To Study How Gef26 Regulates Actin Cytoskeleton Organization In Drosophila

Cytoskeleton plays a very important part during development.It not only play an important role in maintaining the cell morphology,external forces and so on.It also participates in many important life activities.Cell migration,adhesion and division are all dependent on cytoskeleton.F-actin is one of the most important elements of eukaryotic cytoskeleton,which plays an important role in cellular localization and morphology.Previours research reported that Ras superfamily plays essential functions during cytoskeleton organization.Ras superfamily is small GTPase,which is encoded by proto-oncogene.While small GTPase serves as biomolecular switches inside cells to control a variety of essential cellular processes.Small GTPase is known to regulate various cellular functions,including proliferation,survival,growth,migration,differentiation and cytoskeletal dynamics.Activity of small.GTPase is tightly controlled by the coordinated action of two classes of regulatory proteins:guanine nucleotide exchange factors(GEFs),which activate small GTPase by catalyzing the exchange of bound GDP for GTP,enabling them to recognize and activate downstream effectors,and GTPase-activating proteins(GAPs),which suppress small GTPase by enhancing their intrinsic rate of GTP hydrolysis to GDP[1,2].Gef26 is one of the Gef26/Rap signaling.Gef26 activates small GTPase by catalyzing the exchange of bound GDP for GTP,enabling them to recognize and activate downstream effectors.Previous research found GEF family can affect cytoskeleton organization through Abl pathway,thereby further regulates axon pathfinding[3].Our lab's studies have found that loss of Gef26 impaired morphology of neuromuscular junction(NMJ)(unpublished data),but the mechanism is still unkonwn.This research mainly used Drosophila primary culture and NMJ system to analyze how Gef26 regulate cell morphology and its mechanism.Our experimental results showed that,in Gef26 mutant cultures,muscle cells showed abnormal morphology.We further study found that F-actin organization of muscle cells was broken compared with wild type.These data indicated that Gef26 participated in F-actin organization of muscle cells.In Drosophila primary culture,we found that the end of neuron was tortuous.These data indicated that Gef26 may also participate in cytoskeleton organization of neuron.Our experimental results showed that in Gef266/+;Abl4/+ double mutant,F-actin organization of myocyte was rescued.So there were genetic interactions between Gef26 and Abl.We also used Drosophila NMJ system to confirm that there were genetic interactions between Gef26 and Abl in vivo.In Gef266/+ mutant,the bouton number was decreased compared with wild type.In Abl4/+ mutant,the bouton number was increased compared with wild type.While in Gef266/+;Abl4/+ double mutant,the bouton number did not show difference compared with wild type.Our lab's studies have found that loss of Gef26 impaired F-actin of NMJ(unpublished data).These in vivo experiments further demonstrated that there were genetic interactions between Gef26 and Abl during regulating cytoskeleton organization.In Drosophila primary culture,previous studies have found that high temperature(HT,30?)can promote muscle cells differentiation[4].While in high temperature(HT,30?),muscle cells were not seen in Gef26 mutant cultures at embryonic stage 10,so Gef26 may inhibit muscle cells differentiation in high temperature(HT,30?)incubation.While we mixed Gef26 mutant with WT in Drosophila primary culture at embryonic stage 10 in high temperature(HT,30 ?)incubation,muscle cells of Gef26 mutant can differentiate,and interact with cells of WT.This detail mechanism needs to be further studied.Gef26 is conservative in evolution.Gef26 of Droscophila and human has a high degree of homology.We used Drosophila as model organism,and proved that Gef26 regulates cytoskeleton organization.This could provide more clues for future clinical treatment.