Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
BMC Biol. 2012 Aug 10;10:72. doi: 10.1186/1741-7007-10-72.
Fascin-1 is an actin crosslinking protein that is important for the assembly of cell protrusions in neurons, skeletal and smooth muscle, fibroblasts, and dendritic cells. Although absent from most normal adult epithelia, fascin-1 is upregulated in many human carcinomas, and is associated with poor prognosis because of its promotion of carcinoma cell migration, invasion, and metastasis. Rac and Cdc42 small guanine triphosphatases have been identified as upstream regulators of the association of fascin-1 with actin, but the possible role of Rho has remained obscure. Additionally, experiments have been hampered by the inability to measure the fascin-1/actin interaction directly in intact cells. We investigated the hypothesis that fascin-1 is a functional target of Rho in normal and carcinoma cells, using experimental approaches that included a novel fluorescence resonance energy transfer (FRET)/fluorescence lifetime imaging (FLIM) method to measure the interaction of fascin-1 with actin.
Rho activity modulates the interaction of fascin-1 with actin, as detected by a novel FRET method, in skeletal myoblasts and human colon carcinoma cells. Mechanistically, Rho regulation depends on Rho kinase activity, is independent of the status of myosin II activity, and is not mediated by promotion of the fascin/PKC complex. The p-Lin-11/Isl-1/Mec-3 kinases (LIMK), LIMK1 and LIMK2, act downstream of Rho kinases as novel binding partners of fascin-1, and this complex regulates the stability of filopodia.
We have identified a novel activity of Rho in promoting a complex between fascin-1 and LIMK1/2 that modulates the interaction of fascin-1 with actin. These data provide new mechanistic insight into the intracellular coordination of contractile and protrusive actin-based structures. During the course of the study, we developed a novel FRET method for analysis of the fascin-1/actin interaction, with potential general applicability for analyzing the activities of actin-binding proteins in intact cells.
Fascin-1 是一种肌动蛋白交联蛋白，对于神经元、骨骼和平滑肌、成纤维细胞和树突状细胞中细胞突起的组装非常重要。尽管在大多数正常成年上皮细胞中不存在，但 fascin-1 在许多人类癌中上调，并与预后不良相关，因为它促进癌细胞迁移、侵袭和转移。Rac 和 Cdc42 小分子 GTP 酶已被确定为 fascin-1 与肌动蛋白结合的上游调节剂，但 Rho 的可能作用仍然不清楚。此外，由于无法直接在完整细胞中测量 fascin-1/actin 相互作用，实验受到了阻碍。我们使用包括一种新的荧光共振能量转移（FRET）/荧光寿命成像（FLIM）方法来测量 fascin-1 与肌动蛋白相互作用的实验方法，研究了 fascin-1 是正常和癌细胞中 Rho 的功能靶标的假设。
Rho 活性通过一种新的 FRET 方法调节在骨骼肌成肌细胞和人结肠癌细胞中 fascin-1 与肌动蛋白的相互作用。从机制上讲，Rho 调节依赖于 Rho 激酶活性，独立于肌球蛋白 II 活性状态，并且不通过促进 fascin/PKC 复合物来介导。p-Lin-11/Isl-1/Mec-3 激酶（LIMK）、LIMK1 和 LIMK2 作为 fascin-1 的新型结合伴侣，作为 Rho 激酶的下游因子发挥作用，该复合物调节丝状伪足的稳定性。
我们已经确定了 Rho 的一种新活性，即促进 fascin-1 与 LIMK1/2 之间的复合物形成，从而调节 fascin-1 与肌动蛋白的相互作用。这些数据为肌动蛋白结合蛋白在完整细胞中的活性提供了新的机制见解。在研究过程中，我们开发了一种新的 FRET 方法来分析 fascin-1/actin 相互作用，该方法具有分析完整细胞中肌动蛋白结合蛋白活性的潜在普遍适用性。
