Department of Pathology and Cell Biology, Université de Montréal, Canada.
Histochem Cell Biol. 2011 Sep;136(3):245-66. doi: 10.1007/s00418-011-0840-0. Epub 2011 Jul 23.
Wild-type (WT) and myosin heavy chain IIB null [MHCIIB (-/-)] embryonic fibroblasts were used as an experimental model to assess the role of the isoform B of myosin II (MII) in the regulation of the cell shape and intrinsic polarity. Genetic ablation of MHCIIB causes a persistent albeit, unstable protrusive activity in embryonic fibroblasts (Lo et al. in Nonmuscle myosin IIB is involved in the guidance of fibroblast migration. Mol Biol Cell 15:982-989, 2004). Here, we show that MHCIIB-deficient fibroblasts are characterized by a sustained guanine nucleotide exchange factor (GEF)-dependent activation of the small GTPase Rac-1 that is responsible for the continual lamellipodium formation. Moreover, we observed a sustained PKC-ζ activation and an increased association of cortactin with the plasma membrane in the MHCIIB (-/-) cells that were also dependent on GEF-mediated Rac-1 activation. Rac-1 activation and its downstream effects were induced in WT fibroblasts by inhibiting MII ATPase and crosslinking activities, suggesting that an altered actin-MII interaction favours Rac-1 activation, regardless of the MII isoform implicated. In addition, we found MIIB isoform-specific effects that were independent of Rac-1 activation. MHCIIA interacts with cortactin whereas MHCIIB does not. By contrast, MHCIIB interacts with Lgl1, a member of the Scribble/Dlg/Lgl polarity complex, whereas MHCIIA does not. MHCIIB (-/-) fibroblasts exhibited deregulated endogenous levels of the Par polarity complex members, Par3 and Par6. Together, the data show that MHCIIB deficiency causes imbalances in signalling pathways that are responsible for cell polarity determination. The results suggest that these pathways are targets of MIIB in the regulation of the cell's shape and polarity.
野生型（WT）和肌球蛋白重链 IIB 缺失[MHCIIB（-/-）]胚胎成纤维细胞被用作实验模型，以评估肌球蛋白 II 同工型 B（MII）在调节细胞形状和固有极性中的作用。MHCIIB 的基因缺失导致胚胎成纤维细胞中持续但不稳定的突出活性（Lo 等人，非肌肉肌球蛋白 IIB 参与成纤维细胞迁移的指导。Mol Biol Cell 15：982-989，2004）。在这里，我们表明 MHCIIB 缺陷型成纤维细胞的特征是持续的鸟嘌呤核苷酸交换因子（GEF）依赖性小 GTPase Rac-1 的激活，该激活负责持续的片状伪足形成。此外，我们观察到 MHCIIB（-/-）细胞中 PKC-ζ的持续激活和与质膜的增加关联，这也依赖于 GEF 介导的 Rac-1 激活。Rac-1 的激活及其下游效应在 WT 成纤维细胞中通过抑制 MII ATP 酶和交联活性诱导，这表明改变的肌动蛋白-MII 相互作用有利于 Rac-1 的激活，而与涉及的 MII 同工型无关。此外，我们发现了独立于 Rac-1 激活的 MIIB 同工型特异性效应。MHCIIA 与 cortactin 相互作用，而 MHCIIB 则不相互作用。相比之下，MHCIIB 与 Scribble/Dlg/Lgl 极性复合物的成员 Lgl1 相互作用，而 MHCIIA 则不相互作用。MHCIIB（-/-）成纤维细胞表现出失调的 Par 极性复合物成员 Par3 和 Par6 的内源性水平。总之，这些数据表明，MHCIIB 的缺失导致负责细胞极性决定的信号通路失衡。结果表明，这些途径是 MIIB 在调节细胞形状和极性中的作用的靶点。
