Functional Analysis Of Microtubule Severing Protein Fidgetin In Drosophila Neuronal System

The cytoskeleton mainly contains three components,and microtubule is one of them.The microtubule structure is dynamic,and its stability is related to the state of the cell,microtubule-dependent transport,the distribution and function of organelles and biomolecules.Two types of proteins involve in the dynamic regulation of microtubule polymerization and depolymerization.One is that the microtubule stabilizing factors including multiple microtubule associated proteins MAPs(such as Tau,MAP1,and MAP4)and the polymerization promoting proteins at the microtubules-ends(such as EB 1 and CLIP 170);the other is that the microtubule depolymerizing factors(such as Fidgetin and Kinesin),which participating in microtubule severing and terminal depolymerization.Mutations in the encoding genes of those proteins regulating microtubules dynamics lead to some genetic diseases,Mutations in TUBB3(?-tubulin isotype III)may cause serious neurological TUBB syndrome.Hyperphosphorylated Tau proteins detach from microtubules,consequently result in microtubule instability,weakened transport capacity,and nervous system disorders.In the past few years,the studies on Fidgetin have focused on cultured cells,C.elegans and parts of mouse tissues.A comprehensive analysis of Fidgetin function in vivo is still lacking.This research uses Drosophila as a model to explore the effects of Fidgetin on development.Fidgetin,the coding gene is CG3326,locating in the long arm 23E3 region of chromosome 2 of Drosophila.CG3326encodes a protein with the highest homology to human Fidgetin-like 1(the identity and similarity is30%and 48%,respectively).As a depolymerizing factor,Fidgetin is involved in microtubule severing and terminal depolymerization.In this research,we used the CRISPR/Cas9 system to construct a fidgetin non-functional mutant fidgetin^null and an endogenously expressed fidgetin:GFP in Drosophila.We used the att B-att P system to construct a UAS fidgetin to overexpress the fidgetin.It was found that fidgetin:GFP enriched in the ovary of adult Drosophila,showing the expressed characteristics of fidgetin tissue.Loss of fidgetin led to an increase in the number of neuromuscular junctions(NMJ)and class I dendritic branches.Moreover,the field area of class I dendritic arborization neurons is decreased in Fidgetin mutant.The above results provide a powerful tool for further resolving the function of Fidgetin in vivo.