制御

田中, 洋希 名古屋大学

2021.04.30

概要

セマフォリンシグナルは動物細胞の形態変化を制御する主要なシグナルのひとつであり、脊椎動物では神経軸索誘導や血管形成での役割がよく知られている。セマフォリンシグナルによる細胞形態制御には細胞骨格や細胞膜動態の調節が関わることが示されているが、詳細については不明な点が残されている。

線虫C. elegans はセマフォリンシグナル伝達の遺伝学的解析に有用な実験系である C. elegans の膜結合型セマフォリン(SMP-1、SMP-2)は受容体であるプレキシン(PLX-1) を介して ray や陰門を形成する表皮細胞の形態制御を担う。ray は雄成虫尾部に存在する感覚器であるが、plx-1 変異体では幼虫期の ray 前駆体表皮細胞の形態異常が原因となって、一部の ray の位置が異常となる。plx-1 変異の ray 表現型に対する抑圧変異を手がかりとして、これまでに、セマフォリンシグナルが mRNA 翻訳や細胞骨格を制御することが明らかになっている。

以前の研究において、哺乳類 stonin2 相同分子をコードする unc-41 遺伝子変異が ray 表現型を抑圧することが見出されていた。stonin2 は神経軸索末端における神経伝達物質放出後のシナプス小胞膜の細胞内への再取り込みにおいて、synaptotagmin I と共同して働く因子である。synaptotagmin I は、シナプス小胞からの神経伝達物質放出における Ca2+センサーとして働き、シナプス小胞のエンドサイトシス・エキソサイトシスにまたがるリサイクリングを調節することが知られている。C. elegans synaptotagmin I をコードする snt-1 遺伝子変異によって plx-1 変異の ray および前駆細胞での表現型が抑圧されることを確認できたため、本研究ではセマフォリンシグナルが SNT-1 - UNC-41 系を抑制するという仮説に基づいて、遺伝学的・細胞生物学的解析をおこなった。

まず、plx-1 変異体ではエンドサイトシスマーカーである HGRS-1::GFP の顆粒数が野生型と比較して増加しており、セマフォリンシグナルによるエンドサイトシス抑制が示唆された。また、plx-1 変異体ではエキソサイトシスマーカーである GFP::SNB-1 の細胞膜への局在が野生型より増加したことから、セマフォリンシグナルによってエキソサイトシスも抑制されることが示唆された。

次に、SNT-1::mCherry と各細胞内小器官マーカーとの共局在解析から、分解経路を構成する後期エンドソームとリソソームでの SNT-1 の局在が、plx-1 変異体では野生型に比較して減少し、一方、初期エンドソームとリサイクリングエンドソームでの SNT-1 の局在は plx-1 変異体で増加することが明らかになった。また、ライブセルイメージングによって、SNT-1::mCherry の分解経路での輸送は、野生型では plx-1 変異体に比べて輸送速度が速く移動距離も長いことが明らかとなった。さらに RNAi によるノックダウン実験によって、この SNT-1 の輸送は微小管(TBB-2)・ダイニン(CHE-3)系に依存することが示された。

前述の plx-1 変異体におけるエンドサイトシスマーカー発現の増加、およびエキソサイトシスマーカーの細胞膜への局在上昇は、snt-1 変異によって抑圧された。また、 UNC-41 の RNAi ノックダウンは、plx-1 変異体での SNT-1 の分解経路への輸送低下を抑圧した。これらの結果から、セマフォリンシグナルは SNT-1 - UNC-41 系依存的なエンドサイトシスとエキソサイトシスを抑制し、このことが SNT-1 の分解経路への輸送を促進すると考えられる。

本研究によって、C. elegans の SNT-1-UNC-41 系が神経細胞だけでなく表皮細胞でも機能し、細胞形態制御に関わることが明らかになった。近年、脊椎動物神経系の研究において、セマフォリンシグナルがエンドサイトシスを促進することが報告されているが、その分子機構には未解明な部分が多く、セマフォリンシグナルと synaptotagmin I-stonin2 系との関係についてはこれまで報告がない。本研究の結果は、セマフォリンシグナルによる SNT-1 - UNC-41 系抑制を介した新規な細胞形態制御機構の存在を示唆するものである。

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