The Functional Study Of α-ACTN4 In Dorsal Ruffles

Dorsal ruffles, also known as circular ruffles or waves, are ring-like, F-actin-rich, membrane protrusions, which extend from the apical cell surface in response to a variety of growth factors including epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF) and vascular endothelial growth factors (VEGF). Dorsal ruffles form only transiently in response to stimulation. Several physiological roles have been demonstrated for dorsal ruffles, including macropinocytosis and cell motility.α-ACTN4 is primarily distributed in ruffles of migrating cells. EGF stimulation could improve the cell motility and cytokinesis through phosphorylation ofα-ACTN4. It has been speculated that the tyrosine phosphorylation on specific site ofα-ACTN4 might weaken its actin binding ability and disrupted cytoskeleton so that facilitating the cell movement.Our previous study suggested thatα-ACTN4, Mtss1, WASF2 may co-exist in the molecular complex involved in the formation of dorsal ruffling. In this thesis, we investigated the interaction betweenα-ACTN4, Mtss1 and WASF2 by using pull-down and co-immunoprecipitation strategies. As the result, Mtssl-GFP was co-precipitated by withα-ACTN4, but,do not depend on the stimulation of PDGF. For pull down assay, His-tagged Mtss1 and its mutants was recombinant expressed in E.Coli. and further affinity purified by Ni2+ agarose. However, pull down assay showed thatα-ACTN4 could not pull down Mtssl and vice versa. This result indicated that the interaction betweenα-ACTN4 and Mtss1 could be indirect. WASF2 complex is the key component of ruffling. We tested whether the WASF2 complex and Mtss1 could interacted with each other. The original data indicated that it could be true but the details remains to be studed,Additionally, our previous data indicated that the expression of specific F-actin binding proteins may be regulated in the development of cerebellum. Here we tested the expression profile of some candidates including Mtss1, Hugl-1, drebrin and coronin during the postnatal development of cerebellum of SD rat. The result suggested that these proteins may relate to the postnatal development since they had expression peak in the postnatal stages.