The Study Of MTSS1 Functional Complex In The Ruffle Formation

The cytoskeleton is the fiber network in eukaryotic cells which can maintain the cell morphology and cell movement. It consists of the structural system known as the cytoskeleton system.Cell movement is usually accompanied by changes in cell morphology with local protrusions of plasma membrane in the shape of waves which are called ruffling.MTSS1 (Metastasis suppressor 1, also known as Missing in Metastasis, MIM) is considered to be associated with malignant transformation of some particular tumors. The expression levels of actin-binding protein influence the formation of lamellipodia and similar filopodia structures. At the meanwhile, studies have shown that over-expression of the MTSS1 can promote the formation of ruffling when induced through PDGF (Platelet derived growth factor).Our previous work showed that MTSS1 and the WASP family members (Wiskott-Aldrich Syndrome Protein) together with a member of the spectrin family can co-immunoprecipitated when stimulated with the growth factors, which suggested it may be related to these two molecules in cell motility.As a member of the WASP family, WASF2 plays an important role in the formation of lamellipodia and even the cell migration. It has been reported that WASF2 in the form of a complex is responsible for the cell signal transduction from the receptor tyrosine kinase and a GTPase of Rac to the Arp2/3 complex-mediated actin polymerization, which can influence the reconstruction of cytoskeleton, and then regulate actin polymerization, promote the cytoskeletal rearrangement, and ultimately promote the formation of lamellipodia.In this work, two genes interacting with MTSS1 were constructed into the pcDNA3.1-2×Myc vector. Then they were transfected into the 293T cells. The cells were lysed respectively. Finally we got two kinds of protein lysate rich in targeted protein. After that the MTSS1 protein were expressed in the form of prokaryotic expression, purified and cross-linked to Affi-Gel 10 beads with BSA as a control. Pull down assay and western blot analysis showed that there was no direct interaction between WASF2 complex and MTSS1; however, there was direct interaction between MTSS1 and the spectrin family protein.The immunofluorescence assay demonstrated that there was only a little ruffle formation in the front of the cells transfected with GFP and Myc-WASF2; while there was a large mount of ruffle formation around the cells transfected with GFP-MTSS1 and Myc-WASF2; on the meanwhile, the two proteins were colocalized in the ruffle structures.Furthermore, we found that MTSS1 cooperated with ACTN4 to promote the F-actin bundle formation and the dorasl ruffle formation through the in vitro functional analysis of F-actin bundling assay.Then, how did MTSS1 and WASF2 interact with each other? According to current studies, WASF2 can play an important role in the formation of pentamer complex including Sral, Nap1, Abi1 and HSPC. While activated Racl can activate the WASF2 protein complex then playing its role through interaction with the Sral protein. Some studies showed there was a direct interaction between the N-terminal domain IMD of MTSS1(IRSp53-MTSS1 homology domain) and Rac1. Similarly, our immuno-precipitation results showed that WASF2 can co-precipitated with the activated Rac1.We speculate that the indirect interaction between WASF2 and MTSS1 may be mediated through downstream signaling pathways of Rac1 which promote the formation of dorsal ruffling. However, the spectrin family protein promotes the formation of dorsal ruffling through direct interaction with MTSS1.In summary, this paper has showed some explanation for the MTSS1 promoting the formation of ruffling from the point of molecular mechanisms on the basis of current studies, which provides some clues for further research for the mechanisms of MTSS1 promoting the formation of ruffling in the cell motility.