Kronenberg Nils M, Tilston-Lunel Andrew, Thompson Frances E, Chen Doris, Yu Wanjia, Dholakia Kishan, Gather Malte C, Gunn-Moore Frank J
Centre of Biophotonics and SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, United Kingdom.
Centre for Nanobiophotonics, Department of Chemistry, University of Cologne, Cologne, Germany.
Front Cell Neurosci. 2020 Sep 4;14:552213. doi: 10.3389/fncel.2020.552213. eCollection 2020.
Willin/FRMD6 is part of a family of proteins with a 4.1 ezrin-radixin-moesin (FERM) domain. It has been identified as an upstream activator of the Hippo pathway and, when aberrant in its expression, is associated with human diseases and disorders. Even though Willin/FRMD6 was originally discovered in the rat sciatic nerve, most studies have focused on its functional roles in cells outside of the nervous system, where Willin/FRMD6 is involved in the formation of apical junctional cell-cell complexes and in regulating cell migration. Here, we investigate the biochemical and biophysical role of Willin/FRMD6 in neuronal cells, employing the commonly used SH-SY5Y neuronal model cell system and combining biochemical measurements with Elastic Resonator Interference Stress Micropscopy (ERISM). We present the first direct evidence that Willin/FRMD6 expression influences both the cell mechanical phenotype and neuronal differentiation. By investigating cells with increased and decreased Willin/FRMD6 expression levels, we show that Willin/FRMD6 not only affects proliferation and migration capacity of cells but also leads to changes in cell morphology and an enhanced formation of neurite-like membrane extensions. These changes were accompanied by alterations of biophysical parameters such as cell force, the organization of actin stress fibers and the formation of focal adhesions. At the biochemical level, changes in Willin/FRMD6 expression inversely affected the activity of the extracellular signal-regulated kinases (ERK) pathway and downstream transcriptional factor NeuroD1, which seems to prime SH-SY5Y cells for retinoic acid (RA)-induced neuronal differentiation.
Willin/FRMD6是具有4.1埃兹蛋白-根蛋白-莫厄蛋白（FERM）结构域的蛋白质家族的一员。它已被确定为Hippo信号通路的上游激活因子，其表达异常时与人类疾病和功能紊乱相关。尽管Willin/FRMD6最初是在大鼠坐骨神经中发现的，但大多数研究都集中在其在神经系统以外细胞中的功能作用，在这些细胞中，Willin/FRMD6参与顶端连接细胞间复合物的形成并调节细胞迁移。在此，我们利用常用的SH-SY5Y神经模型细胞系统，将生化测量与弹性谐振器干涉应力显微镜（ERISM）相结合，研究Willin/FRMD6在神经元细胞中的生化和生物物理作用。我们提供了首个直接证据，证明Willin/FRMD6的表达会影响细胞的机械表型和神经元分化。通过研究Willin/FRMD6表达水平升高和降低的细胞，我们发现Willin/FRMD6不仅影响细胞的增殖和迁移能力，还会导致细胞形态变化以及神经突样膜延伸的形成增加。这些变化伴随着生物物理参数的改变，如细胞力、肌动蛋白应力纤维的组织以及粘着斑的形成。在生化水平上，Willin/FRMD6表达的变化反过来影响细胞外信号调节激酶（ERK）通路和下游转录因子NeuroD1的活性，这似乎使SH-SY5Y细胞为视黄酸（RA）诱导的神经元分化做好准备。
