Mechanistic Studies Of The Effect Of Rab-10 On AREs Distribution In The C. Elegans Intestines

The maintenance of cell polarity play a pivotal role in life activities,such as cell migration and cell division.Polarized epithelial cells have basolateral membrane and apical membrane.The cargo can be sorted to corresponding membrane domain to maintain stable membrane component and cell polarity by basolateral recycling endosomes(BREs)and apical recycling endosomes(AREs).However,the regulatory mechanisms on spatial distribution of different polarized endosomes is unknown.Phosphatidylinositol-4,5-bisphosphate PI(4,5)P2,mainly enriched in plasma membrane and recycling endosomes,can regulate endocytosis and recycling transport.The research on C.elegans intestine did by Grant found that small GTPase RAB-10 can recruit CNT-1,the GTPase-activating protein of ARF-6,to endosome membrane.Then CNT-1 inactivate ARF-6 and further make PIP5KI fail to activated,which finally reduce the level of PI(4,5)P2.But,RAB-10 functions on whether regulating AREs distribution and apical traffic in polarized cells are not explicit.The C.elegans intestine with property of polarized epithelial cells can be used as a model for our experiment.We confirmed that ARF-6 regulatory mechanism about PI(4,5)P₂ on BREs.We also found that RAB-10 may regulate PI(4,5)P₂ on BREs and AREs via different pathways.Experiments results demonstrate that dominant negative RAB-10(RAB-10 T23N)not only fail to make ARF-6 on BREs and AREs overexpressed to produce more PI(4,5)P₂,but also it make the level of PI(4,5)P₂ on BREs and AREs greatly reduced.Constitutively activated RAB-10(RAB-10 Q68L)cause PI(4,5)P₂ on AREs distribute close to apical membrane,but have no effect on PI(4,5)P₂on BREs.To further explore the mechanism on RAB-10 affect AREs steady distribution in polarized cells,we do researches on two aspects:RAB proteins are reported that they can mediate phosphatidylinositol phosphate metabolism by regulating related the kinases and the phosphatases.Via RNAi screen,we found that knockdown of I phophatidylinositol-4-phosphate 5 kinase PPK-1/PIP5KI can reduce the level of PI(4,5)P2 on basolateral membrane and AREs,but have no effect on apical membrane.The results demonstrate PPK-1/PIP5KI is the key to production of PI(4,5)P2 on basolateral membrane and AREs.RAB-10(T23N)make PPK-1 on AREs nearly disappear,and RAB-10(Q68L)cause PPK-1 on AREs distribute close to apical membrane,which is similar to influence on PI(4,5)P2 on AREs.So,we speculate that RAB-10 can regulate the distribution homeostasis of AREs binding with PI(4,5)P2 via regulating PPK-1/PIP5KI.In addition,interacting with motor proteins is an important way for RAB protein participating in regulating membrane traffic.So,we hypothesis that RAB-10 may affect the transport direction of AREs along the microtubules through motor proteins.Based on small-scale RNAi screen about kinesin proteins affecting vesicles transport to plus-end(C.elegans basolateral membrane),we found that KLP-14/KIF11 can recover the defect that AREs distribute to basolateral membrane caused by RAB-10(Q68L),which indicates that KLP-14may be the potent effector of RAB-10 to regulate AREs distribution.In conclusion,we preliminary found RAB-10 may mediate the level of PI(4,5)P₂ on AREs by regulating PPK-1/PIP5KI and finally affect AREs distribution and function.Besides,KLP-14/KIF11 may be the potent effector of RAB-10 to regulate AREs steady distribution.Our results expand the former cognition that RAB-10 mainly regulate basolateral recycling transport and found new function about RAB-10 in polarized traffic,which provide the research basis of cell polarity maintenance and pathogenesis of related diseases.