CRMP5Interacts With Tubulin To Promote Growth Cone Development In Neurons

Objective：The experimental purpose is to investigate whether CRMP5can interact with tubulinor not, and the development of hippocampal neuron growth cone can be influenced bytheir interaction.Methods：Firstly, the prokaryotic plasmid of CRMP5-GST was constructed and the fusionprotein of CRMP5-GST was expressed. Then GST pull-down assays were used todetect whether CRMP5binding tubulin or not in vitro; Secondly, the eukaryoticplasmids of CRMP5-FLAG and tubulin-GFP were constructed, then they wereco-transfected in293T cells. Indeed its cell lysates, rat brain lysates and the growthcone extracted by rat brain lysates were used to analysed whether CRMP5interactwith tubulin or not in vitro by coIP assays; Thirdly, after immunofluorescence stained,confocal microscope was used to observe distribution of CRMP5and tubulin in293Tcells and neurons; At last, the morphology of growth cones in hippocampal neuronswere observed by over expression or knockout CRMP5.Results：(1) The results of recombinant plasmid digested by restriction enzyme show that thesizes of target genes were consistent with the anticipation, and the DNA sequencingshowed no mismatch and mutation existing in CRMP5and tubulin;(2) The fusion protein of CRMP5-GST was expressed successfully. And its molecularweight was about90KD, which consistent with the expected size of the sum of targetprotein and GST protein size;(3) GST pull-down experiment results showed the fusion proteins of CRMP5-GSTcan bind with tubulin in rat brain lysate in vitro;(4) CoIP assays results showed CRMP5can interact with tubulin in vitro by293T celllysates co-transfected with CRMP5-FLAG and tubulin-GFP, rat brain lysate and the growth cone extracted rat brain lysate;(5) Immunocytochemistry results showed CRMP5and tubulin coexist in the293T cellbody and lamellipodia, and they also coexist in cell body projections and growth coneof neuron; these results were similar to the above experiment results in vitro and vivo.(6) The growth of neuronal growth cone was inhibited when CRMP5gene wasknockout in primary cultured hippocampal neurons;(7) Overexpression of CRMP5gene can significantly promote the growth conedevelopment in primary cultured hippocampal neurons.Conclusions:(1) Prokaryotic expression vector of CRMP5-GST and the eukaryotic plasmids ofCRMP5-FLAG and tubulin-GFP have been constructed successfully;(2) The fusion protein of CRMP5-GST was expressed successfully in vitro;(3) CRMP5can interact with tubulin in vitro and vivo;(4) CRMP5and tubulin coexist in the293T cell body and lamellipodia, and they alsocoexist in cell body projections and growth cone of neuron;(5) CRMP5interact with tubulin may participate in the development of growth conein neuron.