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Browsing Undergraduate Works by Subject "Actin"

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    Investigating the role of NPFs in the Arp3ΔC phenotype
    Kiemel, Jonathan; Rodnick-Smith, Max; Liu, Su-Ling; Nolen, Brad; Wagner, Andrew
    The Arp2/3 complex is an assembly of seven protein subunits that nucleates branched actin networks involved in cellular functions such as endocytosis. Previous work has determined the complex is intrinsically inactive, and can be turned “on” by activators like ATP or proteins called nucleation promotion factors (NPFs). It has been hypothesized that the complex remains in an auto-inhibited state due to the c-terminus of the Arp3 subunit. Deletion of the c terminus (Arp3ΔC) results in a hyperactive Arp 2/3 complex in a purified in-vitro system. Strikingly, this complex is inhibited by the canonical NPF wiskott-aldrich syndrome protein (wsp1). These contrary phenomena are complicated further by the observation of endocytosis in-vivo. In S. Pombe fission yeast, Arp3ΔC generates endocytic patches that have a reduced internalization percentage compared to wild type cells but assemble at nearly the same abundance. This suggests preferential binding of a single NPF to Arp2/3 that polymerizes actin, but in an incorrect manner for endocytosis. Here, we will investigate involvement of dip1 amongst other NPFs in the Arp3ΔC phenotype. Dip1 has been shown to be involved in the temporal regulation of actin polymerization during endocytosis; deletion of this activator results in both decreased patch density and longer but more stochastic patch lifetimes before internalization. Utilizing S. pombe as a model organism, Arp3ΔC will be combined with NPFΔ constructs to determine which activator is responsible for actin polymerization in the absence of the Arp3 c-terminus. Analysis of crosses is accomplished primarily with spinning disk confocal microscopy.