Talin Contributes To The Growth And Regeneration Of Axons In Structural Tension Dependent Manner

Objective: Intracellular tension is involved in neuron differentiation, and play pivotal parts in the growth and regeneration of axons. Similar to chemical and electrical signals, force signals are also involved in mechanisms underlying the growth and regeneration of axons. However, the activities of such forces(especially structural forces through cytoskeletal conduction) are relatively unknown because methods to measure them in real time are lacking. To better understand this process, we created F?rster resonance energy transfer(FRET)-based tension probes that can be incorporated into microfilaments to provide a real-time measurement of forces in neuron cytoskeletons. The aim of this study was to investigate the mechanism underlying intracellular tension in the growth and regeneration of axons.Methods: Rat cell line PC12 cell were treated with β-NGF(50ng/ml) for corresponding time to construct rodent models. Real-time RT-PCR and western blot were applied to detect the m RNA and protein level of Talin,vinculin and E-cadherin in PC12 cells treated with scar inhibitors including aggrecan and CSPG, as well as its downstream signal molecular p-ROCK protein. What’s more, we created F?rster resonance energy transfer(FRET)-based tension probes that can be incorporated into microfilaments to provide a real-time measurement of forces in neuron cytoskeletons. Changes in structural force could be detected and analyzed according to changes in optical signals. We co-transfected the probe and E-cadherin or Talin plasmids, and used immunofluorescence analyses to investigate their effects on the growth and regeneration of axons. In addition, Talin knockdown was achieved using specific small interfering RNA(si RNA). Real-time RT-PCR and western blot analysis wereemployed to detect the m RNA and protein levels of E-cadherin. After pretreatment with Bay11-7082(NF-κB inhibitor), cells were transfected with si-Talin and then stimulated with NGF. m RNA and protein levels of E-cadherin, and transcription factors(snail, ZEb1, ZEb2, SLUG, SIP1, twist, HIF-1, ATF-α, maf) were determined by real-time RT-PCR analyses and western blot.Results: We found that the FRET probe could be used to assess the structural tension of neuron polarity. Nerve growth factor(NGF) upregulated structural forces, whereas the glial-scar inhibitors chondroitin sulfate proteoglycan and aggrecan weakened such forces. Notably, the tension across axons was distributed uniformly and remarkably stronger than that in the cell body in NGF-stimulated neurons. The mechanosensors Talin/vinculin could antagonize the effect of glial-scar inhibitors via structural forces. However, E-cadherin was closely associated with glial-scar inhibitor-induced downregulation of structural forces. Talin/vinculin was involved in the negative regulation of E-cadherin transcription through the nuclear factor-kappa B pathway.Conclusion: Collectively, this study clarified the mechanism underlying intracellular tension in the growth and regeneration of axons which, conversely, can be regulated by Talin and E-cadherin.