Talin Modulates The Growth Of Neurite Through Intracellular Tension

Objective:Neurite growth and extension are regulated by intracellular chemical and mechanical signals.Talin in the cytoplasm plays a key role in polarization and growth of neurons due to its special structure,and can be regulated by both chemical signals and mechanical activities.Talin is composed of two functional domains,head and rod.However,it is not clear how the functional domains affect the neurite growth by regulating chemical signals and mechanical activities.At the same time,traditional Chinese medicine can also effectively promote nerve repair and regeneration,and whether they can also participate in nerve growth through the chemical signals and mechanical activities of Talin remains to be explored.The purpose of this study was to reveal the mechanism of Talin in neurite polarization and growth through chemical signal and mechanical activity by the application of FRET tension probe.Methods:In PC12 and SH-SY5Y cells,NGF and glia scar inhibitor aggrecan,were used to establish the models of neurite growth and inhibition.The overexpression of Talin,Talin head and Talin rod was achieved by liposome transfection.Western blot was performed to detect Talin,Integrin,FAK and p-FAK protein levels.The Integrin activity,Talin and DOK1 protein distribution and cytoskeletal structure changes were observed by immunofluorescence.Talin tension probe(Talin-M)was transfected into cells,and FRET technique was used to measure the change of Talin rod tension at different stimuli.Osmotic pressure of cells was measured by freezing osmotic pressure meter.mRNA levels of competing proteins DOK1,SHARPIN,actinin and filamin were detected by real-time quantitative RT-PCR.Small interfering RNA was used to down-regulate the expression levels of Talin,DOK1,SHARPIN and vinculin.GAP43 was used to mark the neurites in the cells,and MAP2 and TAU were used to mark the dendrites and axons of neurons in the cerebral cortex respectively,and their growth was observed by immunofluorescence.Results:1.After overexpression of Talin head,only the number of neurite increased;2.When full length of Talin was overexpressed,the number and the length of neurite was increased significantly;2.NGF can up-regulate Talin tension,but when myosin is inhibited or actin filaments depolymerize,the upward tension trend is reversed;3.Talin tension and osmotic pressure increased significantly after Integrin activation;4.Aggrecan can weaken Talin tension and up-regulate the expression of DOK1 mRNA and protein;5.When DOK1 expression is down-regulated,Integrin activity,Talin tension were both increased,and cell protuberance and axon length increases;6.Ginsenoside RD can up-regulate Talin tension and promote the increase of neurite length.Conclusion:Full length of Talin is necessary for neurite extension.NGF can increase the tension in Talin rod by increasing the microfilament force and participate in the polarization and growth of nerve.Aggrecan can up-regulate the competitive protein DOK1 of Talin,thus leading to the down-regulation of Integrin activity and the weakening of intracellular mechanical activity.Intracellular Talin tension can interact with osmotic pressure to promote neuritic growth and extension,and this regulatory process can be up-regulated by Integrin activation,and down-regulated by Talin competitive protein DOK1.Ginsenoside RD can participate in the growth and extension of neurite by Talin tension.The exploration of intracellular mechanical activity can reveal the new regulatory mechanism of neurite growth and help to evaluate the medicinal value of various drugs for the repair of nerve injury.