Actin filaments-stabilizing and -bundlingactivities of cofilin-phosphatase Slingshot-1

著者 栗田 宗一号 4学位授与番号 89URL http://hdl.handle.net/10097/36953

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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

くりだそういち

栗田宗一

博士(生命科学)

生博第89号

平成19年3月27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)分子生命科学専攻

ActiRfila}nents-stabilizingand-bun(ilingactivitiesof

cofi生in-phosp}1ataseSlingshot-1

(コフィリンホスファターゼSlingshoHのアクチンフィラ

メント安定化及び東化活性)

(主査)教授水野健作

教授山本和生

教授牟田達史

岨曇榔

馨

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Actin cyioskeleton plays an essential role in various intracellular events, including cell migration,

cyiokinesis, endocyiosis, and cell polarity forrnation. Actin filament dynamics and reorganization are

spatiotemporally regulated by a large number of actin-binding proteins and upstream sib"naling molecules.

Cofilin, a key regulator of actin filament dynamics, promotes depolymerization and severing of actin

filaments to reorganize actin cyioskeleton. Cofilin is inactivated by phosphorylation of Se~~3 by LIM-kinase

and testicular protein kinase, and reactivated by Slingshot (SSH) family of protein phosphatases. Slingshot- 1

(SSH1), an isoform of SSH, is known to be involved in the regulation of actin filament dynamics through

cofilin dephosphorylation. SSHI binds to filamentous actin (F-actin) and its cofilin-phosphatase activity is

highly activated through association with F-actin. Although SSHI has the F-actin-binding property, most

previous studies have not noted the role of SSHI as an F-actin-binding protein. On the other hand, several

reports showed that ovefexpression of phosphatase-dead or wild-type SSHI induced aberrantly thick bundles

or accumulations of F-actin in cultured cells. These observations suggested that SSHI has the potential to

promote F-actin stabilization and bundling.

To examine whether SSHI stabilizes actin filaments, I analyzed the effect of SSHI on dilution-induced

actin filament depolymerization by measuring the fluorescence intensity of pyrene-labeled actin.

Fluorescence intensity of pyrene-actin is known to decrease along with actin depolymerization (Fig. I A).

When F-actin was preincubated with SSHI before initiation of depolymerization, the rate of spontaneous

actin depolymerization was decreased in an SSH I dose-dependent mamer. SSH I also suppressed

cofilin-induced rapid actin depolymerization (Fig. IB). These results suggest that SSHI has

F-actin-stabilizing activity. Depolymerization assays using deletion mutants of SSHI revealed that both the

N- and C-terminal regions of SSHI were required for its F-actin-stabilizing activity against cofilin.

A

3e5 nfn 407 nfn 385 nm

~~~~Ll ~' /~M~M~ 407 nm ~ v~ ¥ f F-actin ~ C)epelyme zatlon

pyrene~t~b8ied ae Pytene-l~beied {* e-Botin

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Fig. i . Actin depolymerization assay. (A) A scheme of pyrene-actin assay. Fluorescence intensity at 407 nm of pyrene-actin decreases along with depolymerization. (B) SSHI suppresses cofilin-induced rapid actin

depolymerization. Fluorescnece intensity (a. u.: arbitrary unit) versus time (s) after initiation of depolymerization is shown.

In addition to F-actin-stabilizing activity, SSHI displayed F-actin-bundling activities in low-speed

centrifugation assays and microscopic analyses. Centrifugation at a low-speed ( I 0,000 x g) precipitates only

crosslinked F-actin. When G-actin was polymerized in the presence of SSH I , both F-actin and SSH I were

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recovered in the precipitates (Fig. 2A), indicating that SSHI bundles F-actin in vitro. I next observed the

F-actin bundles crosslinked by SSH I with fluorescence microscopy. Actin was visualized by using

AlexaFluor546-labeled actin. Full-length of SSH I induced thick and massive actin bundles (Fig. 2B).

Electron microscopic analysis further revealed that SSH I -induced thick actin bundles are composed of

transversely-ordered actin filaments (Fig. 2C). in both low-speed centrifugation assay and fluorescence

microscopic analysis, deletion of the N- or C-terminal region of SSHI significantly decreased

F-actin-bundling activity, although the C-terminal fragments (C. PC) exhibited higher activity than the

N-teuninal fragments (NP, N461, N698). These results suggest that both the N- and C-ternlinal regions of

SSHI were involved in F-actin bundling and the C-tenninal region of SSH I seems to play a predominant

role in Factin bundling.

Fig. 2. F-actin-bund}ing activity of SSH I . A - (A) Low-speed centrifugation assay. G-actin

B In~ut {*D*i 1 2 3 4 5 e 7

~~~~j at I OOOO >< g. Resulting Precipitates (ppt) <c') ;ottppt was polyinerized with SSHI and centrifuged L SsHi *" T5

{~~D5*")1 *o +~ ~~~ Acti~ *oo

,;;lls::Hi ' and supematants (sup) were resolved by 3'

ctl~ ~~j:j:li:i~Bii::1:; ~ polymerized with SSH I oi rts deletlou 75

~ , SDS-PAGE, followed by CBB-staining. (B) 5~ * oo. 3' AlexaFluor546 Iabeled G actm was

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C - .~ ~'=~~i~i~~s:'~iiti=i;,. ~'"'.'~'.,"'- "**"'~ mutants, and analyzed by fluorescence e'~ ~*M SSH1

4 Acti~ ' o~)5 ~'~ ssN~ microscopy. (C) G-actin was polymerized 5 Actin ' 0.1~M SSHI with SSHI and analyzed by electron 7 Actin'0.4 ~M SSHI I *'* 200~~~ microscopy.

To investigate whether F-actin-stabilizing and -bundlin~~cF activities of SSH I participate in actin

cyioskeletal organization in cultured cells, Expression of endogenous SSHI was suppressed by short

interfering RNA (siRNA) in C2C12 mouse myoblast cells, which bear well-organized actin stress fibers.

Knockdown of SSH~ expression significantly suppressed stress fiber formation, whereas controi

siRNA-transfccted cells retained stress fibers (Fig. 3). These results indicate that SSHi is critically involved

in stress fiber formation or maintenance in C2C 1 2 cells.

Fig. 3 . Knockdown of SSH I expression suppressed stress fiber fonrlation in C2C12 celis. C2C12 cells were co-transfected with siRNA vectors and one-tenth the amount of CFP vector. F-actin was stained with

rhodamine-phalloidin. Arrowheads indicate CFP-expressing cells. Percentages of stress fiber-tol~Tling cells in total CFP-expressing cells are shown in the bar graph (right). Results are shown as the means ~ S.D. of four independent experiments

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In conclusion, I showed direct evidence that SSHI possesses F-actin-stabilizing and -bundling

activities in cell free assays. I also demonstrated that knockdown of SSH I expression induced a loss of stress

fibers in cultured cells. These findings suggest a novel cellular function of SSHI in regulating actin

cyioskeletal organization through its F-actin-stabilizing and -bundling activities, in addition to

cofilin-phosphatase activity.

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論文審査結果の要旨

馨婁垂醤慧

アクチン細胞骨格の再編成は、細胞の運動、形態、接着などにおいて重要な役割を担

っており、多くのアクチン結合蛋白質によって制御されている。Slings姦oH(SSB1)

は、アクチンフィラメントの切断・脱重合因子であるコフィリンを脱リン酸化して活性

化することで、アクチン細胞骨格の再編成に関与している。SS班はアクチンフィラメ

ント結合能をもち、そのコフィリンホスファターゼ活性はアクチンフィラメントヘの結

合によって著しく活性化される。本論文は、SSH1がアクチンフィラメントの安定化・

束化因子として機能することを明らかにした。ピレン標識アクチンを胴いたf刀vπ即

でのアクチン動態解析において、SSH1は自発的な、あるいはコフィリンによって誘導

されるアクチンフィラメントの脱重合を抑制し、アクチンフィラメントの安定化活性を

もつことを示した。また、SS田存在下でアクチンを重合させると、アクチンフィラメ

ントの束が形成され、SS則はアクチンフィラメントを束化する活性をもつことを示し

た。SSH1のこれらの活性には、そのN末端領域とC末端領域の両方が重要であること

を明らかにした。マウス筋芽細胞株C2C12細胞におけるSSHlの発現をRNA干渉法によ

って抑制すると、アクチンストレスファイバーをもつ細胞が減少し、こ.の表現型は脳A

干渉の標的配列を持たないヒトSS瓢の発現によって回復することを示し、SS膿のアク

チンフィラメント安定化及び束化活性が、C2C12細胞におけるストレスファイバーの安

定化に寄与していることを明らかにした。以上の結果から、SS田はコフィリンホスフ

ァターゼとしての活性に加えて、アクチンフィラメントを安定化、東化する活性によっ

て細胞内のアクチン細胞骨格を制御していることを明らかにした。

これらの研究成果は,本人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、栗田宗一提出の論文は、博士(生命科学)の

博士論文として合格と認める。

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