Maffioli Elisa, Schulte Carsten, Nonnis Simona, Grassi Scalvini Francesca, Piazzoni Claudio, Lenardi Cristina, Negri Armando, Milani Paolo, Tedeschi Gabriella
Department of Veterinary Medicine, Università degli Studi di Milano, Milan, Italy.
Centre for Nanostructured Materials and Interfaces, Università degli Studi di Milano, Milan, Italy.
Front Cell Neurosci. 2018 Jan 4;11:417. doi: 10.3389/fncel.2017.00417. eCollection 2017.
Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be "interpreted" by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD) allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calcium signaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive mechanotransductive signaling hubs participating in neuronal differentiation were dissected.
神经元细胞能够精确感知其微环境的纳米拓扑特征。所感知的微环境信息将通过机械转导过程进行“解读”，并影响神经元的功能和分化。通过设计模拟适当细胞外基质（ECM）拓扑结构的底物来影响神经元分化的尝试，因机械传感/转导复杂性的深层细节仍难以捉摸这一事实而受阻。将组学方法引入这些生物材料方法中，有可能更深入地了解机械传感/转导背后的分子过程和信号级联反应，但它们在细胞材料中的应用往往受到主要底物自上而下制造方法的技术限制。相反，超声簇束沉积（SCBD）允许在大面积上自下而上制造纳米结构底物，其具有定量可控的类ECM纳米粗糙度，最近已证明这种粗糙度可促进神经元分化和成熟。利用SCBD的这种能力，我们用不同的纳米拓扑结构和/或改变其生物力学状态来挑战类神经元PC12细胞的机械传感/转导和分化行为，并分析了这些情况下它们的磷酸化蛋白质组图谱。与机械转导信号序列中的重要过程相关的多种蛋白质，即细胞/细胞相互作用、糖萼和ECM、膜/丝状肌动蛋白连接和整合素激活、细胞/底物相互作用、整合素粘附复合物、肌动球蛋白组织/细胞力学、核组织和转录调控，都受到了影响。磷酸化蛋白质组数据还表明，ILK、mTOR、Wnt和钙信号参与了这些与纳米拓扑结构和/或细胞力学相关的过程。总之，剖析了参与神经元分化的潜在纳米拓扑结构敏感的机械转导信号枢纽。
