CRMP4 Interacting With Microfilament Mediates For The Growth Of Neurites And Formation Of The Dendritic Spine In Hippocampal Neurons

Objective:The aim of the experiment is to explore whether can CRMP4 interact with actin and to investigate where can CRMP4 interact with actin,and how to influence the growth of neurites and formation of the dendritic spine in hippocampal neurons by their interaction.Methods:Firstly,the prokaryotic plasmid of CRMP4ΔN323,CRMP4ΔC471 were constructed and the fusion proteins of them were expressed.The GST pull-down assays were to detect which part of the CRMP4 can bind with actin in vitro; Secondly,the eukaryotic plasmids of CRMP4 transfect to the neuron and observe the growth of neurites and formation of the dendritic spine in hippocampal neurons.Results:1) The results of recombinant plasmid digested by restriction enzyme show that the sizes oftarget genes were consistent with the anticipation,and the DNA sequencing showed nomismatch and mutation existing in GST-CRMP4,GST-CRMP4ΔN323,GST-CRMP4ΔC471.2) The fusion proteins of GST-CRMP4,GST-CRMP4ΔN323,GST-CRMP4ΔC471 wereexpressed successfully.And their molectular weight were about 90.7k Da,53 k Da,77k Da,which consistent with the expected size of the sum of target protein and GST proteinsize.3) GST pull-down experiment results shows the fusion proteins of GST-CRMP4,GST-CRMP4ΔN323 can bind with actin in rat brain lysate in vitro,but GST- CRMP4ΔC471 can’tbind with actin.4) The results of recombinant eukaryotic plasmid digested by restriction enzyme show that thesizes of target genes were consistent with the anticipation,and the DNA sequencing showedno mismatch and mutation existing in CRMP4ΔN323,CRMP4ΔC471.5) When transfected the CRMP4,CRMP4△N323 and CRMP4ΔC471 in the neuron,we findthat they can obviously promote the growth of neuritis and the formation of the dendriticspine.Conclusions:1) The prokaryotic expression vectors and the eukaryotic plasmids of CRMP4ΔN323 andCRMP4ΔC471 have been constructed successfully.2) The fusion proteins of GST-CRMP4,GST- CRMP4ΔN323 and GST- CRMP4ΔC471 wereexpress successfully in vitro.3) The result of GST pull-down was that the protein of GST-CRMP4,GST-CRMP4ΔN323were interaction with actin.On the contrary,the protein of GST-CRMP4ΔC471 was notinteraction with actin. So the CRMP4 combined with the actin of binding site was 471-570amino acid.4) Overexpression of CRMP4,CRMP4△N323 gene can promote the growth of neuritis and theformation of the dendritic spine.