Rearrangements of actin cytoskeleton are important for normal cell functioning and their deregulation leads to various pathologies (e.g. cancer cell invasiveness). The members both ITSN (ITSN1, ITSN2) and verprolin families (WIP, CR16, WIRE) play an important role in actin cytoskeleton remodeling. ITSNs are associated with the progression of several neurodegenerative pathologies and are implicated in cancer cell survival and migration. The members of the mammalian verprolin protein family are well known interactors with actin and nucleation factors such as cortactin and N-WASP, and can influence the Arp2/3-dependent actin polymerization machinery. Thus, SH3-containing ITSNs and proline-rich verprolins could act as partner proteins because of participation in the same processes such as endocytosis and actin cytoskeleton rearrangement and possessing putative interaction motifs.
The presented work is devoted to the study of ITSN interaction network in actin remodeling by identifying new partner proteins WIP and CR16. Immunoprecipitation and in vitro binding experiments revealed that ITSNs bind WIP and these interactions are mediated by the SH3A, SH3C and SH3E domains of ITSNs. Our results show that interactions between ITSN1 and WIP are direct. For the mapping of ITSN1-interacting regions of WIP structure, WIP deletion constructions have been used, which led to the identification of the importance of 318-450 aa of WIP proline-rich domain for the above mentioned interactions. In addition, we have demonstrated that ITSN1 forms a complex with WIP and N-WASP. The immunofluorescence analysis revealed partial co-localization of ITSN1 and ITSN2 with WIP in mammalian cells.
Recent studies denote that WIP is one of the key components of podosomes and invadopodia. While analyzing proteolytic degradation of extracellular matrix, it has been shown that endogenous ITSN1 and ITSN2 as well as ITSN1/WIP complex are localized in invadopodia of MDA-MB-231 breast cancer cell line. It is known that Cdc42/N-WASP/WIP-dependent actin polymerization via the Arp2/3 complex is required for invadopodia formation. Given the ability of ITSNs to activate Cdc42 and directly interact with N-WASP and WIP, we suggest that ITSNs could be involved in actin polymerization during invadopodia formation.
In the immunofluorescence experiments, co-localization between ITSN1, WIP and clathrin has been demonstrated, indicating their possible ability to participate in clathrin-mediated endocytosis. An analysis of transferrin receptor internalization revealed defects of intracellular transferrin transport in 293 cells with overexpressed ITSN1-L or WIP whereas co-expression of ITSN1-L and WIP normalized transferrin internalization suggesting participation of the complex in the regulation of vesicular transport. Intracellular redistribution of transferrin occurs by several ways including RAB4-dependent early endosomes. Partial co-localization of WIP and ITSN1 proteins with a marker of recycling endosomes, RAB4, has been demonstrated. Moreover, overexpressed ITSN1 promoted significant co-distribution of WIP to RAB4-positive vesicles in MCF-7 cells. Together, these findings suggest that WIP/ITSN1-L complex is involved in the cytoplasmic vesicle trafficking, in particular, in the fast recycling of the transferrin receptor.
The immunofluorescence analysis revealed that both ITSN1-L and WIP co-localize at the filopodia sites and actively and synergistically induce filopodia formation.
For the first time, it has been demonstrated that both members of the ITSN family, ITSN1 and ITSN2, interact with another member of the verprolin family, CR16. In vitro binding experiments showed that the interaction of ITSNs with CR16 is mediated predominantly by neuron-specific isoform of ITSN1 SH3A domain and the SH3E domains of ITSN2. Moreover, overexpressed CR16 promoted the association between ITSN1 and F-actin.
Thus, this manuscript presents the data of new molecular components of invadopodia, ITSN1 and ITSN2, and their interactions with one of key invadopodia proteins, WIP. Obtained results of interactions between intersectins and regulators of actin polymerization, CR16 and WIP, can be used for further study of the functioning of N-WASP/Arp2/3-mediated rearrangements of actin cytoskeleton, which defects of regulation are linked to the development of malignant tumors.
