Mechanisms of APC-Diaphanous mediated actin assembly. Olivia Molinar¹, Richa Jaiswal², Aneliya Rankova², Vince Stepanik¹, Bruce L. Goode², Brooke M. McCartney¹. 1) Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA; 2) Department of Biology, Brandeis University, Waltham, MA.

   Adenomatous polyposis coli (APC) negatively regulates Wnt signaling and stabilizes microtubules, and most recently has been implicated in actin assembly. Both vertebrates and Drosophila have two APC proteins, vAPC and vAPC2, and dAPC1 and dAPC2 respectively. In Drosophila, dAPC1 contains the basic domain necessary for actin assembly function in vertebrates, while dAPC2 does not. vAPC can bundle and nucleate actin filaments through its basic domain, and collaborate with the formin Diaphanous (Dia) to efficiently nucleate actin in vitro. To understand the in vivo function of this activity, our lab uses the assembly of actin caps and pseudocleavage furrows in the syncytial embryo as a model. We have shown that dAPC2 and Dia are required for pseudocleavage furrow extension, but the role of dAPC1 is not well understood. Taken together, it is clear that APC proteins contribute to actin assembly, but significant gaps exist in our understanding of both mechanism and physiological function. To address these gaps, we are taking a coupled in vitro-in vivo approach. in vitro, we have shown that dAPC1 shares a common mechanism of actin assembly with vAPC by exerting its activity through the basic domain and collaborating with Dia via the C-terminal DAD domain. In contrast, dAPC2 does not promote actin assembly alone, but instead inhibits the actin assembly activity of Dia through multiple dAPC2 domains that interact directly with Dia. dAPC2 does not inhibit the activity of other formins, arguing against the possibility that dAPC2 inhibits through a non-specific mechanism. Currently, we are working to understand the mechanism of dAPC2 dependent inhibition in vitro. In parallel, we are assessing the phenotypic consequences of loss of dAPC1 alone and together with dAPC2 and Dia to determine the in vivo role of APC-Dia interactions in actin assembly in the early embryo.