Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2010 Nov 11;5(11):e13921. doi: 10.1371/journal.pone.0013921.
α-Actinins cross-link actin filaments, with this cross-linking activity regulating the formation of focal adhesions, intracellular tension, and cell migration. Most non-muscle cells such as fibroblasts express two isoforms, α-actinin-1 (ACTN1) and α-actinin-4 (ACTN4). The high homology between these two isoforms would suggest redundancy of their function, but recent studies have suggested different regulatory roles. Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin.
METHODOLOGY/PRINCIPAL FINDINGS: Using molecular, biochemical and cellular techniques, we probed the cellular functions of ACTN4 in fibroblasts. Knockdown of ACTN4 expression in murine lung fibroblasts significantly impaired cell migration, spreading, adhesion, and proliferation. Surprisingly, knockdown of ACTN4 enhanced cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. These results, except the increased contractility, are consistent with a putative role of ACTN4 in cytokinesis. For the transcellular tension, knockdown of ACTN4 significantly increased the expression of myosin light chain 2, a element of the contractility machinery. Re-expression of wild type human ACTN4 in ACTN4 knockdown murine lung fibroblasts reverted cell spreading, cellular and nuclear cross-sectional area, and contractility back towards baseline, demonstrating that the defect was due to absence of ACTN4.
These results suggest that ACTN4 is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion.
α-辅肌动蛋白将肌动蛋白丝交联，这种交联活性调节着黏着斑的形成、细胞内张力和细胞迁移。大多数非肌肉细胞，如成纤维细胞，表达两种同工型，α-辅肌动蛋白-1（ACTN1）和α-辅肌动蛋白-4（ACTN4）。这两种同工型之间的高度同源性表明它们的功能具有冗余性，但最近的研究表明它们具有不同的调节作用。有趣的是，ACTN4 在生长因子刺激下发生磷酸化，从而使其与肌动蛋白的相互作用减弱。
方法/主要发现：我们使用分子、生化和细胞技术，探究了 ACTN4 在成纤维细胞中的细胞功能。鼠肺成纤维细胞中 ACTN4 表达的敲低显著损害了细胞迁移、铺展、黏附和增殖。令人惊讶的是，ACTN4 的敲低增强了细胞的紧凑性和收缩力，并增加了细胞和核的横截面积。这些结果（除了收缩力增加外）与 ACTN4 可能在细胞分裂中的作用一致。对于跨细胞张力，ACTN4 的敲低显著增加了肌球蛋白轻链 2 的表达，肌球蛋白轻链 2 是收缩机制的一个元素。在 ACTN4 敲低的鼠肺成纤维细胞中重新表达野生型人 ACTN4 使细胞铺展、细胞和核的横截面积以及收缩力恢复到基线，表明该缺陷是由于 ACTN4 的缺失。
这些结果表明，ACTN4 通过维持跨细胞收缩性和细胞-基底黏附之间的平衡，对于维持鼠肺成纤维细胞的正常铺展、运动、细胞和核的横截面积以及收缩性是必不可少的。
