Clugston Joshua D, Fox Sarah, Harden James L, Copeland John W
Department of Physics, Faculty of Science, University of Ottawa, STEM Complex, 150 Louis-Pasteur Private, Ottawa, ON, K1N 6N5, Canada.
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 3155 Roger Guindon Hall, 451 Smyth Rd, Ottawa, ON, K1H 8M5, Canada.
BMC Mol Cell Biol. 2025 Apr 29;26(1):13. doi: 10.1186/s12860-025-00538-8.
Cells constantly sense and respond to changes in their local environment to adapt their behaviour and morphology. These external stimuli include chemical and mechanical signals, and much recent work has revealed the complexity of the cellular response to changes in substrate stiffness. We investigated the effects of substrate stiffness on the morphology and motility of A2058 human melanoma cells. FMNL2, a formin protein associated with actin cytoskeleton dynamics, regulates melanoma cell morphology and motility, but its role in stiffness sensing remains unclear. This study examines how A2058 cells respond to substrates of varying stiffness and evaluates the impact of FMNL2 depletion on these responses.
We found that with increasing substrate stiffness the cells transitioned from a rounded cell morphology to progressively more elongated morphologies with a concomitant increase in actin stress fiber alignment. Depletion of FMNL2 expression amplified these morphological changes, with knockdown cells showing consistently greater elongation and more pronounced stress fiber alignment compared to controls. Notably, the orientational order parameter (S) revealed higher alignment of actin filaments along the cell's long axis in knockdown cells. Substrate stiffness also affected cell motility, indicated by an apparent optimal stiffness that maximized motility followed by a notable decrease in distance travelled during migration on progressively stiffer substrates. This decrease was largely attributable to a decrease in the time the cells spent in motion as the substrate stiffness increased. FMNL2 depletion significantly exacerbated this effect, with knockdown cells traveling shorter net distances and spending less time moving across all substrates.
This study demonstrates that substrate stiffness profoundly influences A2058 melanoma cell morphology and motility, with FMNL2 playing a pivotal regulatory role. Our observations suggest that FMNL2 is critical for maintaining motility and morphological adaptability under increased stiffness. Loss of FMNL2 enhanced stress fiber alignment and cell elongation while impairing motility, particularly on stiff substrates, revealing FMNL2 as a mechanosensitive effector. This work highlights the need to study metastatic cell behaviour on substrates with biologically relevant properties and provides the foundation for future effort to determine the mechanism by which FMNL2 participates in the melanoma cell response to substrate stiffness.
细胞不断感知并响应其局部环境的变化，以调整自身行为和形态。这些外部刺激包括化学和机械信号，近期的许多研究揭示了细胞对底物硬度变化反应的复杂性。我们研究了底物硬度对A2058人黑色素瘤细胞形态和运动性的影响。FMNL2是一种与肌动蛋白细胞骨架动力学相关的formin蛋白，它调节黑色素瘤细胞的形态和运动性，但其在硬度感知中的作用仍不清楚。本研究探讨了A2058细胞如何响应不同硬度的底物，并评估了FMNL2缺失对这些反应的影响。
我们发现，随着底物硬度增加，细胞从圆形形态转变为逐渐更细长的形态，同时肌动蛋白应力纤维排列增加。FMNL2表达的缺失放大了这些形态变化，与对照相比，敲低细胞显示出持续更大的伸长和更明显的应力纤维排列。值得注意的是，取向序参数（S）显示敲低细胞中肌动蛋白丝沿细胞长轴的排列更高。底物硬度也影响细胞运动性，表现为存在一个明显的最佳硬度，该硬度使运动性最大化，随后在逐渐变硬的底物上迁移过程中行进的距离显著减少。这种减少主要归因于随着底物硬度增加，细胞运动时间减少。FMNL2缺失显著加剧了这种效应，敲低细胞在所有底物上的净行进距离更短，运动时间更少。
本研究表明，底物硬度深刻影响A2058黑色素瘤细胞的形态和运动性，FMNL2发挥关键的调节作用。我们的观察结果表明，FMNL2对于在增加的硬度下维持运动性和形态适应性至关重要。FMNL2的缺失增强了应力纤维排列和细胞伸长，同时损害了运动性，特别是在硬底物上，这表明FMNL2是一种机械敏感效应器。这项工作强调了研究具有生物学相关特性的底物上转移细胞行为的必要性，并为未来确定FMNL2参与黑色素瘤细胞对底物硬度反应的机制的努力奠定了基础。
