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Functional roles of Rho-GEF PLEKHG4B in regulation of actin remodeling and cell-cell junction formation

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Functional roles of Rho-GEF PLEKHG4B in

regulation of actin remodeling and cell-cell

junction formation

著者

二宮 小牧

18

学位授与機関

Tohoku University

学位授与番号

生博第427号

URL

http://hdl.handle.net/10097/00131555

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氏 名 ( 本 籍 地 ) 学 位 の 種 類 学 位 記 番 号 学 位 授 与 年 月 日 学 位 授 与 の 要 件 研 究 科 , 専 攻 論 文 題 目 博士論文審査委員 にのみや こまき 二宮 小牧 博士(生命科学) 生博第427号 令和3年3月25日 学位規則第4条第1項該当 東北大学大学院生命科学研究科 (博士課程)分子化学生物学専攻

Functional roles of Rho-GEF PLEKHG4B in regulation of actin remodeling and cell-cell junction formation (Rho-GEF PLEKHG4B によるアクチン骨格再構築と細胞間接 着形成における機能)

(主査) 大橋 一正 倉永 英里奈 福田 光則

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論文内容の要旨

Cell-cell junctions regulate various structural and functional characteristics of cells, including morphology, polarity, motility, proliferation and differentiation, and thus play essential roles in maintaining correct tissue architecture, development, and homeostasis. Adherens junctions (AJs) are cadherin-based cell-cell junctions, whose formation requires dynamic actin cytoskeletal

reorganization. Rho family small GTPases (Rho GTPases) are key molecules for actin cytoskeletal remodeling and they are activated by Rho guanine nucleotide exchange factors (Rho-GEFs). Although previous studies showed that Rho GTPases and several Rho-GEFs are involved in the formation of epithelial cell-cell adhesions, the complexity of the actin cytoskeletal regulation at junctions has been still elusive.

Considering that genes of Rho-GEFs are identified more than four times as many as those of Rho GTPases in human genome, Rho-GEF genes appear to have diversified for the precise actin cytoskeletal remodeling in various cellular events by spatiotemporal regulation of Rho GTPases. PLEKHG4B (pleckstrin homology domain-containing family G member 4B) is a Dbl-like Rho-GEF, whose structure is closely related to Solo (also known as ARHGEF40) and PLEKHG4 (also known as puratrophin-1). These three Rho-GEFs share a common domain structure with an N-terminal Solo domain, a CRAL/TRIO domain and spectrin repeats in the medial region, and a C-terminal DH-PH domain, except that PLEKHG4 lacks the N-C-terminal Solo domain. While the understanding of the cellular roles of Solo and PLEKHG4 have been recently advanced, the function of PLEKHG4B remains to be investigated.

In this study, I first found that overexpression of PLEKHG4B induced F-actin-rich membrane protrusions in epithelial cells through Cdc42 and Rac1 activation. The protrusions preferentially extended toward adjacent cells, indicating that PLEKHG4B-mediated actin remodeling is cell-cell contact-dependent. To investigate which Rho GTPases are the targets of PLEKHG4B, I conducted pulldown assays and revealed that knockdown of PLEKHG4B significantly decreased Cdc42 activity and tended to increase RhoA activity in the cells. I also

Figure 1. Effects of PLEKHG4B knockdown on cell-cell adhesion in A549 cells. Control cells (top) and PLEKHG4B knockdown cells (bottom) were stained for actin cytoskeleton and β-catenin (AJ). Right panels show magnified views of boxed areas. F-actin β-catenin si C o nt ro l 20 µm si P L E K H G 4 B F-actin

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examined PLEKHG4B knockdown on actin filaments organization and cell-cell adhesions. Control A549 epithelial monolayer cells showed the 'closed junctions' with closely packed actin bundles along the cell-cell contacts, but PLEKHG4B knockdown suppressed closed junction formation and exhibited the 'open junctions' with split actin bundles located away from the cell-cell boundary (Fig. 1). In calcium-switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and -catenin accumulation at cell-cell junctions, suggesting that PLEKHG4B is involved in cell-cell adhesion maturation. Further, I demonstrated that PLEKHG4B knockdown abrogated the reduction in myosin activity normally seen in the later stage of junction formation. The aberrant myosin activation and impairments in closed junction formation in PLEKHG4B-knockdown cells were reverted by ROCK inhibition or LARG and PDZ-RhoGEF PLEKHG4B-knockdown. These results suggest that PLEKHG4B enables actin remodeling during epithelial cell-cell junction maturation, probably by reducing myosin activity in the later stage of junction formation, through suppressing LARG, PDZ-RhoGEF and their downstream RhoA-ROCK pathway.

To gain insights into the mechanisms governing the localization and function of PLEKHG4B, proteomics analysis was conducted to search for its binding partners (Ohta, master thesis, 2020). Among several proteins identified, I focused on annexin A2 (ANXA2), a phospholipid- and actin-binding protein that mediates the organization of membrane microdomains and regulates actin dynamics at specific membrane sites. I showed that ANXA2 binds to PLEKHG4B and is involved in the localization of PLEKHG4B to cell-cell junctions.

Collectively, these results highlight a crucial role of PLEKHG4B, the Rho-GEF targeting Cdc42, in the reorganization of the actin cytoskeleton and formation of cell-cell adhesions through regulating other RhoA-targeting GEFs and associating with ANXA2 (Fig. 2). Further studies on the spatiotemporal regulation of Rho-GEFs and their

interplay with the components of the actin cytoskeleton, cell adhesion molecules, and cell polarity complexes, will advance our understanding of the mechanisms underlying the formation and remodeling of cell-cell adhesions, and their roles in epithelial development and homeostasis, as well as in cancer

invasion and metastasis. Figure 2. Functional model of PLEKHG4B in the

cell-cell junction formation.

PLEKHG4B

Cdc42⤴ PDZ-RhoGEFLARG, RhoA-ROCK⤵ Actin reorganization

Annexin A2

Open junction Closed junction

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論文審査結果の要旨

上皮細胞の細胞間接着構造のAdherens junction (AJ)は、極性化した細胞層を形成させ、組織の機 能発現、恒常性の維持、形態形成等に寄与する重要な構造であり、カドヘリンを介した細胞間接 着から細胞内アクチン骨格の再構築による多段階の過程を経て形成される。この時、アクチン骨 格の主要な制御因子である低分子量G 蛋白質 Rho ファミリー分子群の働きは時空間的に制御され るが、その分子機構は未だ不明な点が多い。

本論文では、Rho ファミリー蛋白質の活性化因子 Rho-GEF の一つである PLEKHG4B に注目し、 AJ 形成過程における機能解析を行った。まず、PLEKHG4B の基本的な細胞機能を探索し、 PLEKHG4B が Cdc42 の Rho-GEF として働くこと、ヒト肺上皮癌 A549 細胞における PLEKHG4B の過剰発現が、細胞間接着部位特異的に突出構造を形成させることを発見した。そのため、A549 細胞に対するPLEKHG4B の発現抑制の効果を解析した結果、AJ の裏打ちとなるアクトミオシン 構造が、正常では AJ 構造と重なる状態(Closed junction)となるのに対し、PLEKHG4B の発現抑制 により細胞間接着部位から離れた構造(Open junction)になることを見出した。Ca2+スイッチ解析の

結果、これは AJ が未熟な状態の形態であること明らかにした。さらに、アクトミオシン構造が Open junction から Closed junction へ変化していく際に、ミオシン II の活性が低下して Closed junction へ移行すること、その過程に PLEKHG4B が RhoA-ROCK 経路を抑制して寄与することを見出し た。加えて、そのRhoA の活性抑制に、PLEKHG4B が RhoA の GEF である LARG, PDZ-RhoGEF を介して働くことを明らかにした。また、PLEKHG4B の結合蛋白質として同定した Annexin A2 が、 PLEKHG4B の細胞間への局在に寄与し、AJ 形成に共役して働くことを見出した。これらの結果 は、AJ の形成過程の詳細な分子機構の解明につながる重要な成果である。 以上のように、本論文は、著者が自立して研究活動を行うに必要な高度の研究能力と学識を有 することを示している。したがって、二宮小牧氏が提出する論文は、博士(生命科学)の博士論文 として合格と認める。

Figure 1. Effects of PLEKHG4B knockdown on  cell-cell adhesion in A549 cells. Control cells (top)  and PLEKHG4B knockdown cells (bottom) were  stained for actin cytoskeleton and β-catenin (AJ)

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