Abstract
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.
Highlights
Cells can sense nanotopographical cues deriving from the extracellular matrix (ECM), predominantly through integrin adhesion complexes (IAC), and the microenvironmental information is converted into alterations of the cytoskeletal organization and mechanics
The cells on ns-Zr15 were found to have small IAC (predominantly focal contact (FC) size), few to none stress fibers and a low cell rigidity, contrary to the large IAC (focal adhesion (FA) size), stress fibers and an increased cellular rigidity on flat zirconia surfaces (flat-Zr) (Schulte et al, 2016a). Besides these two conditions, the proteomic analyses in this study comprise instead PC12 in more versatile experimental conditions including a surface nanotopography with higher roughness, the biochemically NGFinduced canonical neuronal differentiation and manipulations that affect the biomechanical status of the PC12 cell
The presently adopted quantitative proteomic approach challenges PC12 cells with diverse experimental situations that address the impact of substrate nanotopography and/or cellular biomechanics on neuronal cell fate
Summary
Cells can sense nanotopographical cues deriving from the extracellular matrix (ECM), predominantly through integrin adhesion complexes (IAC), and the microenvironmental information is converted into alterations of the cytoskeletal organization and mechanics. Using PC12 cells as a broadly accepted model system for neuronal differentiation, we demonstrated that appropriate biophysical stimuli; provided by the cellular interaction with nanotopographical cues of titania or zirconia surfaces produced by SCBD, promote neuronal differentiation processes (Tamplenizza et al, 2013; Schulte et al, 2016a) This potential of the cluster-assembled surfaces was confirmed in hippocampal neurons (Schulte et al, 2016b). The cells on ns-Zr15 were found to have small IAC (predominantly focal contact (FC) size), few to none stress fibers and a low cell rigidity, contrary to the large IAC (focal adhesion (FA) size), stress fibers and an increased cellular rigidity on flat-Zr (Schulte et al, 2016a) Besides these two conditions (ns-Zr15 and flat-Zr), the proteomic analyses in this study comprise instead PC12 in more versatile experimental conditions including a surface nanotopography with higher roughness, the biochemically NGFinduced canonical neuronal differentiation and manipulations that affect the biomechanical status of the PC12 cell. The precise analysis of biomolecular events triggered by cell/nanotopography interaction, combined with the technical capacities of SCBD, constitutes the necessary foundation for efficient near-future exploitation of SCBD for versatile bio-applications
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