Cofilin cooperates with fascin to disassemble filopodial actin filaments
Cofilin cooperates with fascin to disassemble filopodial actin filaments
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DOI：
10.1242/jcs.086934
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发表时间：
2011-10-01
影响因子：
4
通讯作者：
Faix, Jan
中科院分区：
文献类型：
--
作者：
Breitsprecher, Dennis;Koestler, Stefan A.;Faix, Jan
细胞使用大量的蛋白质来重塑肌动蛋白细胞骨架。根据所涉及的蛋白质，F-肌动蛋白被组织在专门的突起中，如板状伪足或丝状伪足，它们在细胞迁移和传感中发挥不同的功能。虽然在丝状伪足中负责定向细丝组装的因素已被广泛表征，但在这些结构中细丝拆卸的机制大多是未知的。我们研究了肌动蛋白解聚因子cofilin-1如何影响体外和活细胞中fascin交联肌动蛋白丝的动力学。通过全内反射荧光显微镜和荧光测定，我们发现，cofilin介导的切断增强fascin交联束相比，孤立的细丝，和fascin和cofilin的作用协同细丝切断。免疫标记实验首次证明，除了其已知的定位在板状伪足和膜皱褶，内源性cofilin也可以积累在丝状伪足的尖端和轴。荧光标记的蛋白质的活细胞成像显示，cofilin是专门针对丝状伪足停滞后的突出和在其缩回。随后的电子断层扫描建立丝状伪足肌动蛋白丝和/或束碎片精确相关的cofilin积累。这些结果确定了一个新的机制涉及fascin和cofilin的丝状伪足拆卸。
Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascin-crosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.