江奇峰,蔡紹皙,晏小清
1.重慶大學(xué)生物工程學(xué)院,“生物流變科學(xué)與技術(shù)”教育部重點(diǎn)實驗室(重慶大學(xué)),重慶400044;
2.波士頓生物醫(yī)學(xué)研究所,波士頓02472,美國
鈣調(diào)蛋白結(jié)合蛋白通過調(diào)節(jié)細(xì)胞骨架穩(wěn)定性影響腫瘤細(xì)胞運(yùn)動能力
江奇峰1,2,蔡紹皙1,晏小清1
1.重慶大學(xué)生物工程學(xué)院,“生物流變科學(xué)與技術(shù)”教育部重點(diǎn)實驗室(重慶大學(xué)),重慶400044;
2.波士頓生物醫(yī)學(xué)研究所,波士頓02472,美國
輕鏈鈣調(diào)蛋白結(jié)合蛋白(light-chain Caldesmon,l-CaD)是一種重要的肌動蛋白結(jié)合蛋白,普遍存在于眾多非肌肉細(xì)胞中。體外研究證明,l-CaD能通過與肌動蛋白的結(jié)合起到促進(jìn)原肌動蛋白(G-actin)聚合、穩(wěn)定肌動蛋白纖維(F-actin)結(jié)構(gòu)的作用。在磷酸化作用下,l-CaD能從肌動蛋白纖維上脫離并促進(jìn)肌動蛋白纖維的解聚。該研究擬考察l-CaD在細(xì)胞內(nèi)對細(xì)胞肌動蛋白骨架的調(diào)節(jié)作用,闡明l-CaD對細(xì)胞運(yùn)動能力的影響,作者將天然低表達(dá)l-CaD的人源性乳腺癌細(xì)胞MCF-7作為細(xì)胞模型,在MCF-7胞內(nèi)以基因轉(zhuǎn)染的方式高表達(dá)外源野生型l-CaD及其磷酸化突變株A1234-CaD(不可磷酸化CaD)、D1234-CaD(完全磷酸化CaD)。首先,通過激光共聚焦掃描,探討了l-CaD對細(xì)胞骨架重排的調(diào)節(jié);其次,通過細(xì)胞遷移transwell陣列,檢測了l-CaD對細(xì)胞遷移能力的影響;最后,在單細(xì)胞層次上測定了細(xì)胞基底牽張力、胰酶刺激下的細(xì)胞基底脫附能力,并進(jìn)一步檢測了l-CaD對細(xì)胞遷移子過程中細(xì)胞伸張、收縮的影響。研究結(jié)果顯示,l-CaD在胞內(nèi)對細(xì)胞骨架的形成有顯著的調(diào)控作用。非磷酸化l-CaD主要富集在細(xì)胞骨架上,增強(qiáng)了細(xì)胞骨架的強(qiáng)度,導(dǎo)致細(xì)胞基底牽張力以及對胰酶的耐受性增強(qiáng),但對細(xì)胞的遷移能力有顯著的抑制作用;磷酸化l-CaD跟細(xì)胞骨架結(jié)合能力很弱,對細(xì)胞的運(yùn)動能力沒有顯著影響。通過磷酸化,l-CaD起到了一個“蛋白開關(guān)”的作用,通過控制細(xì)胞骨架的解聚、重排來調(diào)節(jié)細(xì)胞的運(yùn)動能力。
鈣調(diào)蛋白結(jié)合蛋白;細(xì)胞骨架;細(xì)胞遷移;細(xì)胞基底牽張力
細(xì)胞的運(yùn)動能力是組織形成及器官發(fā)育的關(guān)鍵因素,細(xì)胞運(yùn)動涉及到生物體眾多的生理過程,其中包括組織修復(fù)、免疫應(yīng)答及腫瘤的浸潤和轉(zhuǎn)移[1~3]。細(xì)胞的肌動蛋白骨架作為胞內(nèi)信號傳遞的關(guān)鍵途徑,是細(xì)胞運(yùn)動的重要引擎。作為維持細(xì)胞正常形態(tài)的主要組成部分,細(xì)胞骨架通過有序的解聚和重排為細(xì)胞提供重要內(nèi)力,促進(jìn)細(xì)胞運(yùn)動[4,5]。細(xì)胞骨架的有序調(diào)整不是僅涉及肌動蛋白重排的單一過程,而是在很多細(xì)胞骨架結(jié)合蛋白協(xié)同參與下進(jìn)行的復(fù)合行為。近些年的研究證實了多種細(xì)胞骨架結(jié)合蛋白參與了細(xì)胞骨架調(diào)控[4],其中就包括鈣調(diào)蛋白結(jié)合蛋白(Caldesmon,CaD)[6~8]。
CaD存在于幾乎所有脊椎動物細(xì)胞中,它有由同一基因通過選擇剪接產(chǎn)生的兩種亞型[9]:只存在于平滑肌細(xì)胞的重鏈鈣調(diào)蛋白結(jié)合蛋白(h-Caldesmon,h-CaD)和細(xì)胞中普遍存在的輕鏈鈣調(diào)蛋白結(jié)合蛋白(l-Caldesmon,l-CaD)。一般認(rèn)為,h-CaD是調(diào)節(jié)平滑肌細(xì)胞收縮的重要組件,l-CaD是細(xì)胞骨架單位[6~8]。l-CaD以一個類似于訂書針結(jié)構(gòu)的方式與肌動蛋白結(jié)合,一方面能夠穩(wěn)定胞內(nèi)應(yīng)力纖維[10~12],另一方面通過與肌動蛋白結(jié)合,抑制肌動球蛋白與肌動蛋白的結(jié)合,從而抑制細(xì)胞骨架的調(diào)整。通過與鈣調(diào)蛋白(Calmodulin)結(jié)合或者在磷酸化作用下,l-CaD能夠從肌動蛋白上解離下來,并恢復(fù)肌動球蛋白與肌動蛋白的結(jié)合。已有研究證明,1-CaD能夠抑制凝溶膠蛋白與肌動蛋白結(jié)合,降低凝溶膠蛋白對肌動蛋白纖維的剪切作用[13~15],1-CaD或者它的C末端片段CaD39,可以加強(qiáng)原肌球蛋白與肌動蛋白絲的結(jié)合[16,17]。因此人們認(rèn)為在體外蛋白層次上,l-CaD能夠通過與肌動蛋白的結(jié)合來調(diào)節(jié)肌動蛋白纖維的結(jié)構(gòu)。在細(xì)胞內(nèi),l-CaD主要集中在應(yīng)力纖維、前端板狀偽足和細(xì)胞膜褶皺中[18~20]。研究顯示,通過向細(xì)胞中顯微注射l-CaD抗體,可以導(dǎo)致細(xì)胞內(nèi)微粒運(yùn)動的瞬時速度和躍移距離顯著減少,胞內(nèi)肌動蛋白纖維解聚,應(yīng)力纖維消失[21,22]。進(jìn)一步研究發(fā)現(xiàn)[23],細(xì)胞分裂間期的靜態(tài)細(xì)胞中,l-CaD以非磷酸化的形式出現(xiàn),并主要位于細(xì)胞骨架較為穩(wěn)定的應(yīng)力纖維上;當(dāng)細(xì)胞開始分裂或遷移時,l-CaD主要以磷酸化的形式位于細(xì)胞骨架調(diào)整活躍的細(xì)胞運(yùn)動前沿,并參與細(xì)胞粘附斑的形成。CaD跟cortactin、Arp2/3 complex、WASP等肌動蛋白結(jié)合蛋白一起存在于細(xì)胞骨架結(jié)構(gòu)足體(podosome)中,足體被認(rèn)為是細(xì)胞粘附、遷移和浸潤細(xì)胞外基質(zhì)的主要方式,因為足體的中心部分是金屬基質(zhì)蛋白酶MMP-9的重要分泌場所[24,25]。近來的研究證明,通過激活細(xì)胞周期依賴性激酶(cell division cycle 2,Cdc2)對l-CaD的磷酸化作用,將顯著影響內(nèi)皮細(xì)胞和血管平滑肌細(xì)胞足體的形成,因此我們猜測l-CaD的分布和磷酸化可能是調(diào)節(jié)細(xì)胞運(yùn)動能力的重要因素。
為了進(jìn)一步明確l-CaD及其磷酸化作用對細(xì)胞運(yùn)動能力的影響,我們選取人源性乳腺癌細(xì)胞MCF-7作為載體,通過基因轉(zhuǎn)染的方式,在MCF-7細(xì)胞內(nèi)高表達(dá)連接了增強(qiáng)型綠色熒光蛋白的外源野生型l-CaD(EGFP-wtCaD)及其磷酸化突變株:包括不可磷酸化l-CaD突變株(EGFP-A1234CaD)、磷酸化l-CaD突變株(EGFP-D1234CaD)。MCF-7天然低表達(dá)l-CaD,因此內(nèi)源性l-CaD對外源高表達(dá)l-CaD的拮抗影響可忽略。結(jié)合細(xì)胞骨架熒光染色、細(xì)胞基底牽張力及遷移能力的測定,我們發(fā)現(xiàn)l-CaD能夠通過穩(wěn)定細(xì)胞骨架,增加細(xì)胞骨架強(qiáng)度、細(xì)胞的基底牽張力和胰酶耐受性,并對細(xì)胞遷移有顯著的抑制作用,磷酸化的l-CaD與細(xì)胞骨架的結(jié)合能力相對較弱,對細(xì)胞遷移能力無顯著影響。我們的研究不僅明確了l-CaD磷酸化可以通過對細(xì)胞骨架的調(diào)節(jié)作用影響細(xì)胞的運(yùn)動能力,而且為通過l-CaD作為靶向手段治療腫瘤提供了新的思路。
人源性乳腺癌細(xì)胞MCF-7(ATCC#HTB-22TM)用添加了10%胎牛血清和1%抗生素的DMEM培養(yǎng)基(CellgroTM)培養(yǎng);人源性上皮細(xì)胞(human mammary epithelial cell,HMEC)用添加了10 ng/mL人源性上皮生長因子(hEGF,CellgroTM)、10 μg/mL胰島素、5%胎牛血清和1%抗生素的DMEM-F12基礎(chǔ)培養(yǎng)基(CellgroTM)培養(yǎng)。所有細(xì)胞均在37℃,5%CO2條件下培養(yǎng)。
通過NcoⅠ將含有l(wèi)-CaD DNA的片段從人源性成纖維細(xì)胞l-CaD(Gene Bank #M64110)的pCB6hx質(zhì)粒上酶切下來,經(jīng)過T4DNA聚合酶平整末端后,用BamHⅠ處理獲得BamHⅠCaD插入片段。將BamHⅠCaD插入哺乳動物表達(dá)載體pEGFPC1 (Clontech)BspE和BamHⅠ多克隆位點(diǎn)之間,獲得野生型l-CaD(EGFP-wtCaD),并通過測序確認(rèn)EGFP開放閱讀框和終止密碼子的正確性。通過定點(diǎn)突變的方式[23],突變ERK跟PAK位點(diǎn)的氨基,獲得了PAK和ERK磷酸化位點(diǎn)完全被封閉的不可磷酸化l-CaD突變株(EGFP-A1234,PAK和ERK位點(diǎn)Ser都更換為Ala)和模擬PAK和ERK位點(diǎn)完全磷酸化的突變株(EGFP-D1234,PAK和ERK位點(diǎn)Ser都更換為Asp),對照組質(zhì)粒為不含l-CaD序列的EGFP空質(zhì)粒載體。
MCF-7細(xì)胞以5×105的密度種在6孔板內(nèi),孵育過夜。800 μl無血清DMEM培養(yǎng)基饑餓細(xì)胞2 h,隨后加入93 μl含有2 μg質(zhì)粒DNA以及5 μl FUGENE轉(zhuǎn)染試劑(Roche)的無血清DMEM培養(yǎng)基。孵育5 h后用完全培養(yǎng)基換液。48 h后通過Western-blot檢測蛋白表達(dá)量。
通過Western blot檢測細(xì)胞中內(nèi)外源性總l-CaD表達(dá)量。將細(xì)胞以1×105細(xì)胞/孔的密度種在6孔板(Becton-Dickinson,Rutherford,NJ)中,孵育24 h。移除培養(yǎng)基,冰預(yù)冷的PBS沖洗細(xì)胞兩次。向每孔加入100 ml裂解液制備總蛋白,裂解液中含有1 mmol/L苯甲磺酰氟(Sigma)、10 mg/mL亮肽素(Sigma)、30 mg/mL胰蛋白酶抑制劑(Sigma)和1 mmol/L NaVO3(Sigma)。冰上孵育30 min,刮凈。細(xì)胞提取物都通過SDS-PAGE分離,并用親和純化的多克隆抗體anti-CaD和anti-β-actin單克隆抗體(Sigma)進(jìn)行免疫標(biāo)記,再用親和純化的、分別連接有IRDyeTM 700和800的抗兔并抗鼠的熒光二抗標(biāo)記。用Odyssey紅外圖像系統(tǒng)(Li-COR,Biosciences,Lincoln,NE)檢測,可以直接定量表征紅外熒光,熒光強(qiáng)度則代表蛋白表達(dá)量。
細(xì)胞種植在表襯有人纖連蛋白的玻璃蓋玻片上(Becton Dickinson),孵育24 h直至細(xì)胞完全鋪展。PBS清洗兩次,4%多聚甲醛固定15 min,0.3%TritonX-100透膜5 min。F-actin用rhodamine phalloidin(1∶100)染色并孵育1 h,所有溶液均為PBS制備。PBS清洗3× 5 min,Mowiol(Sigma)封片。所有圖像均通過Nikon Eclipse TE300顯微鏡的BioRad Radiance 2000激光共聚焦系統(tǒng)獲得,并通過Laser Sharp 2000 BioRad軟件處理。
基于細(xì)胞趨化原理,使用8μm孔徑濾膜的Transwell24孔板(Becton Dickinson)進(jìn)行細(xì)胞遷移實驗。濾膜的內(nèi)、外壁用5 μg/mL的人纖連蛋白(R&D,Minneapolis)在37℃表襯2 h,PBS清洗。200 μl的細(xì)胞懸液以2×104cells/孔的密度種在Transwell的內(nèi)腔。外腔加入800 μl含有10%FBS的完全培養(yǎng)基。37℃孵育24 h,選取9個隨機(jī)視野,用ZEISS-AXIO熒光顯微鏡分別在相差視野和熒光視野下計數(shù)總的遷移細(xì)胞數(shù)和轉(zhuǎn)染了綠色熒光的遷移細(xì)胞數(shù)。具有綠色熒光的遷移細(xì)胞數(shù)與總的具有綠色熒光的細(xì)胞數(shù)之間的比率則作為轉(zhuǎn)染細(xì)胞的相對遷移能力。
細(xì)胞種植于均勻包埋了200 nm紅色熒光微珠的聚丙烯酰胺凝膠基底表面。完全鋪展的細(xì)胞由于細(xì)胞骨架產(chǎn)生的應(yīng)力使凝膠產(chǎn)生一定程度的形變,該形變將導(dǎo)致凝膠內(nèi)部均勻包埋的微珠產(chǎn)生一定的位移,通過Fourier-Transform Traction Microscopy(FTTM)則可以將細(xì)胞從基底脫附前后微珠位移的變化,轉(zhuǎn)算為細(xì)胞施加在凝膠表面的牽張力。
1.7.1 聚丙烯酰胺凝膠的制備
聚丙烯酰胺凝膠基底的制備主要根據(jù)前人的方法[26,27]。具體步驟概括為:1)0.1 mol/L NaOH表面改性玻璃底培養(yǎng)皿(MatTek)的玻璃區(qū)域,過夜風(fēng)干;2)滴加97%的3-aminopropyltrimethoxylsilane于步驟1)中NaOH沾染的區(qū)域,清洗,風(fēng)干,隨后加入0.5%的戊二醛,然后將培養(yǎng)皿清洗并過夜風(fēng)干。3)在培養(yǎng)皿中央加入20 μl含有0.03%雙丙烯酰胺、5%丙烯酰胺(Bio-Rad)、0.6%200 nm紅色熒光微珠(Invitrogen)、0.5%過硫酸銨以及0.05%TEMED(Bio-Rad)的超純水。隨后用25 mm蓋玻片均勻覆蓋直至聚合。移去蓋玻片,加入200 μl溶液〔1 mmol/L Sulfosuccinimidyl-6-[4-azido-2-nitrophenylamino] hexanoate(Sulfo-SANPAH;Pierce)溶解在200 mmol/L HEPE溶劑中〕進(jìn)行表面活化。紫外線照射5 min,0.1 mol/L HEPES清洗兩次,每次15 min,PBS清洗15 min,加入200 μlⅠ型膠原溶液(0.1 mg/mL;Inamed Biomaterials)表襯,4℃過夜。隔天,PBS洗滌并用2 ml無血清培養(yǎng)基水合,于37℃、5%CO2的孵箱中儲存至使用。
1.7.2 細(xì)胞基底牽張力的測定
將1×104個細(xì)胞種植在制備好的聚丙烯酰胺凝膠上,孵育24 h,無血清DMEM培養(yǎng)基饑餓細(xì)胞過夜。由于細(xì)胞高表達(dá)連接了綠色熒光蛋白基因的質(zhì)粒,通過熒光顯微鏡可分別確定單個綠色熒光細(xì)胞以及細(xì)胞下方的紅色熒光微珠,在相差顯微鏡和熒光顯微鏡下對細(xì)胞和細(xì)胞下微珠拍照。胰酶消化細(xì)胞后,獲取另一張細(xì)胞下同一位置的微珠熒光照片。將所得的3張照片轉(zhuǎn)入Matlab,則可確定細(xì)胞基底下微珠的位移[28]。細(xì)胞的區(qū)域通過軟件描繪實驗開始時獲得相差圖片中細(xì)胞輪廓的方法來計算。應(yīng)用Butler等人[29]的方法計算細(xì)胞牽張區(qū)域內(nèi)凝膠基底的形變和位移?;贔ourier-Transform,基底的位移量可轉(zhuǎn)算為由于細(xì)胞骨架鋪展、收縮而施加在基底上的牽張力(PJ表示)。
MCF-7細(xì)胞以5×104密度種植于24孔板中,孵育48 h。PBS洗滌2次,加入標(biāo)準(zhǔn)0.25%胰酶/EDTA溶液(CellgroTM)。在Nikon Eclipse TE300熒光顯微鏡熒光光路下用100X觀測轉(zhuǎn)染細(xì)胞,計數(shù)細(xì)胞脫離基底的速度和比率。
通過Western blot,我們首先檢測了MCF-7細(xì)胞內(nèi)源l-CaD的表達(dá),以正常上皮細(xì)胞HMEC作為對照,我們發(fā)現(xiàn)內(nèi)源性l-CaD在MCF-7細(xì)胞中表達(dá)量很低,并且顯著低于HMEC細(xì)胞(圖1A),因此我們在MCF-7細(xì)胞中通過基因轉(zhuǎn)染高表達(dá)的外源野生型l-CaD及其磷酸化突變株,將不會受到內(nèi)源蛋白的影響;進(jìn)一步,我們在MCF-7細(xì)胞以質(zhì)粒轉(zhuǎn)染的方式高表達(dá)外源野生型l-CaD基因(EGFP-wtCaD)、不可磷酸化CaD突變株(EGFPA1234CaD)、磷酸化CaD突變株(EGFP-D1234CaD)以及空質(zhì)粒載體(EGFP),通過Western blot,我們確定外源l-CaD在MCF-7細(xì)胞中獲得了顯著高表達(dá),因此可以認(rèn)為外源高表達(dá)l-CaD在胞內(nèi)占主導(dǎo)作用(圖1B)。
圖1Western blot檢測細(xì)胞中內(nèi)、外源l-CaD及其磷酸化突變株的表達(dá)通過親和純化的多克隆抗體anti-CaD和anti-β-actin單克隆抗體對細(xì)胞提取物進(jìn)行免疫標(biāo)記。(A)MCF-7內(nèi)源l-CaD (Endogenous l-CaD)表達(dá)量很少,顯著低于對照組人源上皮細(xì)胞HMEC。(B)外源蛋白連接有EGFP,所以外源l-CaD(Exogenous l-CaD)在SDS-PAGE上的位置明顯高于l-CaD標(biāo)準(zhǔn)蛋白(l-CaD STD)。上排從左至右分別為高表達(dá)的外源不可磷酸化CaD突變株(A1234CaD)、磷酸化CaD突變株(D1234CaD)、空質(zhì)粒載體(EGFP-Control)以及野生型l-CaD(wtCaD),下排分別為同一細(xì)胞提取物樣本的內(nèi)源β-actin,可以看出外源l-CaD在MCF-7胞內(nèi)獲得了顯著高表達(dá),EGFP空質(zhì)粒載體沒有連接外源l-CaD,因此對照組細(xì)胞內(nèi)僅能檢測到內(nèi)源l-CaD而沒有外源l-CaD表達(dá)Fig.1Western blot analysis of EGFP-tagged CaD variants and endogenous CaD expressed in MCF-7 human breast cancer cellsThe total extracts from cells transfected with various constructs wereimmunoblottedwithpolyclonalanti-CaDandmonoclonalanti-β-actinantibodies.(A)The expression level of endogenous CaD(Endo-CaD)in MCF-7 cells was much lower than in Human mammary epithelial cells(HMEC).(B)The upper roll was exogenous CaD(Exo-CaD)variants.The samples were,respectively,cells transfected with A1234,D1234,EGFP control,and wtCaD.Because the EGFP tagged with the exogenous CaD,the exogenous CaD was higher than the standard CaD marker.The lower roll was the β-actin which was from the same cell sample.The expression of exogenous CaD was much higher than the endogenous CaD,the endogenous CaD would have little competing effect on the exogenous CaD
圖2 高表達(dá)外源l-CaD及其磷酸化突變株的MDA MB-231細(xì)胞在激光共聚焦掃描下所得的細(xì)胞骨架熒光染色圖片所有的外源l-CaD均為EGFP標(biāo)記,顯示為綠色熒光(左列);F-actin通過rhodamine phalloidin染色顯示為紅色熒光(中間列);右列為合并照片。細(xì)胞均通過轉(zhuǎn)染高表達(dá)EGFP空質(zhì)粒(對照組,EGFP)、EGFP-A1234(A1234)、EGFP-D1234(D1234)和EGFP-wtCaD(Wt)。A1234轉(zhuǎn)染細(xì)胞顯示了最為明顯、牢固的細(xì)胞骨架結(jié)構(gòu);wtCaD轉(zhuǎn)染細(xì)胞的細(xì)胞骨架也比D1234轉(zhuǎn)染細(xì)胞和對照組細(xì)胞(EGFP)的更為明顯。D1234轉(zhuǎn)染細(xì)胞與對照組細(xì)胞表現(xiàn)出相似的細(xì)胞骨架。標(biāo)尺:100 μmFig.2Confocal images of transfected MCF-7human breast cancer cellsAll CaD constructs were EGFP-tagged(left panels);actin was stained with red(middle panels);Merged images are shown on the right panels.Cells were transfected with EGFP(control),EGFP-wtCaD(wild-type CaD)and CaD mutant,including EGFP-A1234 and EGFP-D1234.A1234 transfected cells had most robust cytoskeleton structure,the wtCaD transfected cells also had more robust structure than D1234 and control cells(EGFP).The D1234 transfected cells exhibited similar cytoskeleton structure to the control cells.Scale bar=100 μm
l-CaD作為一種肌動蛋白結(jié)合蛋白,它不僅可以穩(wěn)定肌動蛋白絲,并且能通過抑制肌動球蛋白的結(jié)合,在調(diào)節(jié)肌動球蛋白聚合和重排中起分子剎車的作用。通過磷酸化和脫磷酸化,CaD可以脫離或結(jié)合到肌動蛋白纖維上,促進(jìn)細(xì)胞骨架的形成或重排。為了闡明l-CaD及其磷酸化在MCF-7細(xì)胞骨架中的作用,我們在MCF-7細(xì)胞內(nèi)高表達(dá)了外源野生型wtCaD及其磷酸化突變株A1234、D1234,以EGFP空質(zhì)粒轉(zhuǎn)染細(xì)胞作為對照,通過rhodaminephalloidin熒光染色以及激光共聚焦掃描(圖2),我們發(fā)現(xiàn),盡管MCF-7細(xì)胞內(nèi)沒有明顯的應(yīng)力纖維結(jié)構(gòu),但絕大部分A1234質(zhì)粒轉(zhuǎn)染的細(xì)胞邊緣位置顯示了最為明顯的細(xì)胞骨架結(jié)構(gòu),并且外源性l-CaD與細(xì)胞骨架有著明顯重疊(圖2-A1234);wtCaD細(xì)胞也顯示出強(qiáng)于對照組細(xì)胞的細(xì)胞骨架,外源性l-CaD與細(xì)胞骨架的重疊也非常明顯(圖2-wt);與之相對的是,由于D1234-CaD模擬磷酸化l-CaD,與細(xì)胞骨架的結(jié)合能力很弱,因此轉(zhuǎn)染了D1234的細(xì)胞,表現(xiàn)出顯著弱于A1234及wtCaD轉(zhuǎn)染細(xì)胞的細(xì)胞骨架(圖2-D1234),其細(xì)胞骨架結(jié)構(gòu)與對照組細(xì)胞(圖2-EGFP)類似,但外源性D1234與細(xì)胞骨架也有一定程度的重疊。
圖3 高表達(dá)外源l-CaD及其磷酸化突變株的MCF-7細(xì)胞相對遷移能力(n=9)(A)熒光顯微鏡下計數(shù)的遷移細(xì)胞,每一個熒光亮點(diǎn)代表透膜的轉(zhuǎn)染細(xì)胞。熒光顯微照片顯示A1234轉(zhuǎn)染細(xì)胞透膜數(shù)最少,Wt細(xì)胞透膜細(xì)胞也明顯少于EGFP對照組細(xì)胞,D1234細(xì)胞透膜數(shù)表現(xiàn)出與對照組細(xì)胞類似。(B)細(xì)胞計數(shù)顯示A1234轉(zhuǎn)染細(xì)胞的遷移能力受到了最大程度的抑制,其相對遷移率約為對照組EGFP轉(zhuǎn)染細(xì)胞的25%;wtCaD轉(zhuǎn)染細(xì)胞的遷移能力也受到抑制,其相對遷移率約為對照組細(xì)胞的60%;D1234細(xì)胞遷移的影響不顯著,相對遷移率與對照組細(xì)胞類似Fig.3Results of transwell migration assays of cells that were transfected with EGFP(control), EGFP-wtCaD,EGFP-A1234 and EGFP-D1234 to perform migration assay(A)The images of migrated cells under fluorescence microscope,The light spots represent the transfected cells that migrated through the membrane.(B)The migration ratio for the A1234 transfected cells was about 25%of the control cells(EGFP),and the ratio of wtCaD was about 60%of the control cells.D1234 mutant didn't have significant inhibitory effect on the mobility of MCF-7 cells
為了檢測l-CaD及其磷酸化對MCF-7細(xì)胞遷移能力的影響,基于趨化實驗的原理,我們使用了8 μm孔徑的transwell進(jìn)行細(xì)胞遷移實驗,應(yīng)用遷移過膜的綠色熒光細(xì)胞數(shù)與總的綠色熒光細(xì)胞數(shù)之間的比率表征轉(zhuǎn)染細(xì)胞的相對遷移能力(圖3A)。實驗結(jié)果顯示,在所有的轉(zhuǎn)染細(xì)胞中,A1234轉(zhuǎn)染細(xì)胞的遷移能力受到了最大程度的抑制,其相對遷移率(0.19±0.03)只有對照組EGFP空質(zhì)粒轉(zhuǎn)染細(xì)胞(0.80±0.05)的25%(圖3B);轉(zhuǎn)染wtCaD同樣抑制了細(xì)胞的遷移能力,其相對遷移率(0.45±0.04)約為對照組細(xì)胞的60%;D1234細(xì)胞跟對照組細(xì)胞的遷移能力相近,無顯著差異(0.73±0.045)。很明顯,l-CaD及其磷酸化參與了細(xì)胞的遷移行為,完全阻斷l(xiāng)-CaD的磷酸化將顯著抑制細(xì)胞的遷移。
圖4 細(xì)胞基底牽張力測定及其原理示意圖(n=10)(A-1)在熒光顯微鏡下找到單個表達(dá)外源質(zhì)粒的綠色熒光細(xì)胞(左下方),在熒光顯微鏡和相差顯微鏡下記錄所得細(xì)胞圖片。(A-2)Matlab分析所得的凝膠基底中熒光微粒的位移模擬圖,以及細(xì)胞下方牽張力場的分布。箭頭方向與顏色分布分別代表微粒運(yùn)動的方向與強(qiáng)度。(A-3)經(jīng)過軟件分析得出的凝膠基底中熒光微粒位移的具體強(qiáng)度。標(biāo)尺:50 μm。(B)高表達(dá)外源l-CaD及其磷酸化突變株的MCF-7細(xì)胞的基底牽張力。A1234細(xì)胞展現(xiàn)出最強(qiáng)的基底牽張力;與對照組細(xì)胞相比,wtCaD細(xì)胞的牽張力也顯著增加,D1234突變株對細(xì)胞基底牽張力沒有顯著影響。標(biāo)尺:100 μmFig.4Fouriertransformtranctionmicroscopymeasurementsandtheimagesofa representative MDA MB231 cell gathered in the measurement process(A-1)The single green cellwasfirstidentifiedunderfluorescencechannel(lowerleft)andthenrecordedtheboth fluorescence and phase-contrast images.(A-2,A-3)The computed traction field basedonthe micropatterned beads.The magnitude and the direction of the vectors indicate the beads movement which was used to compute the contractile moment.(B)Results of traction force measurements of MCF-7 cells transfected with wtCaD and phosphomimetic mutants of CaD(n=10)
細(xì)胞對基底的牽張力是影響細(xì)胞遷移的關(guān)鍵因素之一。為了分解l-CaD及其磷酸化突變株對整個細(xì)胞遷移行為的影響,我們使用Fourier-Transform Traction Microscopy,在單細(xì)胞水平上,對l-CaD及其突變株對MCF-7細(xì)胞基底牽張力的影響進(jìn)行了檢測(圖4)。轉(zhuǎn)染了EGFP、wtCaD、A1234、D1234的細(xì)胞種植于均勻包埋了紅色熒光微珠的聚丙烯酰胺凝膠基底表面。通過相差及熒光顯微鏡,獲取胰酶消化前后的細(xì)胞及細(xì)胞下熒光微珠的照片(圖4A-1),通過計算機(jī)分析,獲得細(xì)胞下熒光微珠的位移大小和方向(圖4A-2,3),通過Fourier-Transform將熒光微珠的位移轉(zhuǎn)換為細(xì)胞對基底的平均牽張力。結(jié)果顯示,遷移受到最大抑制(參照圖3)的A1234轉(zhuǎn)染細(xì)胞表現(xiàn)出了最大的牽張力〔(6.7±0.7)pJ〕(圖4B),約為對照組細(xì)胞牽張力〔(2.3±0.6)pJ〕的3倍;與對照組細(xì)胞相比,wtCaD轉(zhuǎn)染細(xì)胞的牽張力(4.5±0.9)也顯著升高,約為對照組細(xì)胞的2倍;相對而言,D1234轉(zhuǎn)染細(xì)胞的牽張力(2.7±0.87)與對照組細(xì)胞相近,無顯著影響。數(shù)據(jù)顯示l-CaD及其磷酸化突變株對細(xì)胞基底牽張力有顯著影響,并與細(xì)胞骨架的結(jié)構(gòu)一致。
細(xì)胞遷移過程中另一個關(guān)鍵步驟就是細(xì)胞從基底脫附。在胰酶作用下,細(xì)胞內(nèi)部肌動蛋白束從粘附斑位置迅速脫離、解聚,從而導(dǎo)致細(xì)胞變圓并從基底上脫附。根據(jù)這樣的原理,我們通過胰酶刺激,對EGFP、wtCaD、A1234以及D1234轉(zhuǎn)染后的細(xì)胞從基底上脫附下來的動力學(xué)過程進(jìn)行了比較。結(jié)果顯示(圖5),與對照組細(xì)胞相比,wtCaD轉(zhuǎn)染細(xì)胞表現(xiàn)出對胰酶處理的延遲型應(yīng)答,A1234轉(zhuǎn)染的細(xì)胞其變圓速率更加緩慢;D1234對細(xì)胞在胰酶刺激下的應(yīng)答影響不大,D1234轉(zhuǎn)染細(xì)胞表現(xiàn)出與對照組細(xì)胞相近的變圓速率。
圖5 胰酶刺激下細(xì)胞基底脫附行為檢測(n=6)在胰酶刺激下,細(xì)胞會由于細(xì)胞骨架的收縮、解聚而變圓并從基底脫附。我們以EGFP空質(zhì)粒轉(zhuǎn)染細(xì)胞為對照組,檢測了外源l-CaD高表達(dá)對細(xì)胞在胰酶刺激下脫附能力的影響。細(xì)胞種植于24孔板內(nèi),滴加標(biāo)準(zhǔn)0.25%胰酶/EDTA溶液,在相差顯微鏡和熒光顯微鏡下實時觀測細(xì)胞收縮、變圓、脫附的過程。記錄0~6 min各組細(xì)胞中圓形細(xì)胞的比例。A1234轉(zhuǎn)染細(xì)胞表現(xiàn)出了對胰酶最顯著的耐受能力;高表達(dá)wtCaD的細(xì)胞對胰酶的響應(yīng)也有所延遲,D1234細(xì)胞跟對照組細(xì)胞對胰酶刺激的響應(yīng)類似Fig.5Detachment of transfected MCF-7 cells upon trypsinizationCells from each plate were trypsinized and monitored under the phase-contrast and fluorescence microscope for time-dependent retraction,rounding and detachment.Percentage of round cells within the designated period was plotted for each type of cells.The percentage of rounded cells in each plate was reported.A1234 cells show the greatest tolerance against enzyme;the response of wtCaD high-expressing cells to enzyme was delayed,D1234 cells display the same response to enzyme as the control(n=6)
眾多研究已經(jīng)證明,l-CaD可以通過與肌動蛋白的相互作用參與到細(xì)胞正常的生理活動中[6~8]。在體外條件下,l-CaD能夠通過與肌動蛋白結(jié)合來穩(wěn)定肌動蛋白纖維[16,17],從而抑制凝溶膠蛋白對肌動蛋白纖維的剪切作用[13~15]。在細(xì)胞內(nèi),一方面人們發(fā)現(xiàn)通過顯微注射l-CaD抗體能導(dǎo)致細(xì)胞骨架解聚,從而降低胞內(nèi)微粒轉(zhuǎn)運(yùn)速度[21,22];另一方面,在磷酸化作用下,l-CaD將從肌動蛋白纖維上解離下來,從而促進(jìn)肌動蛋白纖維的解聚和重排[10~15],并且磷酸化與非磷酸化的l-CaD有不同的分布區(qū)域,非磷酸化l-CaD富集在穩(wěn)定的細(xì)胞骨架上,而磷酸化l-CaD主要集中在細(xì)胞骨架調(diào)節(jié)頻繁的細(xì)胞運(yùn)動前沿及足體內(nèi)[23~25]。這些研究結(jié)果無一不預(yù)示著,l-CaD作為一種重要的肌動蛋白結(jié)合蛋白,在細(xì)胞正常生理活動中起著關(guān)鍵作用。但是,l-CaD在體外對肌動蛋白纖維的穩(wěn)定作用以及通過顯微注射l-CaD抗體的方式所得的結(jié)果,并不能直觀地證明細(xì)胞內(nèi)l-CaD及其磷酸化對細(xì)胞骨架重排和解聚的調(diào)節(jié)作用;在體觀測l-CaD胞內(nèi)分布也不能直觀顯示l-CaD及其磷酸化如何調(diào)控細(xì)胞的運(yùn)動能力,因此在我們的研究中,將l-CaD及其磷酸化突變株作為“分子探針”,通過在胞內(nèi)高表達(dá)外源l-CaD,干擾胞內(nèi)細(xì)胞骨架調(diào)節(jié)進(jìn)程和l-CaD磷酸化程度,證明了l-CaD是通過調(diào)節(jié)細(xì)胞骨架的解聚和重排而影響細(xì)胞的運(yùn)動能力。
為了證實l-CaD及其磷酸化對細(xì)胞骨架的調(diào)節(jié)作用,我們制備了野生型l-CaD(wtCaD)及其磷酸化突變株。將野生型l-CaD的ERK和PAK磷酸化位點(diǎn)定點(diǎn)突變?yōu)楸彼?,獲得了不能通過磷酸化作用從肌動蛋白上脫離的l-CaD不可磷酸化突變株A1234-CaD;將野生型l-CaD的ERK和PAK磷酸化位點(diǎn)定點(diǎn)突變?yōu)樘於彼?,獲得了模擬l-CaD磷酸化狀態(tài)的D1234-CaD。在胞內(nèi)高表達(dá)外源蛋白應(yīng)考慮到內(nèi)源蛋白的拮抗作用,我們之前的研究顯示,l-CaD在人源性乳腺癌細(xì)胞MCF-7內(nèi)表達(dá)量非常低,因此MCF-7是一個進(jìn)行高表達(dá)外源l-CaD研究的優(yōu)良細(xì)胞模型。體外實驗證明了l-CaD通過與肌動蛋白結(jié)合從而穩(wěn)定肌動蛋白纖維的作用,而通過磷酸化,l-CaD將從肌動蛋白上脫附,從而促進(jìn)肌動蛋白纖維的解聚[16,17],我們通過在MCF-7細(xì)胞內(nèi)高表達(dá)外源l-CaD來調(diào)控細(xì)胞骨架的形成,獲得了與體外實驗一致的結(jié)果。我們發(fā)現(xiàn),與對照組細(xì)胞相比,高表達(dá)外源野生型l-CaD(wtCaD)的細(xì)胞擁有更為牢固的細(xì)胞骨架結(jié)構(gòu),外源l-CaD主要富集在細(xì)胞骨架上,與細(xì)胞骨架重合明顯;不可磷酸化l-CaD(A1234)轉(zhuǎn)染的細(xì)胞則顯示了甚至強(qiáng)于wtCaD細(xì)胞的骨架系統(tǒng),由于不能通過磷酸化從細(xì)胞骨架上脫離,絕大部分A1234-CaD集中在細(xì)胞骨架上,顯著增強(qiáng)了骨架強(qiáng)度;D1234-CaD模擬磷酸化l-CaD,與細(xì)胞骨架的結(jié)合作用很弱,不能通過穩(wěn)定細(xì)胞骨架增強(qiáng)骨架的強(qiáng)度,因此D1234高表達(dá)細(xì)胞顯示了與對照組細(xì)胞相似的細(xì)胞骨架系統(tǒng)。這個結(jié)果說明,在胞內(nèi)條件下,l-CaD對細(xì)胞骨架形成有顯著的調(diào)節(jié)作用,非磷酸化l-CaD能夠明顯促進(jìn)細(xì)胞骨架形成并維持其穩(wěn)定性,而磷酸化l-CaD對細(xì)胞骨架的結(jié)合能力很弱,對細(xì)胞骨架的形成、穩(wěn)定性沒有顯著影響。
細(xì)胞遷移由多個步驟組成[30],包括肌動蛋白骨架重構(gòu)、粘附、基底脫附、細(xì)胞牽張,其中,肌動蛋白骨架的解聚跟重排是細(xì)胞遷移的首要步驟。為了進(jìn)一步探明l-CaD是否能通過對細(xì)胞骨架的調(diào)節(jié)作用影響細(xì)胞的運(yùn)動能力,一方面通過細(xì)胞遷移小室,我們考察了l-CaD對細(xì)胞遷移能力的整體影響;另一方面,通過基底牽張力分析及細(xì)胞脫粘附陣列,我們考察了l-CaD對細(xì)胞遷移的子過程——細(xì)胞收縮及脫附的影響。數(shù)據(jù)顯示,細(xì)胞骨架結(jié)構(gòu)最為牢固的A1234-CaD高表達(dá)細(xì)胞整體遷移能力受到了最顯著的抑制,細(xì)胞基底牽張力顯著增強(qiáng),對胰酶刺激的響應(yīng)明顯延遲。這個結(jié)果進(jìn)一步說明了細(xì)胞骨架的解聚和重排對細(xì)胞運(yùn)動能力的促進(jìn)作用。當(dāng)l-CaD的磷酸化被丙氨酸突變阻斷時,A1234-CaD突變株將無法通過磷酸化從細(xì)胞骨架上脫離,細(xì)胞骨架重排受阻,從而導(dǎo)致細(xì)胞遷移受到顯著抑制,而非磷酸化l-CaD在細(xì)胞骨架上的富集在抑制細(xì)胞整體遷移能力的同時,通過增強(qiáng)細(xì)胞骨架的強(qiáng)度,賦予了細(xì)胞更強(qiáng)的基底牽張力,細(xì)胞骨架對胰酶的耐受性增強(qiáng);與A1234-CaD的作用類似,野生型l-CaD高表達(dá)的細(xì)胞遷移能力受到顯著抑制,而基底牽張力和胰酶耐受性增強(qiáng)。wtCaD在胞內(nèi)高表達(dá),可能超出了胞內(nèi)磷酸化激酶的承受范圍,激光共聚焦圖片顯示富余的過量l-CaD得不到高效的磷酸化,以非磷酸化形式大量富集在細(xì)胞骨架上,導(dǎo)致細(xì)胞骨架增強(qiáng)而解聚受阻,細(xì)胞遷移受到明顯抑制的同時賦予了細(xì)胞更強(qiáng)的基底牽張力和胰酶耐受性;D1234-CaD跟肌動蛋白的結(jié)合能力很弱,不能通過穩(wěn)定肌動蛋白纖維而增加細(xì)胞骨架的強(qiáng)度,因此,D1234-CaD高表達(dá)的細(xì)胞表現(xiàn)出了與EGFP空質(zhì)粒轉(zhuǎn)染的對照組細(xì)胞相似的遷移能力,基底牽張力跟胰酶耐受性也不能通過細(xì)胞骨架強(qiáng)度增加而獲得相應(yīng)的提高。
細(xì)胞骨架通過肌動蛋白有序的解聚和重排,在許多細(xì)胞生理活動的控制中起關(guān)鍵作用,包括:細(xì)胞分裂、遷移、內(nèi)吞和外排作用、凋亡和炎癥、囊泡運(yùn)輸和基因表達(dá)。我們的研究證明,l-CaD作為一種重要的肌動蛋白結(jié)合蛋白,通過與肌動蛋白的相互作用參與調(diào)控了細(xì)胞的運(yùn)動能力。l-CaD在胞內(nèi)以非磷酸化的形式結(jié)合到肌動蛋白上,促進(jìn)了細(xì)胞骨架的形成,并能維持其穩(wěn)定性,如果阻斷l(xiāng)-CaD磷酸化,l-CaD很難從細(xì)胞骨架上脫離,將導(dǎo)致細(xì)胞骨架有序的解聚和重排受阻,細(xì)胞運(yùn)動能力將受到顯著的抑制??偟膩碚f,l-CaD通過磷酸化與去磷酸化起到了一個“蛋白開關(guān)”的作用,通過控制細(xì)胞骨架的解聚和重排來調(diào)節(jié)細(xì)胞的運(yùn)動能力,這也為使用l-CaD作為靶底物治療腫瘤,特別是高轉(zhuǎn)移性腫瘤提供了新的理論依據(jù)。
1.Lauffenburger DA,Horwitz AF.Cell migration:a physically integrated molecular process.Cell,1996,84(3):359~369
2.Schneider IC,Haugh JM.Mechanisms of gradient sensing and chemotaxis:conserved pathways,diverse regulation. Cell Cycle,2006,5(11):1130~1134
3.Dormann D,Weijer CJ.Chemotactic cell movement during development.Curr Opin Genet Dev,2003,13(4):358~364
4.Thomas D Pollard,John A Cooper.Actin,a central player incellshapeandmovement.Science,2009,27: 1208~1212
5.Small JV,Rottner K,Kaverina I,Anderson KI.Assembling an actin cytoskeleton for cell attachment and movement. Biochimica et Biophysica Acta,1998,1404:271~281
6.Matsumura F,Yamashiro S.Caldesmon.Curr Opin Cell Biol,1993,5(1):70~76
7.Marston SB,Huber PAJ(Eds).Caldesmon.Biochemistry of smooth muscle contraction.San Diego,CA:Academic Press,Inc.1996
8.Wang CL.Caldesmon and smooth-muscle regulation.Cell Biochem Biophys,2001,35(3):275~288
9.Bretscher A.Thin filament regulatory proteins of smoothand non-muscle cells.Nature,1986,321(6072):726~727
10.Warren KS,Lin JL,Wamboldt DD,Lin JJ.Overexpression of human fibroblast caldesmon fragment containingactin-, Ca++/calmodulin-,and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.J Cell Biol, 1994,125(2):359~368
11.Pittenger MF,Kistler A,Helfman DM.Alternatively spliced exonsofthebetatropomyosingeneexhibitdifferent affinities for F-actin and effects with nonmuscle caldesmon.J Cell Sci,1995,108(Pt 10):3253~3265
12.Humphrey MB,Herrera-Sosa H,Gonzalez G,Lee R,Bryan J.Cloning of cDNAs encoding human caldesmons.Gene, 1992,112(2):197~204
13.Dabrowska R,Hinssen H,Galazkiewicz B,Nowak E. Modulationofgelsolin-inducedactin-filamentseveringby caldesmonandtropomyosinandtheeffectofthese proteins on the actin activation of myosin Mg2+–ATPase activity.Biochem J,1996,315(Pt 3):753~759
14.Ishikawa R,Yamashiro S,Matsumura F.Annealing of gelsolin-severedactinfragmentsbytropomyosininthe presence of Ca2+.Potentiation of the annealing process by caldesmon.J Biol Chem,1989,264:16764~16770
15.Takiguchi K,Matsumura F.Role of the basic C-terminal half of caldesmon in its regulation of F-actin:comparison betweencaldesmonandcalponin.JBiochem(Tokyo), 2005,138:805~813
16.Novy RE,Sellers JR,Liu LF,Lin JJC.In vitrofunctional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants.Cell Motil Cytoskeleton,1993,26:248~261
17.Yamashiro-Matsumura S,Matsumura F.Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin.J Cell Biol,1988,106:1973~1983
18.Bretscher A,Lynch W.Identification and localization of immunoreactiveformsofcaldesmoninsmoothand nonmuscle cells:a comparison with the distributions of tropomyosin and alpha-actinin.J Cell Biol,1985,100: 1656~1663
19.Nakamura N,Tanaka J,Sobue K.Rous sarcoma virustransformedcellsdeveloppeculiaradhesivestructures along the cell periphery.J Cell Sci,1993,106(Pt 4): 1057~1069
20.TanakaJ,WatanabeT,NakamuraN,SobueK. Morphologicalandbiochemicalanalysesofcontractile proteins(actin,myosin,caldesmon and tropomyosin)in normal and transformed cells.J Cell Sci,1993,104(Pt 2): 595~606
21.Hegmann TE,Schulte DL,Lin JLC,Lin JJC.Inhibition of intracellulargranulemovementbymicroinjectionof monoclonalantibodiesagainstcaldesmon.CellMotil Cytoskeleton,1991,20:109~120
22.Lamb NJ,Fernandez A,Mezgueldi M,Labbe JP,Kassab R,Fattoum A.Disruption of the actin cytoskeleton in living nonmuscle cells by microinjection of antibodies to domain-3 of caldesmon.Eur J Cell Biol,1996,69:36~44
23.KordowskaJ,HetrickT,AdamLP,WangCL. Phosphorylated l-caldesmon is involved in disassembly of actin stress fibers and postmitotic spreading.Exp Cell Res, 2006,312(2):95~110
24.ChenWT.Proteolyticactivityofspecializedsurface protrusions formed at rosette contact sites of transformed cells.J Exp Zool,1989,251:167~185
25.Ochoa GC,Slepnev VI,Neff L,Ringstad N,Takei K, Daniell L,Kim W,Cao H,McNiven M,Baron R,De Camilli P.A functional link between dynamin and the actin cytoskeletonatpodosomes.JCellBiol,2000,150: 377~389
26.Dembo M,Wang YL.Stresses at the cell-to-substrate interface during locomotion of fibroblasts.Biophys J,1999, 76:2307~2316
27.Wang N,Tolic′-N覬rrelykke IM,Chen J,Mijailovich SM, Butler JP,Fredberg JJ,Stamenovic′D.Cell prestress.I. Stiffness and prestress are closely associated in adherent contractile cells.Am J Physiol Cell Physiol,2002,282: C606~616
28.Tolic′-N覬rrelykke IM,Butler JP,Chen J,Wang N.Spatial and temporal traction response in human airway smooth muscle cells.Am J Physiol Cell Physiol,2002,283(4): C1254~1266
29.Butler JP,Toli-N覬rrelykke IM,Fabry B,Fredberg JJ. Traction fields,moments,and strain energy that cells exert on their surroundings.Am J Physiol Cell Physiol,2002, 282(3):C595~605
30.Condeelis J.The biochemistry of animal cell crawling. Motion analysis of living cell.New York:Wiley-Liss,1998. 85~100
Abstract:Light chain(l-CaD)is an important actin binding protein,presents almost all types of vertebrate cells.In vitrostudies showed that l-CaD can bind to G-actin to accelerate the polymerization of the G-actin into F-actin and stabilize the formed F-actin.Upon phosphorylation,the l-CaD will disassociate from the F-acin to facilitate its disassembly into G-actin.In order to explore whether the l-CaD mediate the cell mobility by its effect on cytoskeleton,Human breast cancer cell line MCF-7 was chosen as experimental model in which the expression level of l-CaD is very low.By transfection,wild type l-CaD and its phosphorylationmutant(A1234-CaDunphosphorylatableCaD;D1234-CaDphosphorylatedCaD)were force-expressed in MCF-7 cells.First,the effect of the l-CaD on cytoskeleton structure was tested by confocal images;and then the transwell assay was performed to address the influence of l-CaD on cell migration activities;Furthermore,the cell contractility and cell detachment which are sub-steps of cell migration were addressed by Fourier-Transform traction microscopy on single cell level and trypsin stimulation respectively.The results showed that l-CaD had significant effect on cytoskeleton structure of MCF-7 cells. The unphosphorylated l-CaD concentrated on cytoskeleton,increased its intensity which generated more cell contractility and inhibited the response of the cells to the trypsin stimulation.Because of the stabilization effect on cytoskeleton by unphosphorylated l-CaD,the rearrangement of the cytoskeleton was inhibited which resulted in the decrease of the cell migration activities;The phosphorylated l-CaD has very low affinity to the cytoskeleton,so its effect on cell mobility was not significant compared to the controlled cells.Taken together,upon phosphorylation,the l-CaD acted like a"protein switch",it will mediate the cell mobility by its effect on cytoskeleton disassembly and rearrangement.
Key Words:l-Caldesmon;Cytoskeleton;Cell mobility;Cell contractility
Caldesmon Regulate The Mobility of Tumor Cells by Mediating The Actin Cytoskeleton Stability
JIANG Qifeng1,2,CAI Shaoxi1,YAN Xiaoqing1
1.Key Laboratory of Biorheological Science and Technology(Chongqing University), Ministry of Education,Bioengineering college,Chongqing University,Chongqing 400044,China;
2.Boston Biomedical Research Institute,Boston 02472,USA
Q245,Q319
2009-10-16;接受日期:2010-01-05
國家自然科學(xué)基金項目(10872224)和“111”項目
蔡紹皙,電話:(023)65112097,E-mail:sxcai@cqu.edu.cn
This work was supported by grants from The National Natural Science Foundation of China(10872224)and"111"project
Received:Oct 16,2009Accepted:Jan 5,2010
Corresponding author:Cai Shaoxi,Tel:+86(23)65112097,E-mail:sxcai@cqu.edu.cn