Structural Tension Recognition And Mechanism Analysis Of Microfilament Skeleton And Spectrin-connected Protein In Living Cells

Objective:Three signaling systems play the fundamental roles in modulating cell homeostasis:mechanical, as same as chemical, electrical. Based on principle of fluorescence resonance energy transfer (FRET), we constructed cpstFRET cassete and inserted it into actin and spectrin protein hosts respectively. The probe can transfer cytoskeleton structural forces into spectrual signals, aim to reveal the roles of mechanics in maintaining the normal physiology and regulation of neuronal polarization and axon growth.Methods:Based on the the principle of FRET and molecular cloning technology, we circularly permutated Cerulean and Venus and created cpstFRET cassete. Then inersted it between two β-actin genes or into the middle of spectrin monomer gene respectively to creat AcpA and spectrinM probe. Using gene cloning technology we integrated probes into actin microfilaments and spectrin proteins in cell. As the structural tension in actin actin or spectrin proteins changed, the angular orientation between donnor and acceptor varied from parallel to orthogonal with reduced efficiency of FRET. In this way, mechanical signals were transformed into detectable optical signals. By using FRAP/FLIP,3D scan module of laser scanning confocal microscope, combing hypotonic stimulation (ddH2O), IP3 receptor agonist (Caffeine), we verified the availability of probes. In order to clarify the roles of talin and E-cadherin in axon growth, AcpA probe was well expressed in PC12 cell. Then combined with pEGFP-C1-talin, pcDNA3.1(+)-E-cadherin plasmids, β-NGF, scar inhibitory factor, we created microfilament tension model with growth or inhibition of neuronal axons in vitro to reveal the function of talin and E-cadherin during growth of axons. In the end, we expressed these two probes in MCF-7 cell and used verapamil, vinblastin, camptothecin and taxol to find out the impact of those drugs on structural tensions of actin filaments and spectrin proteins, and attempted to clarify the mechanical effects on skeleton structural proteins with stimulation of different drugs.Results:Based on FRET principle, gene coded fluorescence detection probes AcpA and spectrinM could detect varity of structural tensions in real time. Hypotonic and caffeine stimulation can enhance structural tension of actin and spectrin proteins. In neurons, nerve growth factor (NGF) can up-regulate while scar inhibitor aggrecan and CSPG down-regulate the intracellular structural tensions. In NGF-stimulated PC 12 cell, tension in neuronal axons is significantly higher than soma. In addition, talin can increase tensions of intracellular microfilaments and positively regulate axon growth; while scar inhibitory factor can decrease tensions of intracellular microfilaments through E-cadherin and negatively regulate axon regeneration. However, talin can partially reverse the inhibition on axon regeration of scar inhibitor factors. Calcium channel blocker verapamil can improve the structural tension of actin and spectrin proteins; while camptothecin can significantly enhance tension of actin filaments and spectrin proteins; obviously, vinblastine and paclitaxel, specific drugs to tubulin proteins, can enhance tension of actin filaments while tension of spectrin proteins returned to normal levels gradually after slightly growth.Conclusion:Mechanical signals participate in regulation of varieties of physiological activities in cells, such as neuron polarization, axon regeneration, apoptosis and shape-stability. Overexpression of talin can increase tension of microfilament in neurons, as this enhancement seems to be beneficial to differentiation and axonal extend in neuron cells. Tensions in microfilament cytoskeleton and spectrin protein can be raised in a Ca2+-dependented way. However, the decreasion of Ca2+concentration may not lead to down-regulation of tension in actin and spectrin proteins. When functions of microtubule were inhibited, tension in microfilaments cytoskeleton can partially compensate for the deficiencies to maintain normal physiology in cell. Collectively, this study reveals mechanism of tension in actin filaments and traction force in spectrin proteins, which can regulate morphology and physiological activities in living cell, and also provide a new test tool and an effective method for studying intracellular structural tension.