Effect Of RNA Interference On The MTSS1 Mediated Cell Morphology

Tumor metastasis suppressor protein MTSS1(Metastasis suppressor 1;also known as Missing in Metastasis,MIM),as an actin binding protein,plays important roles in the cell morphology,cell motility,neural development and tumor metastasis.Our previous work showed that MTSS1 over-expression in the cultured cerebellar granule neurons can obviously increase the number of dendrites and the length of total dendrites,but axon extension was significantly perturbed.To confirm this result further,we inhibited the endogenous MTSS1 level by by RNA interference technique.As the result,compared with the control group,axon length was substantially extended,but the total dendritic length decreased,accompanied by a reduction in the number of dendrites.This result verified our previous overexpression data.Simultaneously,in order to further investigate the effect of MTSS1 on dendritic branching,we performed the similar experiment on embryotic hippocampal neuronal culture,which elaborated dendrites structure.In accordance with the over-expressing data in cerebellar granule neuron,dendritic arborization was substantially reduced in MTSS1 knock-down cells compared to controls.It suggested that MTSS1 affects not only the number and the total length of the dendrites,but also the branches of the dendrites.All these data confirmed our previous over-expression data,suggesting the role of MTSS1 in neuronal morphogenesis.In addition,our preliminary work also showed,co-transfection of dominant-negative mutant GTPase Racl with MTSS1,can reverse the morphological effect of MTSS1 on cerebellum granule neurons partially,suggesting that the Racl pathway may be involved in the effect of MTSS1 on neuron morphology.Here,we co-transfected constitutively active Racl with MTSS1 and the result exhibited that the activated Rac could not inhibit the effect of MTSS1.In addtion,we found that MTSS1 over-expression can increase the activity of Racl in neuronal cell line Neuro2a.These results might indicated that the Rac1 activity is important for the morphological changes of neurons induced by MTSS1.Except for the effects on neuronal morphology,MTSS1 also play roles the actin fiber organization in cell-cell contact.Our pervious study indicated there is an interaction between α-ACTN4 and MTSS1,and over-expression of MTSS1 promotes the re-distribution of α-actinin 4 to cell leading edge.To confirm this result further,we used knock down assay to inhibit endogenous MTSS1 level in epithelial cells.The result suggested that the MTSS1 depletion reduce the localization of exogenousα-ACTN4 to the cell-cell contact.Most importantly,this polygonal cell would loss one or more cell contact sides,which were replaced by lamellipodia-like structures.Together,by using knock down technique,we fully proved our previous over-expression data,i.e.,MTSS1 could promote the number and length of neurons dendrites,and inhibit axonal elongation;And MTSS1 could affect the distribution of α-ACTN4 in cell peripheral area,which might improve the actin fiber organization in intercellular junction.