The Interaction Between CDC-42 And PAR Protein And CDC-42 Affect The Life-span Of Caenorhabditis Elegans

Rho-family GTPase play an important role in the development,cdc-42 is a member of small GTPase family.It gets involved in many processes of development. Through cdc-42 RNAi,CDC-42 expression and purification,immunostaining, life-span analysis and Co-Immunoprecipitation,we study the interaction of CDC-42 with polarity gene and daf-2,daf-16.After cdc-42(RNAi),the division of C.elegans one-cell embryo changed. CDC-42 can regulate the location of PAR-2 and PAR-6.CDC-42 may control the position of PAR-3 and has no effect on PAR-5.After expression of recombined CDC-42,optimized the expressing condition and purification,immune the mouse to get CDC-42 antibody,we detect the quantity of CDC-42 on N2 and par mutants by Western blot.The results show CDC-42 expressing decreased in par-4 mutant.After cdc-42(RNAi),the quantity of CDC-42 is decreased in N2 worm.Through the immunostaining of CDC-42 antibody on C.elegans wild type and par mutants,the results show that CDC-42 distributed intensely in the cytoplasm of one cell embryo in wild type.The distribution of CDC-42 became enriched in the cortex of the late embryo till to the morphogenesis.The expressing of CDC-42 in par-2(-) and par-6(-) mutants decreased significantly.It implied par-2 and par-6 may regulate CDC-42 expressing.In par-1mutant,the expressing of CDC-42 decreased till to the morphogenesis.It implied that par-1 can regulate CDC-42 expressing.In par-3(-),par-4(-) and par-5(-)mutants,CDC-42 expressing is increased with the cell number increased in the embryo.At the morphogenesis stage,the quantity of CDC-42 recovered proved that par-3,par-4 and par-5 may affect CDC-42 indirectly. To test the life-span of C.elegans after cdc-42(RNAi),the results show that cdc-42 does not effect the life-span of wild type N2 worm.But in daf-2 mutant,knock down cdc-42,the mean life-span of daf-2 mutant worms shortened 2.9 days.And in daf-16 mutant,knock down cdc-42,the mean life-span of daf-16 mutant shortened 0.75 day.Compared daf-2(-) and daf-16(-) with N2,the life-span of daf-2 mutant is extended and daf-16 mutant is shortened.With cdc-42(RNAi),the life-span of daf-2(-) and daf-16(-) are both shortened,it implied that cdc-42 can effect the life-span of C.elegans depend on Insulin/IGF-1 pathway.Using CDC-42 antibody via Co-Immunoprecipitation in wild type N2 and par mutants,and combined with MALDI-TOF anslysis,it proved a lot of molecules may interact with CDC-42.These proteins include M110.3;hypothetical protein Y87G2A.f;Egg Laying defective family member(egl-4);Serpentine Receptor,class U family member(sru-12);F58B3.7:D2063.2;NanOS related family member(nos-3); C.elegans protein C27H2.2c.