actin filaments-stabilizing and -bundling activities of
TRANSCRIPT
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
無難
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
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
一47一
~;
~* i; ~ ~ ~ ~"'
ij+* ~{ il I
~/+'-'~l~~J~~~~' ~ ~)~; ~
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
~~' ~;
c (o
~~'~ -8 e'~'c Cl¥ (:}o ol~i c"t~-e'
a :~ tL Time
B ~ctin, e~ ~M Hi~ no ~iddition -~- e 5~IM cef *~*. ~O5~M SSH~
500 ~ ~~ O~5uM co~ + e 05 ~[M SS~~
:: 450 aj
q'o 400 c: e)
oo, 350 ~
e ~ 3aO ~L
250 ~ , , O 100 200 300 400 500 600
Tinle (S)
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
~~~
~~
s il
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
so - --- A*tin
37
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
- 49 -
i
~i } i;
~~ {
i=.~
{'~ ~ **~
i *~ I
i i'
~
;! I fl ;{ il I
I
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.
50
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。
一51一
き