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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/119157
Study of cell response over nanopatterned ligands on diblock copolymer surfaces
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[eng] Cells in tissues are exposed to extracellular signals that integrate and appropriately translate into specific responses. Receptors at the cell membrane recognize a variety of soluble ligands, extracellular matrix proteins and molecules presented by the neighboring cells. Ligand-receptor recognition event triggers intracellular signal transduction pathways modulating the resulting cell function. Some receptors do not function individually as signaling units but require interactions and associations with other receptors in multimolecular complexes. This process is known as receptor clustering and is an evolutionarily preserved mechanism responsible for the integration of highly complex signals. Increasing evidences suggest that this exceptional integration is subjected to spatially controlled ligand distribution at the nanoscale. Recent developments in highly sophisticated nanofabrication approaches have allowed to experimentally address this detailed spatial regulation on cell signaling. However, it is still unclear how the nanoscale distribution of ligands can impact on the dynamics of receptor activation and signaling processes.
Herein we present a nanostructured platform to create patterns of ligands in regular nanosized (< 30 nm) clusters. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that tend to segregate into nanodomains. The hexagonal arrangement of the PMMA domains acts as template to be replicated by the ligand distribution. Thanks to the versatile functionalization strategy developed, any amine-bearing molecule can be covalently immobilized. The spatial distribution of ligand was analyzed by Atomic force microscopy (AFM) and stochastic reconstruction microscopy (STORM), unveiling the high level of fidelity between the nanopatterned ligands and the underlying polymeric template.
To validate these substrates as platforms for systematic study of receptor clustering processes, an adhesive peptide which promotes focal adhesion formation, was immobilized on the nanopatterned surfaces. While the overall ligand surface density was maintained constant, the spatial distribution of ligands showed a remarkable impact on focal adhesion formation. Cells on nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that ligand presentation in a clustered format might promote multivalent ligand-receptor interactions which can help to shed light on receptor oligomerization processes.
In addition, the nanopatterned substrates developed were used to investigate the dynamics of the process of Eph receptor assembly into oligomeric clusters upon stimulation with ephrin ligands. It is known that Eph receptor oligomer composition is crucial in the fine-tuning of receptor signaling, as it will trigger intracellular signals feedback which will modulate cell response. Oligomerization processes, which imply resolving the temporal evolution of nanometric size objects in diffusive environments such as cell membranes are beyond the reach of live-imaging tools. We in here resolve the oligomerization kinetics of the Eph receptor in live cells with the required spatial and temporal resolution by using an enhanced version of the Number and Brightness (eN&B) technique, which can discriminate with molecular sensitivity the oligomeric species. The results demonstrated that stimulation through surface-bound ligands with a random distribution was not sufficient to activate the receptor signaling. Conversely, when nanopatterned on our substrates, ligands effectively induced receptor oligomerization. In addition, surface-induced ligand clustering by our nanopatterning approach accelerated the dynamics of receptor oligomerization process when compared to antibody-induced ligand clustering. Such an efficiency was induced even when ligand surface coverage was 9-fold lower in the nanopatterned presentation. Therefore, our ligand presenting platform is thought to induce multivalent ligand-receptor interactions, and might be a useful strategy to precisely tune and potentiate receptor responses. It has promising applications in biotechnology and biomedicine, such as cell culture systems to provide insight into relevant receptor clustering processes and design of new bioactive materials and drug-delivery systems.
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HORTIGÜELA, Verónica. Study of cell response over nanopatterned ligands on diblock copolymer surfaces. [consulta: 10 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/119157]