Study of adhesion and migration dynamics in ubiquitin E3A ligase (UBE3A)-silenced SYSH5Y neuroblastoma cells by micro-structured surfaces

摘要

During neuronal development, neuronal cells read extracellular stimuli from the micro/nano-environment within which they exist, retrieving essential directionality and wiring information. Here, focal adhesions (FAs-protein clusters anchoring integrins to cytoskeleton) act assensors,by integrating signals from both the extracellular matrix environment and chemotactic factors, contributing to the final neuronal pathfinding and migration. In the processes that orchestrate neuronal development, the important function of ubiquitin E3A ligase (UBE3A) is emerging. UBE3A has crucial functions in the brain and changes in its expression levels lead to neurodevelopmental disorders: the lack of UBE3A leads to Angelman syndrome (AS, OMIN 105830), while its increase causes autisms (Dup15q-autism). By using nano/micro-structured anisotropic substrates we previously showed that UBE3A-deficient neurons have deficits in contact guidance (Tonazzini et al, Mol Autism 2019). Here, we investigate the adhesion and migration dynamics of UBE3A-silenced SH-SY5Y neuroblastoma cellsin vitroby exploiting nano/micro-grooved substrates. We analyze the molecular processes regulating the development of FAs by transfection with EGFP-vector encoding for paxillin, a protein of FA clusters, and by live-cell total-internal-reflection-fluorescence microscopy. We show that UBE3A-silenced SH-SY5Y cells have impaired FA morphological development and pathway activation, which lead to a delayed adhesion and also explain the defective contact guidance in response to directional topographical stimuli. However, UBE3A-silenced SH-SY5Y cells show an overall normal migration behavior, in terms of speed and ability to follow the GRs directional stimulus. Only the collective cell migration upon cell gaps was slightly delayed for UBE3Ash SHs. Overall, the deficits of UBE3Ash SHS-SY5Y cells in FA maturation/sensing and in collective migration may have patho-physiological implications, in AS condition, considering the much more complex stimuli that neurons findin vivoduring the neurodevelopment.