2023 29 B2

Analysis of the Mechanism of Filopodia-Based WNT1 Transport

By: Amayrani Villegas-Parra

Department: Biology

Faculty Advisor: Dr. Kai (Laura) Burrus

Wnt gradients regulate cell proliferation and differentiation during embryonic development and adult homeostasis. Uncontrolled production and transport of Wnt proteins can disrupt the Wnt signaling pathway, particularly in cancerous cells. Though much is known about the downstream mechanism of how Wnt signals are received, less is understood about Wnt gradient production and transport. The Wnt production requires lipid modification by the acyltransferase Porcupine (PORCN) followed by the binding of Wntless (WLS), a cargo transporter that facilitates Wnt transport from the endoplasmic reticulum to the plasma membrane. Proposed models explaining Wnt transport from the producing cell to the receiving cell include diffusion, exosomes, and filopodia. Our data has shown that incubation of Wnt-producing cells with Wnt/β-catenin reporter cells induced over 100-fold more signaling than did the Wnt-conditioned media, suggesting cell contact is critical for Wnt signaling. Our observation that co-expression of WNT1 with WLS, increases signaling activity, relocalize WNT1 to the cell membrane, and induces new filopodia projections, further suggests that Wnts could be transported by filopodia, which then contact target cells. As IRSp53, Cdc42, and Ror2 have been implicated in filopodia formation, I hypothesize that the inhibition of either of these proteins in cells overexpressing WNT1 and WLS will cause a reduction in Wnt signaling. To test this hypothesis, I created and expressed IRSp53_4k, Cdc42_T17N, and DN Ror2 mutant constructs along with WNT1 and WLS in HEK293T cells. I then measured Wnt signaling activity using a SuperTopFlash assay. I found that none of the inhibitory proteins significantly decreased WNT1 signaling activity. Concurrently, we found that WLS promotes the signaling activity of WNT1, but not its so-called functionally redundant sister, WNT3A. Following this differential result, I am assessing differences in the binding of WNT1 and WNT3A to WLS using computational and experimental methods. My findings suggest that neither IRSp53, Cdc42, nor Ror2 appears to be required for WNT1 signaling. Ongoing research includes identifying proteins involved in filopodia induction that are required for Wnt signaling and understanding potential differences in the interaction between WLS and different Wnts.