ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion
ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion
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DOI：
10.1152/ajprenal.00210.2002
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发表时间：
2003-04-01
影响因子：
4.2
通讯作者：
Molitoris, BA
中科院分区：
文献类型：
--
作者：
Ashworth, SL;Southgate, EL;Molitoris, BA
缺血损伤诱导肌动蛋白细胞骨架的破坏和聚集，但影响这些变化的机制仍不清楚。为了确定肌动蛋白解聚因子（ADF）/cofilin参与缺血诱导的肌动蛋白细胞骨架破坏的作用，我们利用猪肾培养细胞，LLC-PKA 4.8，和腺病毒含有野生型（wt），组成型活性，和非活性爪蟾ADF/cofilin连接到绿色荧光蛋白[XAC（wt）-GFP]在ATP耗竭模型。LLC-PKA 4.8细胞中的高腺病毒感染性（70%）导致线性增加的XAC（wt）-GFP和磷酸化（p）XAC（wt）-GFP（失活）表达。ATP消耗快速诱导内源性pcofilin以及pXAC（wt）-GFP的去磷酸化，并因此激活，同时形成荧光XAC（wt）GFP/肌动蛋白聚集体和杆。表达GFP或组成型失活突变体XAC（S3 E）-GFP的LLC-PKA 4.8细胞的短期ATP耗竭没有发生显著的肌动蛋白细胞骨架改变，但表达组成型活性突变体的细胞表现出几乎瞬时的肌动蛋白破坏，伴随聚集体和杆的形成。正常和ATP耗尽的LLC-PKA 4.8细胞的共聚焦图像三维体积重建表明，除了XAC-GFP/肌动蛋白聚集体形成之外，25分钟的ATP耗尽诱导XAC（wt）GFP顶端和基底信号的快速增加。这些数据表明XAC（wt）-GFP参与缺血诱导的肌动蛋白细胞骨架改变，并决定这些ATP耗竭诱导的细胞改变的速率和程度。
Ischemic injury induces actin cytoskeleton disruption and aggregation, but mechanisms affecting these changes remain unclear. To determine the role of actin-depolymerizing factor (ADF)/cofilin participation in ischemic-induced actin cytoskeletal breakdown, we utilized porcine kidney cultured cells, LLC-PKA4.8, and adenovirus containing wild-type (wt), constitutively active, and inactive Xenopus ADF/cofilin linked to green fluorescence protein [XAC(wt)-GFP] in an ATP depletion model. High adenoviral infectivity (70%) in LLC-PKA4.8 cells resulted in linearly increasing XAC( wt)-GFP and phosphorylated (p) XAC( wt)-GFP (inactive) expression. ATP depletion rapidly induced dephosphorylation, and, therefore, activation, of endogenous pcofilin as well as pXAC(wt)-GFP in conjunction with the formation of fluorescent XAC( wt) GFP/actin aggregates and rods. No significant actin cytoskeletal alterations occurred with short-term ATP depletion of LLC-PKA4.8 cells expressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, but cells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal and ATP-depleted LLC-PKA4.8 cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC( wt) GFP apical and basal signal in addition to XAC-GFP/actin aggregate formation. These data demonstrate XAC(wt)-GFP participates in ischemia-induced actin cytoskeletal alterations and determines the rate and extent of these ATP depletion-induced cellular alterations.